Systemic sclerosis - scleroderma
Department of Dermatology, University of Leipzig, Germany
Systemic sclerosis is a clinically heterogeneous, systemic disorder which affects the connective tissue of the skin, internal organs and the walls of blood vessels. It is characterized by alterations of the microvasculature, disturbances of the immune system and by massive deposition of collagen and other matrix substances in the connective tissue. This review discusses epidemiology and survival, clinical features including subsets and internal organ involvement, pathophysiology and genetics, microvasculature, immunobiology, fibroblasts and connective tissue metabolism and environmental factors. Early diagnosis and individually tailored therapy help to manage this disorder, which is treatable, but not curable. Therapy involves immunomodulation as well as the targeting of blood vessel mechanics and fibrosis. Physical therapy and psychotherapy are also important adjunctive therapies in this multifactorial disease.
Systemic sclerosis (SSc) is a clinically heterogeneous generalized disorder which affects the connective tissue of the skin and internal organs such as gastrointestinal tract, lungs, heart and kidneys. It is characterized by alterations of the microvasculature, disturbances of the immune system and by massive deposition of collagen. The first detailed description of a scleroderma-like disease was published by Curzio in Naples in 1753. The patient, a young woman suffered from excessive tension and hardness of the skin. Nearly 100 years later, in 1847 Gintrac introduced the term scleroderma, as the skin was the most obvious organ involved. The extensive involvement of internal organs has only been realized in the second half of the 20th century.[3,4,5]
The spectrum of sclerodermatous diseases comprises a wide variety of clinical entities such as morphea (patchy, linear, generalized), pseudo-scleroderma and the overlap-syndromes with similar cutaneous and histopathologic manifestations. (Fig 1) However, these variants will not be discussed further. For the differential diagnosis see Table 1. Due to the complexity of the internal organ involvement SSc has attracted much attention from several disciplines (e. g. rheumatologists, pulmonologists,nephrologists) and therefore, a close cooperation with them is recommended, concerning diagnostic procedures and therapeutic regimens. In addition, the complex pathophysiology of SSc, involving genetic factors, environmental factors, vascular and immune system functions, as well as fibroblasts and matrix substances made SSc attractive to study events leading to autoimmune diseases or connective tissue diseases, in general. Basic functions of various cell types (endothelial cells, T-lymphocytes, monocytes, fibroblasts, mast cells) as well as the production and effects of cytokines, growth factors, and adhesion molecules have been studied and animal models have been developed to give closer insights into the pathophysiology of this disease.
The American College of Rheumatology (former American Rheumatism Association - ARA) has defined criteria, that are 97 % sensitive and 98 % specific for SSc as follows:
proximal diffuse (truncal) sclerosis (skin tightness, thickening, non-pitting induration)
The patient should fulfill the major criterion or two of the three minor criteria.
Raynaud's phenomenon is observed in 90-98 % of SSc patients. It may precede SSc for years and its presence may have predictive value for the subsequent development of SSc, in particular in association with abnormal nailfold capillaries and the occurrence of antinuclear antibodies (ANA).[9,10]
In our experience, the American College of Rheumatology criteria from 1980 urgently need revision, particularly to more adequately incorporate patients with limited SSc. We support arguments that new advances in medical technology provide the opportunity to detect disease in patients who do not meet criteria established in 1980. In accordance with Poormoghin et al. and Lonzeti et al. we support the addition of simple clinical variables such as nail capillary microscopy and anticentromere antibody (ACA) positivity as novel minor criteria. [11,12] With these two new criteria the sensitivity of ARA preliminary criteria was improved from 33 to 97 %.[12,13]
Over the years several attempts have been made to establish a classification system. Such classifications differentiate either different degrees of skin involvement,[14,15] distinct clinical manifestations such as CREST-syndrome (calcinosis, Raynaud's phenomenon, esophagus dysmotility, sclerodactyly, teleangiectasia),[16,17] SSc sine scleroderma, vascular and inflammatory forms, or associations with different autoantibodies.
In the past, acroscleroderma and diffuse scleroderma were distinguished. Acroscleroderma was mainly defined by vascular alterations and by skin sclerosis, limited to acral areas (fingers), while diffuse scleroderma involved both the trunk and the extremities with pronounced inflammation and more rapid progression. On the other hand Barnett et al. and the working group of the German Dermatological Research Community (Arbeitsgemeinschaft Dermatologische Forschung) differentiated three types:
During the last 10 years the majority of researchers have used the classification into limited versus diffuse cutaneous SSc according to Le Roy et al. These categories are described as follows (Table 2):
SSc subsets according to LeRoy at al
Limited Cutaneous SSc
Diffuse cutaneous SSc
More than 50 % of SSc patients belong to the limited SSc. They have a more insidious onset of illness, a long history of Raynaud's phenomenon and swelling of digits, a more benign course, and a lower incidence of renal involvement and restrictive pulmonary disease with a much better prognosis.  Some cases are associated with anticentromere antibodies (ACA).
Patients with diffuse cutaneous SSc have a short history. These patients often have acral sclerosis, arthritis, Raynaud's phenomenon, and rapid progression of skin involvement including arms and trunk. In addition, they have a higher incidence of renal, [21,22] cardiac,  pulmonary disease,  and tendon friction rub.  Antitopoisomerase antibodies (ATA) or antifibrillarin antibodies (against U3 RNA associated protein) may be present. When associated with anti-RNA polymerase, patients with diffuse SSc have the shortest survival time and worst prognosis.
Three phases of dermal involvement can be distinguished:
Compared with other connective tissue diseases SSc is relatively rare. A certain genetic background in combination with the typical immune reactivity determines the susceptibility to express SSc. The true incidence is obviously underestimated since early features are frequently overlooked. The prevalence of SSc is reported to be between 13 to 105 and 13 to 140 per million in North America, Australia and Europe, respectively,[27,28,29] and as high as 290 in South Carolina.
The incidence of SSc is between 2.6 and 20 to 28 per million per year. [29,301,32,33,34] The overall female/male ratio was reported as 3:1..[29,34] However, this ratio is larger in Great Britain (6:1) and in the USA (8:1)...[34,35] The female preponderance is most marked early in adult life (7:1), narrowing toward the fifth decade to 2 - 3:1. The average onset of SSc occurs between 40 and 50 years, but in women it is in the late childbearing years between 30 and 39. Less than 10 % of patients develop SSc before the age of 20.
Several survival studies have indicated a distinct dependency on the internal organ involvement. In general, the survival rate lies between 34 and 73 %. It is, however, shorter with a poorer prognosis in men and older patients than in women and younger patients. Survival differences are also notable in whites as compared to blacks.
The average annual mortality was reported to be between 0.9 and 3.8 per million population per year; in Australia it was 4.1. The overall 5 and 10 year survival rates were 86 % and 69 %, respectively in Sweden. There is a 4.6 fold risk of death compared with the general population, it is even worse among male patients with the diffuse subset. Most patients die of cardiopulmonary or renal disease. There is also an increased cancer mortality, particularly of the lung.
A logistic regression model identified 3 factors: proteinuria, elevated ESR and low carbon monoxide diffusing capacity, that in combination, had an accuracy of 80 % in predicting mortality. The absence of these 3 factors was associated with 93 % survival.
The extent of skin sclerosis (skin score) was revealed to be a useful marker of both severity and prognosis. Sclerodactyly alone is associated with 79 84 % survival at 5 years and 47 % to 75 % survival at 10 years, while truncal SSc at disease onset reduces survival to 48 to 50 % at 5 years and 22 % to 26 % at 10 years. Five year follow-up resulted in the following predictive factors of survival in 264 patients: older age ( 64 years), reduced renal function (blood urea nitrogen 16 mg/dl), anemia (haemoglobin 11 gm/dl), reduced pulmonary diffusing capacity for carbon monoxide ( 50 %), total serum protein level ( 6 gm/dl), and reduced pulmonary reserve (forced vital capacity 80 %).[31,32] Out of 646 patients studied before angiotensin converting enzyme inhibitor treatment was available, none of the 24 patients with kidney disease at onset survived for 6 years and the 1-year survival was only 25 %.
SSc in general affects the connective tissue, predominantly of the skin and vessel wall and, to a lesser extent, of the gastrointestinal tract, heart, lungs and kidneys.
Cutaneous symptoms, often associated or preceded by Raynaud's phenomenon and arthralgias of the fingers, are usually early signs in the course of SSc and therefore helpful for establishing the diagnosis and initiating therapy.
Raynaud's phenomenon is episodic digital ischaemia provoked by cold or emotion. It is characterized by three phases: (1) palor due to vasospasm (2) cyanosis due to ischaemia (reduced oxygen content) and (3) rubor due to reactive hyperemia. Raynaud's phenomenon can be observed in 3-4 % of the general population and as a benign transient condition in 20-30 % of young women. However, it is associated particularly with connective tissue diseases and is seen in 98 % of SSc patients, occurring as the first symptom in up to 70 % of SSc.  Raynaud's phenomenon is associated with the following in SSc:
Sclerosis of the skin is the leading feature of SSc. It starts at acral areas, particularly at the fingers and hands, later at the face including lips and the frenulum of the tongue (Figure 2-7). In several patients skin manifestations remain limited to acral areas for years. In others, however, sclerosis ascends progressively from the extremities to the trunk and finally, affects the whole integument. On the contrary, in a few cases skin alterations appear at the trunk and then spread over the whole body surface within a short period of time. As a rule these patients suffer from severe internal organ involvement and have a poor prognosis.
In the course of the disease sclerosis develops gradually. It starts with edema, turns into sclerosis and ends in an atrophic stage. Thus, fingers are initially swollen with nonpitting edema, and are later hardened. Besides hypo- and hyperpigmented areas, teleangiectasias and trophic painful ulcerations, in particular at acral locations such as fingertips and knuckles (rat bite necroses), and atrophy are found. Hair loss and anhidrosis reflect the degeneration of appendages due to surrounding fibrosis. In some cases calcinosis, with secondary infection of slowly healing ulcerations leading to gangrene and acroosteolysis, may cause articular deformities and dissolution of terminal phalanges. Typical features of the face involvement are shrunken nose, microcheily, reduced mouth aperture and microglossy. In addition, radial furrowing around the lips, teleangiectasias and changes in the pigmentation ( mottled or diffuse hyperpigmentation resembling Addison's disease or focal hypopigmentation as postinflammatory pigment incontinence) are typical. Sclerosis limits expression, leading to a mask-like stiffness of the face.
A subgroup of patients characterized by extensive calcinosis, Raynaud's phenomenon, esophageal dysmotility, sclerodactyly and widespread teleangiectasias is described as CREST-syndrome which has been previously reported as Thibičrge-Weissenbach syndrome. This entity is now classified within the subset of limited SSc.
It is generally accepted that the survival of SSc patients decreases as skin sclerosis extends proximally and progressively, in particular when evaluated within the first 2 to 3 years of disease onset.[31,32,37,40,41] Therefore semiquantitative measures of the skin score are well advised and are performed by clinical palpation and evaluation on a scale of 0 to 3 in 10 to 15 body areas, or more objectively by ultrasonographic measurement of skin thickness[47,48] or by using both wet and dry forearm skin biopsy weight, which correlates well with the forearm skin score (see therapy). 
