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OBJECTIVE: A phase II randomized controlled trial of recombinant human relaxin suggested that a dosage of 25 mug/kg/day was safe and clinically effective in improving skin disease and reducing functional disability in scleroderma (systemic sclerosis; SSc). We undertook a large randomized, double-blind, placebo-controlled clinical trial to compare placebo with 10 mug/kg/day and 25 mug/kg/day recombinant human relaxin, given for 24 weeks in patients with stable, diffuse, moderate-to-severe SSc. METHODS: Men and women ages 18-70 years with diffuse cutaneous SSc (dcSSc) were administered recombinant human relaxin (10 mug/kg/day or 25 mug/kg/day) or placebo for 24 weeks as a continuous subcutaneous infusion. There was a followup safety visit at week 28. RESULTS: The primary outcome measure, the modified Rodnan skin thickness score, was similar among the 3 groups at baseline and at weeks 4, 12, and 24. Secondary outcomes such as functional disability were similar in all 3 groups, while the forced vital capacity decreased significantly in the relaxin groups. The discontinuation of both doses of relaxin at week 24 led to statistically significant declines in creatinine clearance and serious renal adverse events (defined as doubling of serum creatinine, renal crisis, or grade 3 or 4 essential hypertension) in 7 patients who had received relaxin therapy but in none who had received placebo. CONCLUSION: Recombinant relaxin was not significantly better than placebo in improving the total skin score or pulmonary function or in reducing functional disability in patients with dcSSc. In addition, relaxin was associated with serious renal adverse events, the majority of which occurred after stopping the infusion. If relaxin is used therapeutically for any conditions other than scleroderma, close monitoring of blood pressure and renal function must be performed. [ABSTRACT FROM AUTHOR]

OBJECTIVE: To evaluate CAT-192, a recombinant human antibody that neutralizes transforming growth factor beta1 (TGFbeta1), in the treatment of early-stage diffuse cutaneous systemic sclerosis (dcSSc). METHODS: Patients with SSc duration of <18 months were randomly assigned to the placebo group or to 1 of 3 CAT-192 treatment groups: 10 mg/kg, 5 mg/kg, 0.5 mg/kg. Infusions were given on day 0 and weeks 6, 12, and 18. The primary objective of this study was to evaluate the safety, tolerability, and pharmacokinetics of CAT-192. Secondary outcomes included the modified Rodnan skin thickness score (MRSS), the Scleroderma Health Assessment Questionnaire, assessment of organ-based disease, serum levels of soluble interleukin-2 receptor, collagen propeptides (N propeptide of type I [PINP] and type III collagen), and tissue levels of messenger RNA for procollagens I and III and for TGFbeta1 and TGFbeta2. RESULTS: Forty-five patients were enrolled. There was significant morbidity and mortality, including 1 death in the group receiving 0.5 mg/kg of CAT-192 and 3 deaths in the group receiving 5 mg/kg of CAT-192. There were more adverse events and more serious adverse events in patients receiving CAT-192 than in those receiving placebo, although these events were not more frequent in the high-dose treatment group. The MRSS improved in all groups during the study, but there was no evidence of a treatment effect for CAT-192. Improvement in the MRSS correlated with the disease duration (r = -0.54, P = 0.0008). Changes in the PINP level from baseline correlated with changes in the MRSS (r = 0.37, P = 0.027). CONCLUSION: We report the first evaluation of a systemically administered and repeatedly dosed anti-TGFbeta1 drug. In this pilot study, CAT-192, in doses up to 10 mg/kg, showed no evidence of efficacy. The utility of clinical and biochemical outcome measures and the feasibility of multicenter trials of early dcSSc were confirmed. [ABSTRACT FROM AUTHOR]

OBJECTIVE: Mutations in fibrillin 1, a key component of extracellular microfibrils, are associated with connective tissue disorders such as Marfan's syndrome or skin fibrosis in the tight skin mouse model of scleroderma. Previous studies have suggested that fibrillin 1 mediates skin fibrosis via its interface with associated microfibrillar proteins and type I collagen; in particular, microfibril-associated glycoprotein 2 (MAGP-2), an extracellular matrix protein that binds to fibrillins and the alphavbeta3 integrin, is increased in TSK mouse and human scleroderma skin. Because the function of MAGP-2 in the biologic processes of the matrix remains unknown, this study investigated whether MAGP-2 regulates type I collagen. METHODS: Fibroblast cultures conditionally overexpressing MAGP-2 were developed. Cells were analyzed by Western blotting, Northern blotting, pulse-chase analysis, and immunofluorescence to assess the effect of MAGP-2 on type I collagen. RESULTS: Cells overexpressing MAGP-2 formed increased MAGP-2 matrix and showed a 3-fold increase in intracellular type I procollagen. This increase was associated with increased levels of type I collagen in the medium and matrix. Increased type I collagen colocalized with the MAGP-2 matrix. MAGP-2 overexpression had no effect on type I procollagen messenger RNA, but markedly increased the half-life of type I procollagen. MAGP-2 induced type I collagen even under conditions in which no MAGP-2 matrix was detectable, and did not require the presence of the RGD motif of MAGP-2 in its integrin-binding site. CONCLUSION: This study shows that MAGP-2 stabilizes type I procollagen, identifying an important function of MAGP-2 in extracellular matrix homeostasis. It also suggests that MAGP-2 might mediate skin fibrosis in TSK mice and in patients with scleroderma. [ABSTRACT FROM AUTHOR]

Objectives: Ischaemic digital ulcers (DUs) are common in patients with systemic sclerosis (SSc) and are a cause of disease-related morbidity. In an earlier trial, treatment with bosentan, an oral endothelin receptor antagonist, reduced the occurrence of new DUs by 48%. The present study (RAPIDS-2, for 'RAndomized, double-blind, Placebo-controlled study with bosentan on healing and prevention of Ischemic Digital ulcers in patients with systemic Sclerosis') was conducted to more fully evaluate the effects of bosentan treatment on DUs associated with SSc., Methods: This double-blind, placebo-controlled trial conducted at 41 centres in Europe and North America randomised 188 patients with SSc with at least 1 active DU ('cardinal ulcer') to bosentan 62.5 mg twice daily for 4 weeks and 125 mg twice daily thereafter for 20 weeks (n=98) or matching placebo (n=90; total 24 weeks). The two primary end points were the number of new DUs and the time to healing of the cardinal ulcer. Secondary end points included pain, disability and safety., Results: Over 24 weeks, bosentan treatment was associated with a 30% reduction in the number of new DUs compared with placebo (mean ± standard error: 1.9±0.2 vs 2.7±0.3 new ulcers; p=0.04). This effect was greater in patients who entered the trial with more DUs. There was no difference between treatments in healing rate of the cardinal ulcer or secondary end points of pain and disability. Peripheral oedema and elevated aminotransferases were associated with bosentan treatment., Conclusions: Bosentan treatment reduced the occurrence of new DUs in patients with SSc but had no effect on DU healing. Bosentan was well tolerated and may be a useful adjunct in the management of patients with SSc with recurrent DUs.