Again, the usefulness of the skin thickness score has recently been confirmed as a predictor and correlate of the outcome in SSc. In addition, the degree of skin involvement can be assessed by therapists and patients themselves using a questionnaire regarding functional ability and disability, respectively. The neck sign consists of ridging and tightening of the skin of the neck on extension of the head. It is positive in more than 90 % of SSc patients.
Histopathology of the skin
Histopathologic findings are not very characteristic in SSc and similar to morphea. At the early stage one observes mild inflammatory infiltrates consisting of lymphocytes (mostly T helper cells), monocytes, histiocytes and plasma cells around the blood vessels and ducts of the eccrine sweat glands, and partly also in the interstitial tissue and subcutaneous fat tissue. The collagen fibers are edematous.
In the later stage these infiltrates are reduced or disappear completely. The vessel walls are thickened and hyalinized; their lumen is narrowed leading to devascularization. The collagen bundles are thickened and densely packed with eosinophilic staining. Only very few fibroblasts are seen (acellular sclerosis). This is true for the reticular dermis and their septae deeply extending into the subcutis. The eccrine sweat glands are atrophic due to surrounding fibrosis and they are located in the upper part of the dermis.[5,23]
Abundant accumulation of connective tissue and matrix proteins is first seen in the vicinity of blood vessels in the reticular dermis and at the border of the subcutaneous tissue. By electron microscopy, irregular, thin, newly synthesized collagen fibrils with a diameter of 10-30 nm are seen. Later, in addition to the small fibers, large diameter fibers occur as a sign of aging, in a bimodal distribution.[53,54,55] Ultrastructural studies of the vessel revealed vacuolization and damage of endothelial cells, reduplication of the basement membrane and activation of the rough endoplasmic reticulum of pericytes and fibroblasts.
Involvement of internal organs
Prognosis of SSc largely depends on involvement of internal organs, particularly the lungs, heart and kidneys. The gastrointestinal tract, although most frequently affected, is less life threatening. It has been demonstrated that severe organ involvement often occurs early in the course of diffuse SSc. As their survival is markedly reduced, these patients should be monitored very closely during the first three years and potential disease modifying therapies must be initiated early.
Esophagus and gastrointestinal tract
The gastrointestinal tract is frequently involved in SSc. The esophagus in is involved in more than 85 % of cases with resultant dysphagia and phagodynia due to hypomotility, reflux, peptic esophagitis, Barrett's metaplasia and fibrotic strictures. Esophageal involvement is diagnosed by conventional radiography (barium swallow) which shows a stiff glass tube appearance (figure 8), by manometric measurements, and by sensitive scintigraphic procedures that are quantitative and non-invasive. The occurrence of malignancies in the esophagus is low as shown in a follow-up study of long-standing reflux esophagitis and Barrett's esophagus.
Peristaltic abnormalities may delay gastric empting (watermelon stomach) and may affect motility of the small and large intestinum leading to pseudoobstruction or malabsorption due to bacterial overgrowth. In addition, diverticular ulcerations, stenosis, chronic obstipation, megacolon and rectal prolapse have been described.[62,63,64,65,66] The histopathology of the gastrointestinal tract shows mild inflammatory infiltrates of the lamina propria and in certain segments, atrophy and fragmentation of the smooth muscles followed by collagen deposition and changes of the blood vessels.[58,66] Liver is rarely involved primarily in SSc. It may be secondarily affected, a sequela of right ventricular heart disease. However, the association of SSc with primary biliary cirrhosis is more often described. This disorder is seen in 17 % of 189 patients, predominantly in the CREST variant with anticentromere and antimitochondrial antibodies[68,69] Pancreatic exocrine function is often reduced, but is usually of minor clinical impact in SSc. The gastrointestinal manifestations are summarized in Table 3.
The frequency of lung involvement in SSc ranks second to gastrointestinal manifestation; it varies from 40 to 90 %. The risks for severe pulmonary manifestation are the following: male sex, diffuse SSc, presence of ATA and inflammatory signs, and significantly reduced diffusion capacity. Pulmonary hypertension and lung fibrosis are to be differentiated. Pulmonary hypertension is a significant cause of death, thus it decreases the 5 years survival from 90 % to 50-70 % .[72,73] The early clinical features may often be missed and lead to greater morbidity and mortality.
Pulmonary hypertension occurs more frequently in limited SSc, while restrictive fibrotic lung alterations are more frequently observed in patients with diffuse SSc. In the CREST-syndrome, irreversible pulmonary hypertension can develop despite the absence of fibrosis. In pulmonary hypertension arterioles show concentric intima proliferation, media hypertrophy and, in part, myxomatoid degeneration, finally leading to narrowing of the lumen and increase of pressure in the pulmonary circulation. The pulmonary blood pressure is best diagnosed by Doppler echocardiography (Table 4). Pulmonary hypertension with dyspnea and right-sided failure occurs in 5 to 10 % of patients.
Lung fibrosis develops after alveolitis with proliferation of connective tissue by fibroblasts under the influence of growth factors and chemokines such as platelet -derived growth factor (PDGF), insulin growth factor (IGF-1), MCP-1, and MIP-1a..[76,77] The disturbance of lung function is characterized by restrictive changes with reduced respiratory volume and by impaired diffusion capacity. In addition, obstructive changes involving both small and large airways may be present.[78,79] Radiographic findings are characterized by fine-reticular, partly nodular, fibrosis of the lung areas. There are also thickened septae and netlike stripes near the blood vessels. Later, with increased fibrosis, a cystic transformation takes place, representing the so-called ³end-stage-lung² or ³honeycomb lung² (Figure 9). The introduction of high resolution computer tomography has significantly increased the sensitivity of radiographic diagnosis, particularly in terms of the early detection of alveolitis. Bronchoalveolar lavage (BAL) is able to evaluate the activity of florid alveolitis as shown by an increase in the number of alveolar macrophages, neutrophilic and/or eosinophilic granulocytes, and immune complexes. The number of lymphocytes is normal. In prospective studies, BAL have been shown to be a useful predictor of the course of SSc lung disease: the presence of alveolitis is associated with worsening dyspnea, worsening of chest radiographic findings, and a significant decline in both forced vital capacity and carbon monoxide transfer factor.
Cardiac involvement is often present, but rarely significant clinically. Even dyspnea and retrosternal pain are attributed mainly to other organs such as the lung or esophagus. In addition, syncope and angina pectoris may be caused by either endothelial damage of small coronary arteries or myocardial fibrosis due to other basic diseases. It is hard to prove the specificity of this involvement due to SSc itself. The prevalence reported in the literature depends upon the diagnostic methods chosen. Myocardial perfusion scintigraphy, ventriculography and echocardiography are the most sensitive techniques. Clinically manifested forms have been described in 20-25 % with a 70 % mortality after 5 years. However, autopsy revealed alterations such as myocardial fibrosis and pericardial effusion in 30-80 % of patients.[81,82] Myocardial fibrosis occurs as patchy or diffuse forms. Repeated episodes of ischemia and reperfusion lead to the destruction of the myocardium and replacement by connective tissue. In electrocardiographic studies of 80 SSc patients, hypokinetic alterations of the left ventricle were found. Electrocardiographic abnormalities such as conduction system disturbances (27 %), signs of infarction (13.8 %), and non-specific ST and T-wave changes (13.8 %) were observed in agreement with echocardiographic findings. Arrhythmias are common and adversely affect survival. Ventricular tachycardia can occur in up to 19 % of patients. Echocardiography is very sensitive in detecting even small pericardial effusions and heart valve alterations. Very useful also is the thallium scintigraphy. The involvement of coronary arteries may lead to myocardial infarction. In addition to morphological alterations of the coronary arteries, also a cold-induced coronary spasm (cardial Raynaud's phenomenon) is discussed. Clinical pericarditis is present in only 10 to 15 % of patients with SSc and is more common in patients with limited disease.[82,89]
Affection of the kidney has the worst prognosis and highest mortality of all internal organs involved. Clinically 10 40 % are affected, but by autopsy, the figure is 80 %. Patients with diffuse skin involvement carry a high risk of an acute renal crisis characterized by malignant arterial hypertension with headache, vision disturbances, cramps, left ventricular hypertrophy and retinopathy. It generally manifests itself within the first 4 years. However, the chronic form develops slowly over years and leads, in 50 % of affected patients, to a moderate reduction of kidney function, often clinically inapparent. Diagnostic criteria are proteinuria (< 1 g/24 h), azotemia (blood urea nitrogen (BUN) > 25 mg/100 ml), arterial hypertension (> 140/90 mmHg) and reduction of the glomerular filtration rate. Factors predictive to renal crisis are listed in Table 5.
The pathogenetic events are not completely understood. The primary event seems to be the damage of endothelial cells with thickening and proliferation of the intima with deposits of glycoproteins and mucopolysaccharides in the interlobular and small arcuade arteries. These changes lead to narrowing or even obliteration of vessel lumina and ultimately to infarction of glomeruli and tubuli. This is followed by platelet aggregation, adhesion and liberation of growth factors.
These morphological changes in combination with functional vasospasm due to cold (renal Raynaud's phenomenon) reduce the cortical blood circulation resulting in the liberation of renin, increase in the plasma rennin levels, and hyperplasia of the juxta-glomerular apparatus. The angiotensin converting enzyme(ACE) inhibitors were shown to significantly improve the 5 year survival of SSc patients. While under no treatment only 16 % with renal involvement survived after 1 year; the 5 year survival of the treatment group was 45 %. Thus, ACE inhibitors are able to limit kidney failure and prolong survival.
Muscles, joints and bones
Weakness of muscles is a common (60-80 %) finding in SSc and is associated with minimal elevation of creatinin kinase and aldolase and polyphasic motor unit potentials on electromyography. However, in 6 to 12 % an inflammatory myositis indistinguishable from polymyositis, associated with PM-Scl antibodies, can develop, sometimes accompanied by myocarditis.[94,95,96] The most consistent electromyographic abnormalities are a decrease in amplitude and duration of single potentials with a concomitant increase in polyphasic potentials.
Arthritis is the initial symptom in 2/3 of SSc patients, often preceding the typical skin changes. It may resemble rheumatoid arthritis at the onset of the disease. It is less destructive, however. Contractures are generally dermatogenic due to sclerotic changes of the overlying skin or surrounding connective tissue. Alterations of the bones occur in 6% of patients and consist of resorption of the tufts of the terminal phalanges, juxta-articular osteoporosis, erosions of the dorsal heads of metacarpal and proximal phalangeal bones. Joint space narrowing with marginal erosions may be present in up to 9 15 % of patients.
Periarticular subcutaneous calcification can be complicated by painful ulcerations which may also be located around the iliac crest, spine protuberances, elbows, and knees. The juxta articular tendons of the fingers, forearms, legs, and neck can be altered by fibrosis causing audible friction rubs as a characteristic clinical sign of SSc.
Sjögren Syndrome is defined as an inflammatory disorder of salivary glands often characterized by circulating antibodies to Ro (SSA) and La (SSB) antigen. Depending on the diagnostic criteria used it occurs in 5 to 90 % of SSc patients, in particular those with limited disease.[100,101]
Neurological manifestations include peripheral neuropathy with reduction in the conduction velocity and patients often show a prolonged response to local anaesthesia. Trigeminal neuralgia is found in about 4 % of patients; carpal tunnel syndrome occurs in 3 % of patients.[103,104]
Hematological abnormalities are mostly related to renal disease, microangiopathic hemolytic anaemia or from bleeding gastrointestinal teleangiectasias.
Although pregnancies are less frequent in SSc, women can safely have healthy deliveries. Steen has prospectively observed 91 pregnancies during a 10-year period. No increase in the frequency of miscarriage was found except in those with long-standing diffuse scleroderma. In 29 % of pregnancies, preterm birth occurred and all but one of the infants survived. While Raynaud's phenomenon improved during pregnancy, esophageal reflux became worse. After delivery some women with diffuse SSc had increased skin thickening. In early diffuse SSc three patients suffered from renal crisis. Therefore patients with early diffuse SSc should wait until their disease stabilizes before becoming pregnant. High risk pregnancy management is required. The higher risk of premature birth is explained by decidual vasculopathy, which is similar to that seen in hypertension and is associated with poor perinatal outcome. A trend toward a reversed ratio of decidual CD4 to CD8 positive T cells is seen in SSc which is not seen in normal or hypertensive pregnancies. PDGF and TGFb do not appear to be involved in the pathogenesis of decidual vasculopathy in SSc.
Pathophysiology of SSc
The course and even the initial events in the pathogenesis of SSc are still poorly understood. The microvasculature (endothelial cells, platelets, capillaries) is one of the first affected systems, sometimes preceding the outbreak of the disease even by years (Raynaud's phenomenon). There is evidence that the disease may be immunologically triggered, again as an early event. T-lymphocytes in collaboration with monocytes, endothelial cells, platelets and mast cells act as mediators and targets in the pathophysiological network. They express and release adhesion molecules, interleukins, and growth factors which act upon fibroblasts. Nevertheless, the excessive tissue fibrosis is due to expansion of fibrogenic clones of tissue fibroblasts, which behave relatively autonomously and overexpress genes encoding extracellular matrix components. [109,110] This leads to excessive deposition of collagen and other connective tissue matrix proteins in the skin and internal organs as well as in the walls of blood vessels.
As in various autoimmune diseases, the pathogenesis is partly based on genetic background and modulated by environmental factors. In the following chapters the three main pathogenetic pathways will be discussed. These include microvasculature abnormalities, abnormal immune response, and dysregulation of fibroblast activity. It is important to remember that these abnormatities occur in the context of the necessary genetic and environmental background. (Figure 10)
The most prominent genetic factor is gender (female:male = 3 to 6:1). Another factor is the human major histocompatibility complex (MHC). An increased frequency of class I and II MHC alleles were found. However, their nature and association were controversial (i.e. HLA Bw35, DR1, DR5 or HLA1-B8-DR3). On the other hand, the linkage of DR5 and DR3 to DRw52 is suggested to be the primary MHC class II allele associated with SSc. In addition, there is an association between the development of lung fibrosis and B8-DR3-DRw52-DQB2. Pulmonary disease can be predicted if DR52a and ATA are present (relative risk 16.7). In addition, HLA DRw11 and a DQ sequence were associated with severe SSc and ATA positivity.
Racial and ethnic origin also plays a role in disease susceptibility; diffuse disease is significantly more likely in black than white women. Takeuchi et al. found an extreme difference of genetic background of Scl-70-positive SSc with regard to HLA-DR betweeen Japanese and other ethnic groups. In particular, the association of TAP1 and TAP2 with DRB1*1502 was increased in Japanese SSc patients with the diffuse form and with ATA. In Mexican patients DR5 (DRB1*1104) plays a role in genetic susceptibility for the disease.
A cluster of structurally unrelated gene products such as complement components (C2, factor B, C4A and C4B), heat shock protein (HSP70), 21 hydroxylase (CYP), and tumor necrosis factor (TNF) exhibit a high degree of polymorphism. In these studies HLA C4A null alleles provide the strongest correlation of the MHC with SSc, and HLA-DQA2 is an additional primary susceptibility marker.
Finally, clastogenic activity has been described in sera and cell extracts from SSc patients. Chromosomal breakage, deletions, and acentric fragments were increased in lymphocytes and fibroblasts (15.5 % versus 1.7 % breaks in healthy controls). Interestingly, bleomycin exerts clastogenic activity and is able to induce a scleroderma-like disease. The increased spontaneous and clastogen-induced chromosomal damage rates indicate that SSc lymphocytes may have a general susceptibility to DNA damage caused by free radicals.
Numerous clinic features of SSc are similar to chronic graft versus host-disease (GvH). Several years ago the hypothesis was discussed, that persistent cellular microchimerism might play a role in the pathogenesis of SSc. Microchimerism results from the movement of fetal cells (perhaps also stem cells) through the placenta during pregnancy into the maternal circulation and their persistent survival even for decades due to HLA class II compatibility or minor differences.[122,123,124]
Among 86 female SSc patients, 16 experienced spontaneous abortion as compared to 9 % in healthy women. In 17 % of women SSc started during their pregnancy. In female SSc patients the presence of fetal CD3 positive T cells in the maternal circulation[121,127] and of fetal cells in the affected tissue has been identified through y chromosome specific DNA sequences by means of quantitative PCR in statistically increased amounts as compared to healthy controls. The persistent microchimerism might cause SSc in certain patients by initiating a fetal anti-maternal GvH like response. Again, other trigger factors are required to prevent tolerance and to convert this type of GvH from the latent into the manifested form. In male SSc patients, allogenic cells from the mother or a twin, or white blood cells after blood transfusion may survive and act in a similar way.
Raynaud's phenomenon, increased vascular wall thickness, vascular occlusion, devascularization, and thickening of the basement membrane are features which were described many years ago. Changes in the nailfold capillaries are one of the first signs in SSc.[130,131] Furthermore, vascular injury is the basis for the major clinical manifestations of SSc including pulmonary hypertension, myocardial dysfunction and renal involvement.
In internal organs, in particular the kidney, arteriols are characterised by intimal proliferation, thinning of the media, and fibrosis of the adventitia, and exhibit accumulation of proteoglycans and collagens, probably produced by myofibroblasts (Figure 11).
In addition, the vascular pathology is associated with altered vascular function, with increased vasospasm, reduced vasodilatory capacity, and increased adhesiveness of the blood vessels to platelets and lymphocytes.
The role of endothelial cells (EC) is still poorly understood. On the one hand, EC are targets of immune activity. On the other hand, they may act as immune costimulators. One of the prevailing hypotheses suggests an origin in repeated insults to the vascular endothelium, in particular after cold exposure. The vascular abnormality may be caused by repeated episodes of vasoconstriction leading to hypoxia, ischemia, and intravascular occlusion
Nail fold capillaries
Prominent SSc vascular abnormalities are noted in capillaries and small blood vessels. Affected capillaries are characterized by distorted and irregular loops. The changes include reduced numbers of capillaries and the presence of avascular areas as shown by nail fold capillaroscopy, even in the preclinical stages (figure 12). On the ultrastructural level the earliest changes consist of large gaps between endothelial cells, vacuolisation of endothelial cytoplasm, an increase in the number of basal lamina-like layers, and disruption of endothelial cell cytoplasmic membranes.[135,136]
The most prominent clinical vascular dysfunction in SSc is related to dysregulation of the vascular tone leading to vascular spasm and reduction in blood flow, best illustrated by Raynaud's'phenomenon. It results from digital arterial closure after cold exposure. In SSc, an imbalance in endothelial signals (increased vasoconstrictory endothelin release), impaired vasodilatory mechanisms (nitric oxide - NO, - endothelial dependent relaxation factor - EDRF), enhanced platelet aggregation and deficient neuropeptide levels lead to the well recognized vasospastic propensity in the disease. Other damaging influences, such as toxic factors, proteases (granzyme 1), lipoperoxides, and IgG anti-endothelial autoantibodies (see autoantibodies) may contribute to this process.[138,139]
The increased level of urinary F2-isoprostanes supports the hypothesis that free radical-catalyzed peroxidation of arachidonic acid occurs in SSc.[140,141] The increased release of endothelin, thromboxane, factor VIII antigen, and thrombomodulin are signs of such injury to EC, partly also mediated by anti-EC-antibodies.[139,142]
Increased plasma endothelin levels have been associated with Raynaud's phenomenon and SSc, particularly diffuse SSc. Endothelin exerts a prolonged vasoconstriction and is profibrogenic as well, enhancing fibroblast proliferation and collagen synthesis. Thus, it may be a major link between vascular pathology and the abundant deposition of connective tissue matrix materials in SSc. Increased endothelin expression in microvascular endothelial cells of the upper dermis in association with an increased number of endothelin-binding sites is also reported in SSc. In addition, this occurrence is related to lung fibrosis.
Endothelial dependent relaxation factor (EDRF), nitroxide (NO)
Deficient endothelial dependent relaxation in SSc is suggested by impaired maximal responses to endothelial dependent vasodilators such as bradykinin and substance P in conjunction with defective endothelial production of the vasodilator NO. Presumably, the endothelial NO synthase gene expression is inhibited, in particular by TGFβ. Thus the impaired NO production may contribute to platelet activation and to oxidative injury of endothelial cells, as well as promote inflammation and enhance the arteriolar internal proliferation in SSc.
Endothelial cell apoptosis
Endothelial cell apoptosis may also be a primary event in scleroderma. It may be related to viral infection, including cytomegalovirus (CMV), in view of the increased levels of anti-CMV antibodies measured in SSc and the remarkable similarities between CMV vasculopathies and SSc vascular disease. On the other hand, endothelial apoptosis may be related to immune reactions to environmental factors, reperfusion injury, or to antiendothelial antibodies.
Angiotensin converting enzyme (ACE)
Angiotensin converting enzyme (ACE) is located on the luminal surface of the endothelium. Decreased plasma ACE activity was reported in SSc patients. ACE levels inversely related to levels of vWF have been proposed as markers of EC injury. It is not clear yet if ACE plasma activity is a reflection of decreased synthesis or inhibition of enzyme activity. Further indicators of vascular injury include increased serotonin-induced platelet aggregation. In addition, platelets release thromboglobulin, platelet factor 4150, cytokines, and growth factors [platelet derived growth factor (PDGF) and transforming growth factor (TGF)], which can themselves activate EC. This indicates an imbalance between endothelial and platelet function.
In SSc, EC expressing increased numbers of ligands of 1-integrins as well as MadCAM1, CD34, ELAM-1, and ICAM-1 facilitate the interaction with lymphocytes, which express 1- and 2-integrins. In this way the transcapillary migration of inflammatory cells is mediated, leading to prominent T-cell infiltrates around blood vessels in early skin lesions. The antiendothelial autoantibodies also induce leukocyte adhesion to EC. Circulating levels of endothelin-1, P-selectin, E-selectin, VCAM-1 and ICAM-1 are useful markers of vascular and fibrotic change in SSc. They correlate well with their in situ activity.