Objective: To examine the validity of a durometer to objectively measure skin hardness in systemic sclerosis (SSc), and to compare digital durometry with the modified Rodnan skin score (MRSS) and ultrasonography., Methods: Patients with SSc and healthy controls underwent durometry measurements in 3 assessments: a Latin square experiment to establish durometry's intra- and interobserver reliability compared with skin scoring (5 SSc, 1 control); a longitudinal cohort to assess sensitivity to change in skin hardness (13 SSc, 5 controls); and an ultrasound cohort to evaluate correlation between durometry, ultrasound-measured skin thickness, and clinical skin scoring (30 SSc, 12 controls)., Results: Intraobserver reproducibility was higher for durometry than for clinical skin scoring (intraclass correlation coefficient [ICC] 0.97 versus 0.85), whereas interobserver reproducibility was similar (0.75 versus 0.73). Interobserver reproducibility of durometry was good for all body areas (ICC 0.61-0.85), but for skin scoring it was moderate in the legs (0.51) and poor in the abdomen (0.08), feet (0.09), and fingers (0.27). Durometry scores correlated with clinical skin scores (Latin square: r = 0.44, P = 0.03; longitudinal cohort: r = 0.81, P < 0.001) and ultrasound-measured skin thickness (hands: r = 0.58, forearms: r = 0.63, upper arms: r = 0.40; P < or = 0.001 for all). Uninvolved skin in patients with SSc was harder than skin from controls (mean +/- SD 23 +/- 7 durometer units [DU] versus 19 +/- 6 DU; P < 0.0001). Finally, there was a strong correlation between change in MRSS and change in durometry score (r = 0.77, P = 0.002)., Conclusion: Durometer-measured skin hardness correlates well with MRSS and ultrasound-measured skin thickness, provides greater reliability than MRSS, and is sensitive to changes in skin hardness over time. Durometry should be considered for use in clinical therapeutic SSc trials.

Cartilage oligomeric matrix protein (COMP) is an extracellular glycoprotein that belongs to the thrombospondin gene family. It is found predominantly in cartilage, tendon, ligament, and bone. Mutations in the COMP gene have been linked to the development of pseudoachondroplasia and multiple epiphysial dysplasia. COMP influences the organization of collagen fibrils by interacting with collagens I, II and IX. Gene expression profiling of cultured skin fibroblasts suggested that COMP mRNA levels were elevated in scleroderma. We therefore examined COMP expression in SSc and normal skin biopsies. Immunohistochemistry confirmed that COMP protein accumulates in SSc but not normal skin, with SSc skin showing striking deposition in the papillary and deeper dermis. Significant staining was also seen in non-lesional skin from patients. Due to its involvement in the development of fibrosis, TGFbeta was examined for a possible role in regulating COMP expression. Cultured SSc fibroblasts demonstrated greater staining for COMP compared to normal controls prior to stimulation, and TGFbeta-1 induced a large increase in mRNA and protein. Murine fibroblasts engineered to overexpress human COMP demonstrated increased levels of fibronectin and collagen in the extracellular matrix. Taken together, these data demonstrate that COMP is overexpressed in SSc skin and cultured fibroblasts possibly due to autocrine TGFbeta stimulation, and COMP overexpression is sufficient to stimulate excess matrix deposition. By interactions with other matrix proteins and cells, COMP may play a role in pathogenic matrix deposition.

Objective: This study compares the responsiveness to change of the Medical Outcomes Study Short Form Health Survey (SF-36), a measure of health related quality of life (HRQOL), and the Health Assessment Questionnaire Disability Index (HAQ-DI), a function instrument, in a randomized clinical trial for treatment of systemic sclerosis (SSc)., Methods: A phase 2/3, multicenter, prospective, placebo controlled trial was conducted to evaluate human recombinant relaxin treatment in patients with diffuse SSc over 24 weeks. At baseline, subjects had stable, moderately severe, diffuse SSc of disease duration < or = 5 years, modified Rodnan skin score > or = 20, serum creatinine < 2.0 mg/dl, percentage forced vital capacity (% FVC) predicted > or = 50%, and % DLCO predicted > or = 40% and were not receiving concomitant disease modifying therapies. Internal consistency reliability of multi-item scales was estimated using Cronbach's alpha. Responsiveness to change of the SF-36 and HAQ-DI was computed between Weeks 0 and 24. Subjects were classified as unchanged or having a meaningful change in 4 different external measures: Change in (1) skin score > or = 30%; (2) % FVC predicted of > or = 15%; (3) self-reported patient global assessment by visual analog scale (VAS) > or = 20%; and (4) physician global assessment by VAS of > or = 20%. Responsiveness indices were computed and Cohen's effect size criteria were used to assess the magnitude of change., Results: A total of 239 patients participated in this trial, with 196 completing the 24 week trial. Cronbach's alpha for the SF-36 scales ranged from 0.76 to 0.93 and for the HAQ-DI ranged from 0.69 to 0.91 (good to excellent). The SF-36 had a larger magnitude of responsiveness in overall disease (patient and physician global assessment) compared to the HAQ-DI, while the HAQ-DI had a larger magnitude of responsiveness in clinical measures (i.e., change in skin score and % FVC predicted) than the SF-36., Conclusion: These data support inclusion of both the SF-36 and HAQ-DI as outcome measures in future clinical trials of diffuse SSc.