Abnormalities in fibrinolysis have often been seen. Evidence for accelerated fibrinogen turnover, fibrin deposition, and altered regulation of plasma fibrinolysis have been obtained. Finally, deficiencies in complement regulatory molecules with a protecting function, such as membrane cofactor protein and decay-accelerating factor, may contribute to the vascular damage.
Increased numbers of mast cells have been described in patients with diffuse SSc. These mast cells are activated and their granules can regulate fibroblast biologic activity.[157,158]. Mast cell heparin is a potent stimulus for bFGF and TGFβ, suggesting the contribution of mast cells to the inflammatory and fibrotic components of SSc.[159,160]
The location of inflammatory infiltrates, mainly CD4-T-cells, around blood vessels and at sites of active connective tissue formation suggests their pathogenetic role.[152,161]. The majority of T-cells are HLA DR-positive. The absolute lymphocyte counts and the relative amounts of lymphocyte subsets in sera are controversially described (CD4+ or CD8+ T-cells, memory and natural killer cells). However, an increased ratio of helper CD4+ T lymphocytes to suppressor/cytotoxic T lymphocytes has been reported in patients with SSc.[163,164] The T-cell function is not uniform as far as the response to various mitogens or the results of the autologous mixed lymphocyte reaction. Skin extracts and collagen act as antigenic stimuli for T cells in scleroderma patients. Vδ1 + γ/δ T-cells are increased in both the blood and lungs and show evidence of antigen-driven selection. Increased expression of c-myc, c-myb and c-ras protooncogenes, as determined by the RNA hybridization technique was found in peripheral T lymphocytes, but not in B cells in SSc patients, indicating early and late activation of T cells. The antibody production to primary immunization and recall antigens is normal.
Lymphocyte as well as monocyte derived cytokines, such as interleukin (IL)-1, IL-2, IL-4, IL-6, and receptors, such as soluble CD4 and IL-2R, were elevated in the circulation. Most IL-2 is secreted by T lymphocytes, while monocytes/macrophages express high affinity IL-2 receptors and respond to exogeneous IL-2. IL-2 can induce up to 40-fold elevations in the secretion of active TGFβ by monocytes. These data suggest that activated T cells produce IL-2 which upregulates TGFβ in monocytes, which in turn activates fibroblasts to secrete and organize the elements of the extracellular matrix. Thus, the scenario for fibrosis is complete. In addition, it has been shown, that type 2 cytokine producing T cells, not only CD4+ T cells but also CD8+ T cells, play important roles in the pathogenesis of SSc, especially in the early phase. Peripheral blood T-lymphocytes showed both TH1 and TH2 activation.
Lymphocyte and monocyte ligands, L-selectin, sialated glycoproteins, LFA-1 (CD11a, CD18), and Mac1 (CD11b, CD18) bind to the EC receptors and modulate the migration of these cells. In addition, lymphocytes respond to chemotactic stimuli, produce activation of fibroblasts via ICAM-1, and bind to non-cellular integrins expressed on collagen and fibronectin via surface VLA-1 (CD49a, CD29) and VLA-4 (CD49d, CD29). These processes explain the reciprocal activation of both immune cells and fibroblasts by direct cell contact as well as by the indirect effects of soluble cytokines.[161,162] Finally, the late phase of graft versus host disease (GVHD) resembles an immune-cell-mediated reaction with scleroderma-like features (see also microchimerism).
The non-specific humoral immunity leads to abnormalities such as hypergamma-globulinemia, polyclonal B-cell stimulation, autoantibody production and immune complex formation.
The various autoantibodies in SSc are not closely related to the pathogenesis. Although the majority of autoantibodies are epiphenomena, some others are quite specific for SSc and its subsets (Table 6).
In the majority of SSc sera, autoantibodies to intracellular antigens are recognized. However, an individual patient's sera contains only a limited number of self antigens, often in a disease specific manner. The particular autoantibody present is often indicative of clinical expression, disease course and overall severity. In SSc with highly variable clinical features such information is a valuable aid to the diagnosis and prognosis of an individual patient.
Anti nuclear antibodies (ANA) have been detected in approximately 85% of SSc patients. With repeated investigations during the course of the disease, they have been found in approximately 98%.
Three main serological subgroups in SSc were described, each characterized by a distinctive pattern of clinical features: sera containing anti-DNA-topoisomerase I (anti-topo I) antibodies, sera containing antibodies recognizing one or more centromere proteins (CENPs-A, -B, -C and -D), and sera containing antibodies to RNA-polymerase III (RNAP III) (Table 6). Each of these ANAs can be detected in roughly 20-25 % of patients, and they are generally considered to be mutually exclusive.[173,174,175,176,177]
Antitopoisomerase antibodies (ATA)
ATA are detected in a speckled staining pattern in HEP2 cells by indirect immunofluorescence in a total of 23% SSc patients. A 70 kD protein found in SDS polyacrylamide gel electrophoresis is the antigenetically active, proteolytic fragment of the native 100 kD molecule. It mediates the relaxation of the supercoiled DNA. ATA occur more frequently in Thai and Japanese patients than in African Americans. ATA are associated with specific amino acid sequences of the first domain of the HLA DQb1 tyrosinase residue at position 30 and HLA DR11. They are directed against at least seven different epitopes; some are homologous to certain mammalian n35gag retroviral proteins. The association of ATA with DR3 and DRW52a means a significantly higher risk of pulmonary interstitial fibrosis. ATA correlate with diffuse cutaneous SSc (dSSc). While anti-topo I positive patients are associated with the highest frequency of pulmonary interstitial fibrosis, risks are intermediate with respect to cumulative survival times and frequencies of dSSc and renal involvement.
Anticentromer antibodies (ACA)
ACA have been described by Moroi et al. in 1980. In indirect immunofluorescence they appear as punctate spots dispersed in the interphase nucleus, localized to the constriction (centromer, kinetochor) on metaphase chromosomes. In the Western blot they reflect 4 different proteins (Cenp A, B, C, D). Cenp B (80 kD) is almost universally found, while A (19 kD) and C (140 kD) are the next most common. They correlate with the presence of polar amino acids at position 26 of the HLA DQb1 first domain. In general, ACA are associated with HLA DR1, DR4, DR8, DR11, DQ7 (DQb1*0301). In combination with Raynaud's phenomenon they predict the evolution of SSc. ACA occur nearly mutually exclusive with ATA. ACA interact with cell division (mitosis) and are closely associated with CREST-syndrome. They occur in a total of 28% of SSc patients. The ACA group has the best prognosis of the three with the longest cumulative survival times and the lowest frequency of dSSc, pulmonary involvement and renal disease.
Anti RNA polymerase antibodies (A RNA PA)
A RNA PA are again of major significance, reflecting the worst prognosis. RNA polymerase represents multiple subunits of three enzymes (Pol I, II, III), which are responsible for the protein biosynthesis by ribosomes. A RNA PA are shown in a punctate fine speckled nucleolar pattern. Patients with anti-RNAP III antibodies exhibit the greatest risk of dSSc, the highest mean maximum skin sickness score, the shortest cumulative survival times, and the greatest likelihood of renal involvement compared with patients in either of the other two groups. These mutually exclusive serological subgroups of SSc may be associated with etiologically distinct disease processes or alternatively, the different antibody patterns may reflect differences in patient's vulnerability.[181,182]
Antibodies to fibrillarin (U3RNP) (AFA)
Fibrillarin represents a 34 kD basic protein associated with U3RNP and is responsible for the processing of the periribosomal RNA. AFA occur in 7 - 9% of SSc patients, more frequently in African Americans (56%). They are of minor significance. AFA can be detected in the fibrillar region of the nucleus by immunoelectronmicroscopy, in a clumpy pattern of nucleolar staining by indirect immunofluorescence, or most reliably, by immunoprecipitation of radiolabelled cell extracts. AFA are associated with diffuse skin and multiple organ involvement, pulmonary arterial hypertension, skeletal muscle involvement, teleangiectasias and early disease onset.
In addition, minor serological subgroups of other antinucleolar antibodies also occur in SSc. For instance, anti-PM/Scl antibodies (4 % to 11 % in SSC) are usually associated with limited SSc or the SSc/polymyositis overlap, sclerodermatomyositis (70% of SSc/PM overlap cases). PM-Scl antigen consists of 11 to 16 polypeptides of which two proteins of 75 and 100 kD have been identified as the major antigenic components. Anti PM-Scl antibodies appear in a homogeneous nucleolar pattern by indirect immunofluorescence. Autoantibodies to PM-Scl bind to an area with homology to the nuclear localization signal found in HIV tat protein and SV40 large T antigen. This indicates that viral antigens, which share epitopes of the host, may initiate an autoimmune response via molecular mimicry. Antibodies against HIV proteins in HIV-negative SSc patients can be explained in this way. They are associated with HLA DR3, A1, B8. Anti PM/Scl positive patients had a favourable outcome in terms of response to corticosteroids or immunsuppressive therapy as well as decreased disability due to muscle weakness. Such patients have a good prognosis. Anti PM/Scl antibodies predict no serious visceral involvement.
On the other hand, anti-To/ThRNP antibodies (4%) are recognized in limited SSc, hypothyroidism, and small bowel involvement. To/Th represent the mitochondrial RNAs responsible for the endoribonucleolytic cleavage of mitochondrial primer RNA involved in the replication of mitochondrial DNA. It is distributed in different cellular components such as nucleus, nucleolus, cytoplasm, and mitochondria and on different proteins as well. It shows a homogeneous nucleolar pattern.
Anti-U1-RNP antibodies are found in about 6% of patients, often associated with the SSc/systemic lupus erythematosus (SLE) overlap syndrome or mixed connective tissue disease (MCTD), arthritis, isolated pulmonary arterial hypertension, and early disease onset.
Anti Ro-antibodies (9%) are indicative of a very severe and rapidly progressive disease course, including renal failure and pulmonary hypertension.
Finally, anti-Ku, anti-Jo1 (anti-histidyl tRNA-synthetase) and anti-PL7 (anti-treonyl-tRNA-synthetase) antibodies occur in a small proportion (5%) of SSc patients, particularly those with SSc/polymyositis overlap syndrome. Anti-Jo1, when associated with dermatomyositis/polymyositis, interstitial lung fibrosis, arthritis and Raynaud's phenomenon, is termed anti-synthetase syndrome. Jo1 represents a family of 20 enzymes, which must properly recognize the tRNA and the amino acid to maintain fidelity of translation.
Anti-agalactosyl IG antibodies were detected in 52 of 70 SSc patients (74%). Elevated levels were associated with the presence of contracture of phalanges, pulmonary fibrosis and with more severe SSc (diffuse ATA positive form).
Spencer Green et al. investigated the test performance of ATA and ACA in SSc in a metaanalysis of 479 articles. Thirty of them fulfilled the inclusion criteria. They evaluated the sensitivity of ACA in 32% (57% in limited SSc) and of ATA in 34% (40% in dSSc). Either test was positive in 58 %. In only 3 patients both antibodies were present. In other connective tissue diseases ACA occured in 5% and ATA in 2 % compared to the controls (< 1%). They concluded that both antibodies are highly specific. As 40 % of SSc patients are likely to have neither antibody (ACA, ATA) present, the negative result does not exclude the diagnosis. When considering anti-RNA polymerase antibodies the percentage of antibody negative SSc patients is only about 20.
The pathogenetic role of these autoantibodies is mostly unknown. Although some of these SSc specific autoantibodies are capable of inhibiting the cellular function of the autoantigens they recognize in vitro, they are unlikely to have access to the intracellular locations of these antigens in vivo. On the other hand, some of the autoantibodies may recognize extracellular antigens or those exposed on the cell surface. These autoantibodies could be involved in the disease pathogenesis.
It has been shown that anti-endothelial cell-antibodies, after binding to vascular endothelial cells, can induce endothelial activation, upregulation of cell adhesion molecules, and consequent monocyte adhesion, possibly via an autocrine IL-1 mediated effect. In addition, these EC-antibodies are able to increase the synthesis and release of coagulation factors such as factor VIII and thrombomodulin as well as induce EC apoptosis. Altogether anti-EC-antibodies may affect microvessels more distinctly than macrovessels. Their prevalence in SSc is between 28 and 85%, approximately 44% in limited SSc (lSSc) and 84% in dSSc. The low affinity anti-EC-antibodies may indicate an epiphenomenon of vascular injury, while the high affinity antibodies are rather pathogenic. For example anti-EC- antibodies have been associated with vascular involvement, digital ischemic ulcers, and alveolo-capillary impairment.[192,193] In addition, they can mediate EC cytotoxicity by direct complement activation.
In SSc certain human lymphocyte antigen (HLA) alleles occur in an increased frequency, with major differences in various ethnic groups (see also genetics). The HLA-DR associations are particularly strong when clinical subsets and/or autoantibody defined subgroups of SSc are considered, thus explaining an increased susceptibility to express the clinical features of SSc in certain subgroups (Table 7). [176,194,195] Furthermore, some autoimmune responses might be due to linkage disequilibrium of certain DR-alleles with particular DQ-alleles.
Finally, an association between anti-topo I positive silica-associated SSc (SI-SSc) and the HLA-A-DR3 alleles has been shown. Anti-topo I positive idiopathic SSc cases were associated with DR2 and DR5. Obviously different pathogenetic mechanisms lead to the production of anti-topo I antibodies in the two groups and a different topo I derived peptide was presented by MHC class II molecules in SI-SSc. Probably different kinds of antigen-presenting cells (APC) containing different proteases are involved or alternatively, the altered antigen-processing was caused by macrophage derived cytokines following silica exposure.
In conclusion, the here described autoantibodies are relatively disease specific for SSc. Although they may not be directly involved in the disease pathogenesis, they are extremely reliably associated with disease specific pathologic phenomena and are therefore valuable predictors of the different subtypes of SSc. The main three antibodies are highly disease specific. However, approximately 20% of SSc patients are likely to have no antibody present which means that negative results do not exclude the diagnosis of SSc. Consequently, it is now possible to identify over 80 % of SSc patients by these main three autoantibodies. Both antigen-driven and molecular mimicry hypotheses have been proposed for ANA induction in SSc. Some autoantibodies are homologous to certain mammalien p30gag retroviral proteins.[179, 185] Autoantibodies are important for the early diagnostics of the specific type of SSc and the initiation of the appropriate therapy. In the future, autoantibody testing may - at least in part - be used to determine the clinical activity of the disease and monitor a patient's response to immunological therapies. Clinical studies are required to show whether the course of the titer or the immunglobulin class of the autoantibodies correlate with the disease activity.
Physiologically, skin fibroblasts synthesize little extracellular matrix (ECM) because of various inhibitory influences and negative feedback through non-cellular matrix components. With activation signals from lymphocytes and monocytes, endothelial cells and platelet fibroblast properties are altered either directly, by cell contact, or indirectly, via specific cytokines, such as IL-1, IL-2, IL-4 (proliferation, collagen synthesis) and IL-6 (matrix metallo-proteinases). Interferons potentially suppress collagen synthesis. The activating factors, PDGF and TGFβ, are also released from platelets. Ultimately, activated fibroblasts release cytokines and growth factors, such as IL-1, prostaglandin E, TGF&beta, connective tissue growth factor (CTGF), PDGF, and IL-6, which may exercise self-activation via an autocrine loop.[200,201] In this way ICAM-1 is expressed on fibroblasts, which augments adhesion and retention of immune cells within the tissue.[202,203] Up to now, there are no data showing apoptosis of fibroblasts in SSc lesions or in culture. This is in agreement with results reporting unchanged numbers of fibroblasts in SSc lesions compared to healthy skin. Therefore, the fibrotic events in SSc would be due to the changed synthesic activity of matrix proteins rather than to changed cell numbers present in the skin.
Cytokines and growth factors
Several cytokines and growth factors, such as IL-1, IL-2, IL-4, IL-6, IL-8, IL-10, IL-13, TGFβ, PDGF, TNFα, interferon (IFN)-γ and particularly the high-affinity IL-2 receptor, can also be found elevated in the serum of SSc patients.[205,206,207] To some extent they are correlated with the degree of organ involvement and disease activity.
IL-2 is produced by activated T-cells. IL-2 receptor is shed from them, particularly in the early active stage of SSc. Collagen promotes IL-2 production and laminin induces IL-2 receptor expression on lymphocytes. Endothelial cell membranes increase c-fos expression in T-cells, which themselves induce IL-2 expression. This may explain the presence of activated T-cells in the perivascular infiltrate of SSc skin.
IL-4 promotes T-cell adhesion to EC, promotes differentiation of lymphocytes, and stimulates fibroblast proliferation and extracellular matrix (ECM) synthesis. In SSc sera, IL-4 is correlated to the extent of skin fibrosis. IL-4 induces TH2 cells, resulting in low levels of IFN-γ, a potent inhibitor of collagen synthesis. Its lack may enhance the fibrotic reaction.
IL-6 is produced by various cells in the skin and is elevated in serum in a high percentage. IL-6 is synthesized by fibroblasts and induces ECM production, also in an autocrine loop. Finally, it regulates the high-affinity IL-2R in lymphocyte cultures, most likely regulating the effect of IL-2 on immune activation in SSc.[201,208]
TGFβ clearly activates fibroblasts to produce increased amounts of ECM components such as collagen I, III, V and VII, and fibronectin. In human fibroblast cultures, TGFβ1 induces its own expression. In involved and uninvolved skin, TGFβ1, was detected by some authors by in situ hybridization and immunohistochemical staining. The presence of TGFβ1 prior to the onset of fibrosis indicates an early involvement of this factor in the pathogenesis of SSc. It was shown that TFGβ1 increases the promoter activity and type I collagen mRNA and protein synthesis in fibroblasts.[210,211] In addition, TNFα, IL-1 and IFN-γ modulate the expression of the collagen I gene, partly by their influence on transcription factors acting on collagen I gene regulatory elements. TGFβ2 was shown to co-localize with enhanced collagen type I gene expression in the perivascular infiltrate of SSc skin. TGFβ is the only known inducer of connective tissue growth factor (CTGF). CTGF gene expression and skin sclerosis are correlated in SSc. Contrary to the results with normal fibroblasts, TGFβ1 does not upregulate tissue inhibitor of metalloproteinases 1 (TIMP-1) in SSc fibroblasts with already elevated spontaneous secretion of TIMP-1; it is suggested to be an autocrine growth factor in SSc.
CTGF is a potential mediator in terms of the maintenance of the fibrotic phenotype of fibroblasts. Besides promoting mitogenic stimulation, CTGF acts chemotactically on fibroblasts and induces the synthesis of extracellular matrix proteins. In addition, it is able to induce fibrosis in mice after subcutaneous injection. CTGF exhibits a permanent overexpression of mRNA in fibrotic lesions of SSc skin and in isolated fibroblasts, indicating its involvement in the pathogenesis of SSc. CTGF maintains fibrosis, as its increased basal expression can not be inhibited in vitro. Its effect on the responsive element within the collagen promoter was shown. Contrary to TGFβ the mRNA expression of CTGF is higher in the fibrotic phase than in the inflammatory one.[212,217]
PDGF acts as a strong mitogen for fibroblasts. As SSc fibroblasts are previously exposed to elevated PDGF in vivo, they respond less to PDGF in vitro when compared with normal fibroblasts. Again, PDGF and its β-receptor subunits are immunohistochemically located in the perivascular inflammatory infiltrates of SSc skin, but not in healthy skin. In vitro TGFβ selectively increases the expression of the β-receptor subunit in SSc fibroblasts and the subsequent incubation with PDGF AA distinctly enhances the proliferation of this "clonally selected" fibroblast type in SSc. In addition, c-myc and c-myb mRNA protooncogene expression is increased in intralesional fibroblasts, as already shown in activated T-cells of SSc.
Matrix protein metabolism
In SSc, abundant deposits of collagen type I are found in the perivascular region of the dermis and at the border between deep dermis and subcutis, as demonstrated by immunohistochemistry and in situ hybridization (using 1 chain-specific cDNAs). In addition, collagen types III, V, VI, VII, fibronectin and tenascin are also overexpressed in the skin.[219,220,221] Tenascin can inhibit the attachment and spreading of fibroblasts. Glycosaminoglycans and decorin, which have also been found in the skin, may serve as cytokine receptors and modulators of collagen fiber diameter. Thus, altered dermatan sulfate proteoglycans may affect the organization of matrix fiber in SSc. In addition, decreased collagenase expression is characteristic of most SSc fibroblasts. Both increased collagen synthesis and decreased collagenase expression may result in excessive accumulation of collagen, indicating that the balance between these synthesizing and degrading processes is crucial and may be modulated by TIMP-1 expression.
Recently, it was shown that elevated TIMP-1 levels in SSc patients support the hypothesis that matrix accumulation occurs in SSc at least in part owing to decreased degradation. Moreover, the increase in TIMP-levels at early stages of SSc explain the early progressive course of dermal fibrosis. Altogether tissue matrix accumulation may be due to increased inhibitors rather than to decreased metalloproteinases.
One of the most interesting phenomena in the pathophysiology of SSc is the persistence of the fibrogenic phenotype. In situ as well as in vitro fibroblasts with different synthesizing capacities for extracellular matrix proteins could be found, indicating the heterogenicity of fibroblasts in SSc. Heterogenity in gene expression in SSc is not limited to collagen. Recently it has been demonstrated that protease nexin1 (PN-1) is overexpressed in SSc. This may play a role in increasing collagen gene transcription. Le Roy characterized the so-called collagen high-producers of fibroblasts isolated from the deep reticular dermis of patients at the early stage of SSc. This altered gene expression may be due to turning on autocrine signals in the fibroblasts, that, once activated, stimulate a continuous feedback loop. Alternatively, a certain subpopulation of fibroblasts may be selected with the preferential property of proliferation and matrix protein synthesis in vivo. On the other hand, fibroblasts from unaffected skin do not show exaggerated matrix production, which indicates that a basic fibroblast defect does not exist.