Recent studies suggest that, in addition to activation and hypersecretion of matrix components, fibroblasts from patients with systemic sclerosis (SSc) are relatively resistant to apoptosis. Transforming growth factor-beta (TGF)-beta is strongly implicated in the pathogenesis of SSc and we and others have shown that TGF-beta can activate Akt, a kinase with potent anti-apoptotic effects. To determine whether Akt was activated in SSc, we quantified phospho-Akt expression in skin fibroblasts in vitro by western blot analysis and a functional kinase assay. In addition, the relative proportion of fibroblasts containing activated Akt in was quantified by immunohistochemistry on skin sections insitu. Analysis of Akt phosphorylation of skin fibroblasts in vitro suggested increased phosphorylation of Akt, and evaluation of skin sections by immunohistochemistry revealed significantly higher percentages of fibroblasts that stained for phospho-Akt compared with controls (78% +/- 14.0% vs 13% +/- 9%, p < 0.001). In addition, co-incident staining of phospho-Akt and alpha-smooth muscle actin was observed in some fibroblasts. These findings indicate that Akt is activated insitu in skin fibroblasts from patients with SSc. Akt activation may contribute to resistance to apoptosis, selection of disease-inducing fibroblasts, and, possibly, myofibroblasts.

Objective: Recurrent digital ulcers are a manifestation of vascular disease in patients with systemic sclerosis (SSc; scleroderma) and lead to pain, impaired function, and tissue loss. We investigated whether treatment with the endothelin receptor antagonist, bosentan, decreased the development of new digital ulcers in patients with SSc., Methods: This was a randomized, prospective, placebo-controlled, double-blind study of 122 patients at 17 centers in Europe and North America, evaluating the effect of treatment on prevention of digital ulcers. The primary outcome variable was the number of new digital ulcers developing during the 16-week study period. Secondary assessments included healing of existing digital ulcers and evaluation of hand function using the Scleroderma Health Assessment Questionnaire., Results: Patients receiving bosentan had a 48% reduction in the mean number of new ulcers during the treatment period (1.4 versus 2.7 new ulcers; P = 0.0083). Patients who had digital ulcers at the time of entry in the study were at higher risk for the development of new ulcers; in this subgroup the mean number of new ulcers was reduced from 3.6 to 1.8 (P = 0.0075). In patients receiving bosentan, a statistically significant improvement in hand function was observed. There was no difference between treatment groups in the healing of existing ulcers. Serum transaminase levels were elevated to >3-fold the upper limit of normal in bosentan-treated patients; this elevation is comparable with that observed in previous studies of this agent. Other side effects were similar in the 2 treatment groups., Conclusion: Endothelins may play an important role in the pathogenesis of vascular disease in patients with SSc. Treatment with the endothelin receptor antagonist bosentan may be effective in preventing new digital ulcers and improving hand function in patients with SSc.