Interestingly, these different properties are maintained ex vivo for several generations under laboratory culture conditions. This phenomenon found in vitro can be explained either by clonal selection of fibroblasts in vivo, by clonal selection during outgrowth from the biopsies or by the influence of artificial culture conditions. Freshly isolated SSc fibroblasts are significantly and selectively insensitive to exposure to PDGF and to deprivation of serum derived factors. Therefore it was proposed that SSc fibroblasts can produce their own autocrine growth factors.
When fibroblasts are cultured in 3-dimensional collagen gels, they change their morphology into elongated bipolar shapes with filopodia due to interaction with the ECM, and they contract the gels into a dense connective tissue matrix. Consequently, fibroblast functions are altered (protein synthesis and, in particular, collagen synthesis at the protein and mRNA level). Gel contraction is mediated via the α2 and α1 subunit of integrins. In SSc several fibroblast lines did not show downregulation of collagen synthesis, particularly when they maintained elevated mRNA stability.[231,232] Finally, the (2 integrin subunits are less expressed in SSc fibroblasts,indicating a disturbance of the feedback between ECM and fibroblasts, which are unable to adjust their collagen synthesis to the amount of surrounding ECM molecules. On the other hand, integrin α2 production by TGFβ stimulation is not impaired in SSc fibroblasts. No significant changes in α1 integrin expression could be observed. In addition, altered fibroblast susceptibility to apoptosis may play a role in the pathogenesis of fibroblast abnormalities. Thus, SSc fibroblasts are more resistent to Fas-mediated apoptosis than normal fibroblasts. While TGFβ1 can induce such resistence in normal fibroblasts, there is an increase in TGFβ1 receptors on SSc fibroblasts. Thus, TGFβ may alter fibroblast sensitivity to apoptosis and have long term effects on collagen synthesis. In addition, endothelin-1, overexpressed in SSc protects fibroblasts from c-myc dependent apoptosis[237,238]. Finally, hypoxia can also select for apoptosis resistent cells by inducing apoptosis in c-myc overexpressing cells.
It is now evident that persistent overproduction of collagen is responsible for the progressive nature of tissue fibrosis in SSc. Upregulation of collagen gene expression in SSc fibroblasts appears to be a critical event in this process, reflecting a fundamental alteration in the regulatory control of gene expression in SSc fibroblasts. Trans-acting nuclear factors which bind to cis-acting elements in enhancer (intronic) and promoter regions of the genes modulate the basal and inducible transcriptional activity of collagen genes.
Concerning regulation of collagen synthesis, there are several candidates to inhibit the fibrotic process, such as antibodies to TGFβ or lysylhydroxylase inhibitors, which interact with cross-link formation between the collagen chains. The most exciting approach might be the direct inhibition of transcription factors by antisense oligonucleotides, which interact with specific DNA elements that control the activity of these genes in fibroblasts.
Fibroblasts subjected to mechanical strain proliferate, elongate, and become bipolar and oriented along the plane of the force. Prominent action stress fibers develop within the cells as in myofibroblasts. These specialized fibroblasts have been found in excess in SSc skin and are required for generation of force in mechanically stressed lattices. They are characterized as "synthetic" phenotype: synthesis of collagen is induced, whereas that of metalloproteinases is repressed. Mechanical stress also regulates the expression of cytokines such as TGFβ1, -β3 and CTGF which have been previously shown to play an important role in the development of fibrosis.
Models of SSc
Several animal models can help to better understand the pathogenesis of SSc, although there are distinct differences in the clinical features. One of the most important models in living humans are the environmentally induced forms of SSc, such as silica-induced SSc, which cannot be distinguished from idiopathic SSc by clinical or laboratory features.
Induced animal models include GvHD, bleomycin-induced fibrosis, injection of glycosaminoglycans from the urine of SSc patients, and fibrosis due to exposure to organic solvents. Hereditary models are the avian SSc of the UCD200 chicken and the tight skin mouse 1 and 2. Among these models, the tight skin-2 mouse seems to be the most promising model because it shows, at the same time, enhanced collagen synthesis and deposition as well as infiltration of mononuclear blood cells in the dermis.
Environmental factors induce SSc-like diseases. Several reports describe chemical compounds within our environment and their ability to induce SSc-like diseases upon exposure[244,245,246] (Table 8). These substances can induce SSc-like diseases that can be distinguished from SSc by the following features:[244,245]
Chlorinated hydrocarbons may induce systemic diseases, but aromatic hydrocarbons are associated with local fibrosis restricted to the areas of direct contact. Other substances such as bis(4-amino-3-methylcyclo-hexyl)-methan, bleomycin, pentazocin, and 5-hydroxy-tryptophan and carbidopa, are not discussed here. Tryptophan, used for insomnia and depression, can trigger an eosinophilic fasciitis similar to Shulman syndrome or the eosinophilia myalgia syndrome, due to a synthetic contaminant.[248,249] In the early 1980s the toxic oil syndrome caused by rape seed oil contaminated with aniline derivatives, attracted a great deal of attention when more than 300 of the 20,000 people affected died.
The clinical and laboratory data reveal that the common markers of idiopathic SSc are also present in silica induced SSc. Due to this, and together with the similar pathophysiology, silica should be accepted as an inducer of SSc. This is in agreement with Rodnan et al., Rustin et al., and Gabay and Kahn.[251,254,255]
Our experimental data concerning the exposure of various cell cultures such as macrophages/monocytes, endothelial cells, and fibroblasts to silica support the hypothesis that silica mediated cell activation can play a role in the pathogenesis of SSc.
However, the long exposure times needed for the onset of silica induced SSc and the fact that not all exposed persons develop both silicosis and SSc suggest that silica alone does not cause SSc. The development of SSc in a silica exposed individual will depend not only on the length of exposure but also on the individual genetic background of the host (see also genetics, autoantibodies).
In addition, exacerbation of SSc or even the new onset of cases of SSc have been observed after X-ray treatment in a generalized form.
In 1996, five cases of SSc which occurred shortly after episodes of physical trauma were described. In addition, physical exertion was reported as a possible precipitating factor in both adult and paediatric eosinophilic fasciitis.[258,259] In 1996, Vancheeswaran et al. noticed a significant association between trauma and childhood SSc. Trauma has been associated with linear scleroderma, which often occurred in the same site as trauma within 6 months.[261,262]
The question of whether the trauma actually caused the disease or was only coincidental has a major legal implication. The most probable hypothesis is that physical trauma and/or emotional stress may activate subclinical or aggravate preexisting disease. The observation of ANA, anticentromere antibodies, and the the particular HLA pattern favours this hypothesis.
Treatment of SSc
SSc is characterised by a variety of clinical features and a long term life threatening course. Therapy has to be planned individually because of inadequate knowledge of the point at which therapeutic action is appropriate and the difficulty of obtaining objective measurements of the treatment results. Beside basic recommendations, physiotherapeutic activities and competent psychological guidance are important. The therapy is directed at 3 pathogenic compartments.
A critical attitude to therapy and a great deal of patience as well as psychological empathy are important for successful guidance of patients, because very often treatment results can only be seen after several months.
In SSc is a rare disease with a chronic, and occasionally also self-limiting course, with a variety of possible internal organs pathologies and different stages of disease. To add confusion to the evaluation of treatment results, most published trials have few patients enrolled. This lack of large well designed, multicenter (multinational), double-blind, and placebo controlled trials leaves a major gap in our efforts to evaluate current recommended therapy, the more so as natural improvement and spontaneous regression confound the picture. Objective measures, such as the skin score which evaluates skin thickness at various locations, must be standardized to allow accurate comparisons. Other measurements related to signs of inflammation and autoimmunity or organ function are also required to adequately evaluate treatments. Therefore clinical trials in SSc must (semi) quantitatively define disease activity, specific stage of disease, joint motility and level of internal organ involvement as published recently by Medsger et al. They developed a severity grading scale from 0 (no documented involvement) to 4 (endstage disease) for 9 potentially affected organ systems and tested it in 579 SSc patients. In addition, they validated this scale by using an independent group of 680 SSc patients and achieved international agreement. The severity scale will assist in the design and conduct of clinical trials and serve as a framework for developing a SSc disease index.
In 1995, the European Scleroderma Study Group initiated a multicenter prospective one year study for this reason. 290 patients were consecutively recruited by 19 different research centers in different countries: 173 with limited and 117 with diffuse SSc. A high degree of variability in epidemiological and clinical features emerged, indicating the necessity to draw up a standardized procedure for the management of patients with SSc. For this reason, three separate 10-point indices of disease activity were constructed: one for patients with diffuse SSc, the second for patients with limited SSc and the third for all patients with SSc. The parameters were revealed to be feasible, reliable and valid preliminary indices to define disease activity in SSc.
Thorough education and councelling is necessary to achieve good compliance.
Arranging patient consultations with skilled psychotherapists is very important. This creates confidence, increases compliance, and assists the patients in their ability to maintain a comfortable quality of life. Furthermore, the positive influence of autogenic training could be shown by the following criteria in the Raynaud's phenomenon: reduction of the number of attacks and increase in the skin temperature, even though the increased blood levels of neuropeptides could not be modified. Relaxation hypnosis can be recommended as additional complementary therapy of SSc. The exchange of experience and councelling is very helpful in self help groups. Their work should be actively supported by dermatologists.
SSc is non curable, but treatable, though therapeutic generally moves slowly.
Vasoactive substances are used to induce vasodilation, normalize platelet function, and eventually, to increase fibrinolysis. The profound upregulation of 2-adenergic receptors responsible for abnormal smooth muscle tone and the repeated episodes of ischaemia-reperfusion and tissue hypoxia with endothelial cell dysfunction have been suggested as targets of therapy.
Prostacyclin (PgI2) and its analogue (Iloprost) mediate vasodilation, inhibit platelet aggregation, α production and adhesion of granulocytes to endothelial cells. In addition, they increase fibrinolysis and exert a cytoprotective effect on endothelial cells. Finally, they mediate the formation of vasodilatory molecules, like endothelial cell dependent relaxation factor (EDRF), and inhibit the production of free oxygen radicals. Besides vascular effects, prostacyclins are thought to regulate immune responses, influence fibrous tissue formation and modulate red cell deformity.
After intravenous infusion for 5 to 10 days the function of blood vessels was improved for several weeks. All together there have been five trials of I.V. iloprost compared with placebo in SSc related Raynaud's phenomenon.[272,273] This therapy can be continued with intervals during the cold season. The initial intravenous dose is increased from 0.5 ng/kg/min. to 2 ng/kg/min. This therapy promoted the healing of ischemic finger tip ulcerations.[274,275]
In our own experience the serum levels of s ICAM-1, s VCAM-1 and soluble E-selectin were initially elevated and significantly reduced after 5 days of iloprost infusion. The measurement of the serum concentrations of VEGF and endothelin-1 revealed decreased levels after therapy, too. These results indicate that the well-known clinical benefit of iloprost infusions on Raynaud's phenomenon is serologically detectable by the reduction of serum levels of endothelin-associated adhesion molecules, cytokines and growth factors reflecting an improvement in endothelial function.
Headache, flushing, vomiting, abdominal pain, fatigue, and water retention have to be considered as these are common side effects. Continuous intravenous or inhalative application of iloprost or epoprostenol was effective in SSc associated pulmonary hypertension.[277,278] Aerosolic iloprost might be potentially useful as treatment for CREST syndrome associated pulmonary hypertension. However, patients have to be carefully selected. 100 µg/d aerosolic iloprost improved the quality of life in all 5 patients, decreased the NYHA functional class, and increased the distance walked within 6 minutes.
Oral application of iloprost (50 µg twice daily) was disappointing. Beraprost sodium, another oral prostacyclin analogue showed a trend towards decreasing digital ulcers in a multicenter double-blind placebo controlled trial over 6-12 months.
Calcium channel blocker
Calcium channel blockers inhibit the intracellular uptake of calcium and consequently the contraction of smooth muscle cells in the vessel wall, mediated by calcium dependent protein kinases. Three doses of 10 mg nifedipin or nicardipin, which exerts less side effects than nifedipin, are able to reduce frequency and severity of Raynaud's attacks, to increase the digital blood flow, and to inhibit the formation of new finger ulcerations as shown in double-blind studies after 6 weeks[284,285]. In addition, there has also been shown a platelet aggregation inhibiting effect. The reduced myocard perfusion due to primary or secondary heart involvement can be improved by nifedipin. Typical side-effects have to be considered: hypotension, headache, flush, tachycardia, and vomiting.
Inhibitors of angiotensin converting enzymes (ACE)
The ACE-inhibitor, captopril, (75-150 mg per day orally) exerts protective effects on the kidney and decreases the blood pressure. In patients suffering from nephrogenic hypertension and kidney involvement it is effective and helps to prolong the interval until kidney transplantation becomes necessary, and may even prevent renal failure or facilitate patients' withdrawal from chronic dialysis. The drastically reduced survival rate of 16% within the first year is significantly increased by captopril to approximately 55% in SSc patients suffering from kidney disease. The increased plasma renin levels are reduced by captopril and indirectly the levels of bradykinin and prostaglandin are increased, leading to improvement of kidney perfusion.
In addition, the healing of finger ulcerations is accelerated and the frequency and severity of Raynaud's attacks are positively influenced. As side-effects, gastrointestinal complaints, bone marrow depression (leucopenia), and hypotensive dysregulation have to be considered.
These drugs including reserpin, prazosin, phenoxybenzamin and methyldopa act by blocking sympathic vasoconstriction. They can improve Raynaud's phenomenon only in higher doses, so that orthostatic side-effects are frequently observed, limiting the application of these drugs.
Pentoxyphyllin is able to reduce the number of rat bite necroses and to improve the peripheral blood circulation. On the other hand, the frequency and severity of Raynaud's phenomenon remains unaltered by pentoxyphyllin. Other properties of pentoxyphyllin are, however, very interesting:
Low molecular dextran
This compound applied as an infusion inhibits the platelet aggregation and increases fibrinolytic activity in plasma. As serious side-effects, anaphylactic reactions due to the formation of Immunoglobulin G antibodies have to be considered.
In SSc patients with reduced fibrinolytic activity in serum application of stanozolol, urokinase, or recombinant tissue plasminogen activator can be tried. The efficacy was ranked as relatively low.[292,293,294,295]
This hormone exerts its vasoactive effect by release of a stable prostacyclin degradation product (6 ketoprostaglandin F1 alpha). After 10 days of infusions of 100 IU per day, 89% of SSc patients reported the long term improvement of acral blood circulation and Raynaud's attacks. Similarly positive effects were seen after application of calcitonin gene-related-peptides.
Endothelin-1 (ET-1) receptor antagonists
The use of an ET-1 antagonist in SSc is an attractive idea, because it may act not only as a vasodilator but also may reduce the pro-fibrotic action of ET-1. Bosentan (Roche Holding AG) is being tested as an ET-1 receptor antagonist in the treatment of primary and secondary forms of pulmonary hypertension.
Nitric oxide (NO)
Despite the evidence of increased NO production in SSc there is also evidence of injury to the endothelium, downregulation of endothelial NOS, and defective production of NO. Attempts to improve blood flow by administering the substrate for NO, L-arginine, significantly decreased laboratory-induced Raynaud's phenomenon in SSc patients but did not alter vascular responses to acetylcholine or sodium nitroprusside. In addition, a NO-generating gel applied topically increases skin blood flow as measured by photoplethysmography. Inhaled NO allows selective pulmonary vasodilation and reduction of pulmonary arterial pressure. Studies of inhalation therapy with NO and prostacyclins (iloprost) are underway.
Inhibitors of angiotensin II
Angiotensin II is a profibrotic hormone. It induces angiotensin II receptor type I (AT1) on fibroblasts and also induces the synthesis of TGFβ1 and of extracellular connective tissue.[302,303] Studies with angiotensin II inhibitors to reduce fibrosis in SSc have not been done yet. In addition, the AT1 receptor antagonist, Losartan, decreases plasma levels of TGFβ1 in chronic allograft nephropathy and reduces heart fibrosis and radiation-induced lung fibrosis in animal models. When comparing Losartan with nifedipin, Losartan led to a greater reduction in the severity of Raynaud's phenomenon and in pro-collagen type I N-terminal propeptide. Finally, the outcome of the high renin state of SSc renal crisis has improved.
Raynaud's phenomenon also occurring in internal organs involved in SSc leads to hypoperfusion and ischaemia with free radical and other reactive oxygen species production. SSc autoantigens are susceptible to fragmentation by oxidative stress, thus potentially promoting the autoimmune process in SSc. An abnormal level of oxidative stress and a deficiency of dietary antioxidants are reported in SSc.[308,309] Intravenous administration of superoxide dismutase inhibits fibrosis in an animal model. The antioxidant probucol exerted beneficial effects in the treatment of Raynaud's phenomenon in SSc. The powerful thiol-containing antioxidant, N-Acetylcysteine, was administered to 22 patients with SSc in a 1-year, parallel, double-blind, placebo-controlled prospective study. Most parameters remained unchanged, however, most patients belonged to the late stage of SSc. In addition, N-acetylcysteine was applied in a multicenter, open clinical trial for 11 weeks as intravenous infusions for 5 days starting with a 2 h loading dose of 150 mg/kg, subsequently adjusted to 15 mg/kg/h. Twenty-two patients completed the 5 day infusion and 20 of them the posttreatment follow-up. Frequency and severity of Raynaud's attacks were significantly reduced; the same was true for ulcerations. The cold challenge test mean recovery time fell by approximately 70 % between the beginning of treatment and day 12 and 61. These preliminary data support the hypothesis that N-acetylcysteine may ameliorate damage induced by reactive oxygen species.
At the early stage of SSc infiltrations of inflammatory and immune cells, such as neutrophils and/or eosinophils, in the alveolar space of lungs, monocytes, T-lymphocytes, and mast cells in lesional skin and involvement of joints and muscles are prominent clinical features of the disease. Traditional anti-inflammatory medication such as non-steroidal anti-inflammatory drugs or corticosteroids provide only little benefit.
Prednisolone (initially: 40-100 mg/d, maintenance dose: 10-15 mg/d) or methylprednisolone in general inhibit inflammation while they exert a catabolic effect on collagen synthesis (atrophy) and stabilize the cross linking of collagen fibrils. In addition, synthesis of other extracellular matrix proteins is inhibited. (Methyl) prednisolone is indicated in the treatment of inflammatory episodes of SSc, sclerodermatomyositis and overlap-syndromes, in particular in arthritis, myositis, alveolitis and vasculitis. However, there is evidence that corticosteroids (> 15 mg/d prednisolone) increase the risk of triggering a SSc renal crisis. In addition, various significant side-effects due to long-term maintenance treatment such as hyperglycemia, osteoporosis, hypertension, peptic ulceration of, muscle atrophy, and suppression of the cortex of adrenal glands have to be considered. Altogether the clinical experience is disappointing for a dramatic effect of corticosteroids.
Non-steroidal antiinflammatory drugs (NSAIDs)
NSAIDs are useful to ameliorate the pains of joints, muscles and tendons symptomatically. Major side-effects are ulceration of the stomach, in particular when combined with corticosteroids.
There is one uncontrolled study in a small group of 11 SSc patients that minocycline (50 mg BID) had beneficial effects. One patient died; two patients dropped out due to non compliance; two patients suffered from an acute renal crisis. Minocycline is suggested to exert anti-inflammatory effects and to inhibit the formation of free oxygen radicals. In our laboratory we were not able to show in-vitro effects on fibroblast function. In addition, there is concern about the tetracycline-induced autoimmune disorders including lupus-like disease.
Cyclophosphamide combined with prednisolone is able to inhibit the progression of lung fibrosis in smaller groups of patients with SSc and idiopathic lung fibrosis more distinctly than after prednisolone monotherapy.[317,318] Patients with early inflammatory changes, such as alveolitis of the lung, showed the most beneficial effects. However, side-effects such as infections, leucopenia, and hemorrhagic cystitis often force dose reductions. Intravenous pulse therapy seems to be better tolerated than daily oral administration.
In a 24 week randomized double-blind trial, methotrexate in a weekly dose between 15 and 25 mg, achieved improvement of skin score, creatinin-clearance and general well-being in 68%.
Azathioprine in a dose of 2-3 mg/kg/d is characterized by a better spectrum of side-effects when compared to other immunosuppressants. In a prospective double-blind, randomized and placebo-controlled study Ragun et al. showed improvement of parameters of lung function and a marginally significant survival advantage due to prednisolone/azathioprine therapy in comparison to prednisolone monotherapy in idiopathic lung fibrosis, which is similar to SSc lung fibrosis.
Cyclosporin A inhibits effects due to T helper cells and IL-2 secretion. It decreased procollagen III levels, improved skin lesions and healed acrosclerotic ulcerations. However, lung function did not improve. Above 3 mg/kg body weight nephrotoxic side-effects have to be taken into consideration. Thus, besides hypertension and increase creatinine levels, fatal outcome due to renal complications have been described.[322,323]
Plasmapheresis is expected to remove immune complexes, mediators and autoantibodies from the blood circulation and to stop the progression of the disease or even to improve the disorder.[324,325] Long-term follow-up studies are rather disappointing.
Other immunosuppressive principles
Antilymphocyte globulin, CD4 antibodies, and IL-2 receptor (CD25) antibodies, have been used in anecdotal cases.[327,328] Concerning CD4 antibodies, reports have been published in psoriasis and rheumatoid arthritis, in particular.[329,330]
Extracorporal photochemotherapy has been introduced in the treatment of malignant T-cell lymphoma and autoimmune diseases. In an interesting study two treatments on two consecutive days per month for 6-10 months were performed and compared to 750 mg/d penicillamine. The improvement of the skin score was relatively small. A recently designed European study ended disappointingly after an enthusiastic beginning, when improvement of the internal organs involved, could not be achieved. In addition, a new controlled trial shows no significant difference in skin score.
Autologous stem cell transplantation
Case reports of allogenic bone marrow transplantation (BMT) performed for malignancy have shown that autoimmune diseases were eradicated concurrently. Due to significant morbidity and mortality, allogenic BMT is no longer utilized for the treatment of human autoimmune diseases. Instead of BMT, autologous stem cell transplantation (ASCT) was performed after immunoablative pretreatment in 64 centers of 20 countries in 70 SSc patients with a mean follow up of approximately 2 years. Inclusion criteria for this trial were diffuse SSc, disease duration < 3 years, progressive course, skin score > 20 and involvement of one or more internal organs such as lung, heart and kidney, however, with minor abnormalities in their function. The mortality rate was 17%, in part caused by interstitial pneumonitis, possibly due to an exaggerated response to total body irradiation. 70% of patients had a > 25 % improvement in their skin score and stabilization of lung function. This is still an experimental therapy, and currently limited to patients with severe, progressive life-threatening disease. In addition, there are also risks of the infusion of untreated auto-reactive lymphocyte clones despite immunoablative pretreatment regimens or defective stem cells reactive to chronic auto-antigenic stimulation. Thus, ATA were not eliminated indicating that no "new or healthy" immune system could be reconstituted. Even high dose cyclophosphamide (4 g/m_) alone not followed by ASCT resulted in improvement of some SSc patients.
Oral tolerance to collagen
A phase I trial using ingestion of bovine type I collagen in SSc patients demonstrates significant reductions in levels of IFN and IL-10 in the in vitro response of peripheral blood monocytes, indicating that oral administration of collagen could induce oral tolerance as measured by T cell reactivity. However, the target of this therapy may only be a secondary mechanism.
Thalidomide is known as an immune modulator which alters cytokine production. Thalidomide was given to 44 patients with chronic GvHD skin disease and a complete response occurred in 14 patients, a partial response in 12 and no response in 18 patients. Ten SSc patients were treated in an open-label dose-escalating trial with improvement of skin fibrosis. However, severe peripheral neuropathy and chronic dermatitis have to be considered.
Inhibitors of fibrosis
The inhibition of geranylgeranyltransferase I causes an inhibition of expression of genes encoding type I and type III collagen without effecting cell viability. This might be an interesting target of therapy. Thus, rotterlin, a protein kinase inhibitor exerted a powerful dose-dependent inhibition of type I collagen synthesis and reduction of collagen A1 mRNA steady-state levels in normal and SSc fibroblasts. Polypeptides including relaxin and interferons may also limit fibrogenesis. Inhibitors of post-translational modification of collagen such as inhibitors of prolyl 4 hydroxylase and lysyl oxidase may also prevent deposition of collagens. However, general manipulation of collagen metabolism may cause adverse effects prohibiting clinical trials with these substances.
Penicillamine has been used for many years as a first choice drug. It labilizes the cross links of lysin/hydroxylysin-aldehyde groups of collagen and maintains collagen in a soluble form, sensitive to degradation. In addition, certain immunomodulating and zytostatic effects have been discussed. In slowly increasing doses from 150 to 750 mg/d for years numerous side-effects such as proteinuria, leucopenia, thrombocytopenia, ulcerations of the mucosa, gastrointestinal problems, dysgeusia, cholestasis, myasthenia, myositis, pemphigus and lupus erythematosus were found in up to 47 % of patients. Thus, therapy had to be stopped in 29 % of patients. The therapeutic effects were moderate: softening of the skin and cessation of the progression of lung fibrosis.[344,345] A recently performed double-blind randomised controlled study has shown that the high dose D-penicillamin therapy (750-1000 mg/d) is not superior in terms of skin softening, frequency of renal crisis and death rate as compared to low dose therapy (125 mg/alternate day). On the other hand, there was no significant clinical difference between penicillamine and placebo, either.
Furst and Clements would use D-penicillamine only in a patient who has been on this drug for a prolonged period of time (without significant adverse events) and who refuses to discontinue the drug. However, recently, Medsger concluded from his experience that D-penicillamine favourably alters the natural history of skin involvement in diffuse SSc even when used in low dose. Furthermore, recurrence of diffuse skin change after discontinuation and improvement in skin thickening after re-initiation of the drug support its effectiveness. Nevertheless, due to its high rate of side-effects and the questionable therapeutic effect, penicillamine should, in our opinion, no longer be used in the treatment of SSc. A: Penicillin G
Penicillin G belongs to the prolylhydroxylase-inhibitors and has been used therapeutically by our group for many years in a dosis of 10 Mega IU/day as an intravenous short-term infusion over 30 minutes for 10-15 days. Although clinical studies do not exist, we use this regimen at the early edematous stage of SSc and have found improvement of the skin score (softening of sclerosis) and a decrease in the frequency and severity of Raynaud's attacks. However, we were not able to influence the collagen metabolism in fibroblast cultures in vitro.
Other prolylhydroxylase inhibitors such as hydralazin, diphenylhydantoin and chlorpromazin as well as L-Dopa and glutamine did not achieve therapeutic relevance.
Bucillamine is a derivative of D-penicillamine. It inhibits the proliferation of TH1 cells and transendothelial T cell migration. Six SSc patients were treated with 200-300 mg/d bucillamine for 12-36 months with some improvement of skin thickening and lung function.
Oral psoralen-UVA light (PUVA) may provide a successful treatment for cutaneous lesions in limited SSc. Kercher et al. used bath-PUVA and achieved improvement in generalized morphea as well as in SSc. UVA increases the synthesis of collagenase in fibroblasts of the skin and generates singulet oxygen. In addition, it exerts anti-inflammatory effects.
Inhibitors of cytokines and growth factors
Agents that decrease the amount of TGFβ or interfere with its action may be of benefit in SSc. For example, antibodies to TGFβ were able to reduce bleomycin-induced fibrosis, prevent chronic GvHD in mice, or inhibit the activation of tissue fibroblasts.[353,354,355]. However, CTGF might be a more reasonable target in the treatment of SSc. On the other hand, an open label trial of entanercept, the TNF inhibitor, in 10 SSc patients demonstrated an improved skin score in 4 of 9 evaluable patients. An IL-1 receptor antagonist prevented development of pulmonary fibrosis in mice. Finally, this anticytokine therapy requires patients at the early inflammatory stages of disease before late stage fibrosis has developed.
IFNγ and α inhibit the synthesis of collagen I, II and III, reduce the expression of collagen mRNA and enhance the production of collagenase by fibroblasts in vitro. Several uncontrolled trials of IFNγ suggest improvement in skin score and other objective parameters.[359,360,361] However, no benefit was shown for skin score or change in the collagen type I mRNA levels in skin tissue from 32 SSc patients in another open-label uncontrolled trial. In a randomized, controlled multicenter trial, 44 SSc patients showed improvement in their skin score, but quality of life measurements were superior in the placebo group. IFN may also activate endothelial cells and upregulate adhesion molecules, thus aggravating inflammation, Raynaud's phenomenon and digital ischaemia. Side-effects are headache, fatigue, muscle pain, sweating at night, fever, vomiting and arthralgia (flu-like syndrome).
Studies with IFNα did not show improvement of skin and may worsen associated lung disease. Finally, IFNβ may be a candidate for clinical trials, but it has not been studied yet.
Relaxin is a member of the insulin-growth factor family with a molecular weight of 5000-6000. Obviously it plays a role in connective tissue remodelling during pregnancy. Relaxin inhibits collagen synthesis and increases production of procollagenase. In addition, it acts as a renal vasodilator improving glomerular filtration. SSc patients with the stable diffuse form for less than 5 years received 25 or 100 µg/kg body weight of recombinant relaxin through continuous s.c. infusion. While the higher dose did not achieve benefit, the lower dose at week 4, 12 and 24 did show some benefit compared to placebo.[367,368] The large nation-wide placebo-controlled follow up study with 25 mg/kg body weight demonstrated no difference compared to placebo in the outcomes of skin score and pulmonary function testing. The dose given was 50 fold higher than achieved during pregnancy. In addition, it should be noted that softening of connective tissue in pregnancy is restricted to the environment of the birth canal and does not occur over the whole body.
Halofuginone is an alcaloid, which reduces collagen synthesis and collagen I (I) gene expression in humans. Its effect in animal models of skin fibrosis (murine GvHD and tight skin (TSK) mouse) demonstrated decreased collagen content and reduced clinical signs of sclerosis. In addition, halofuginone causes a dose dependent inhibition of collagen I (I) gene expression and collagen synthesis in skin fibroblast cultures from patients with GvHD and SSc. The application of a topical formulation achieved clinical improvement in GvHD patients.
(Neo) tigason and cis-retinoid acid showed moderate improvement or even healing of ischemic ulcerations in SSc patients after several weeks of treatment. Effects on the microtubule system and collagen metabolism (inhibition of fibroblast proliferation and collagen synthesis) were discussed. The following side-effects have to be considered: teratogenicity, liver function disturbances and increase in serum lipids.
Cyclophenyl (antagonist of estrogen), colchizin (inhibitor of tubulin polymerisation and microtubulus dependent collagen synthesis) and griseofulvin (inhibitor of fibroblast proliferation in vitro) could not achieve therapeutic relevance. The same is true for factor XIII as an inhibitor of collagen synthesis. Potassium para-aminobenzoate has been used for many years in doses of 4 g TID. However, initial effects appeared to be non-significant after correction for patients groups.
Additional symptomatic treatment
Metoclopramide, 5-10 mg (Paspertin) before each meal and at bedtime, is indicated for the treatment of esophageal dysmotility. In reflux esophagitis the following medications are recommended: H2 antagonists such as cimetidin (400 mg TID), ranitidine (150 mg BID) as well as proton pump inhibitors like omeprazol (20-60 mg/d). Antibiotics are given in gastroenteritis (bacterial overgrowth) and infection of the lung and kidney according to general therapeutic standards.
Cardial involvement is treated with digitalis and diuretics, while renal crisis and malignant hypertension require ACE inhibitors such as captopril or enalapril, and in severe cases, dialysis. In pulmonary hypertension the administration of anticoagulants, calcium channel blockers and prostacyclin analogues are indicated, the latter ones as inhalation therapy.
Hydrocolloid dressings help in the healing of ulcerations. Calcium deposits can be excised or their perforation may be facilitated by adhesive tapes. Colchicine (0.5 mg BID) is able to reduce local inflammation surrounding the calcified lesions.
There is no ideal drug available for the treatment of SSc. Symptomatic treatment helps to improve quality of life. Various disease modifying agents address different pathways of the disease pathogenesis such as vascular disease, autoimmunity and tissue fibrosis. Beside current recommendations (see Table 9) a variety of novel strategies have been reviewed. But more well designed placebo-controlled trials are needed to evaluate the benefit of any new medication, especially as SSc is a challenging disease with a heterogeneous clinical phenotype. In addition, the paucity of objective criteria renders the evaluation of improvement or deterioration more difficult. Physiotherapy to maximize the range of motion of all large joints and psychological counselling are necessary components of the successful management of SSc.
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