The Tight skin (Tsk) mouse is an important model of skin fibrosis that occurs in systemic sclerosis. These mice develop skin tethering and thickening associated with expression of a mutant fibrillin-1 gene. We show that Tsk fibrillin-1 leads to marked alterations in elastic fibers of the hypodermis of Tsk animals. In Tsk mice, a prominent elastic fiber layer found normally at the interface between hypodermal muscle and connective tissue was absent from an early age. The lack of elastic fibers at the hypodermal muscle-connective tissue (M-CT) interface was associated with a loss of staining for fibulin-5 in the same region. These mice also formed disorganized elastic fibers throughout hypodermal connective tissue as they aged. The increased elastic fibers in Tsk hypodermal connective tissue was associated with increased fibrillin-1 and fibulin-2 matrices. These results suggest that Tsk fibrillin-1 causes skin tethering by altering matrix protein composition in Tsk hypodermal connective tissues. The closely parallel alterations in elastogenesis associated with increased fibulin-2 in hypodermal connective tissues and decreased fibulin-5 at the hypodermal M-CT interface suggest that these proteins mediate the effect of Tsk-fibrillin-1 on elastogenesis.

Objective: Skin fibrosis in the TSK mouse, a model of skin fibrosis seen in systemic sclerosis (SSc), is caused by a large in-frame duplication in the Fbn1 gene, tsk-Fbn1. We investigated whether tsk-Fbn1 might cause dermal fibrosis by affecting Fbn1 and associated extracellular matrices. We also studied whether deposition of microfibril-associated glycoprotein 2 (MAGP-2), a protein that is associated with fibrillin 1, was altered in the skin of patients with SSc., Methods: An in vitro model of the TSK mouse was created by conditionally expressing tsk-Fbn1 in mouse embryonic fibroblasts (MEFs). Cell cultures were examined by immunofluorescence and Western and Northern blotting to determine the effect of tsk-Fbn1 on the structure, expression, and deposition of fibrillin 1 (Fbn-1), type I collagen, and MAGP-2. The skin of TSK mice and SSc patients was analyzed by immunohistochemistry for MAGP-2 expression., Results: Expression of tsk-Fbn1 in cultured MEF cells altered the morphology of Fbn-1 fibers and increased the deposition of type I collagen into the extracellular matrix (ECM) without concomitantly changing messenger RNA expression, secretion, or processing of type I procollagen. Moreover, MEF cells expressing tsk-Fbn1 showed increased MAGP-2 matrix. MAGP-2 was increased in the dermis of TSK mice. Fibrotic SSc skin also showed higher levels of MAGP-2 in the dermis than nonfibrotic SSc skin and normal skin., Conclusion: Tsk-Fbn1 altered ECM organization and caused fibrosis by affecting the deposition of MAGP-2 or other Fbn-1-associated proteins. Alterations in microfibril structure or deposition might contribute to fibrosis in SSc.

Objectives: Transforming Growth Factor-beta (TGFbeta) is the predominant cytokine in all forms of fibrotic reactions. As well as being secreted by immune modulators of fibrosis such as macrophages, it is involved in an autocrine feedback loop of fibroblast stimulation whose regulation is still poorly understood. We wished to gain some insight into the mechanisms of the fibroblast response to TGFbeta., Methods: We undertook an exhaustive transcript profiling experiment using a widely validated restriction enzyme based method for identifying differentially expressed genes (GeneCalling). Transcriptional responses throughout a 24-hour time course were examined at multiple time points and classified., Results: By 24 hours of TGF treatment over 1000 bands, representing a large number of transcripts, were down- or upregulated greater than 2-fold. All of the known genes responsive to TGFbeta, such as collagen and connective tissue growth factor, were upregulated., Conclusions: This encyclopedic method revealed many unknown transcriptional responses to TGFbeta including the upregulation of a variety of less expected cytoskeletal and matrix components, as well as interactions between the TGFbeta and tumor necrosis factor (TNF) pathways and alterations in cell death-related pathways. These may in part explain the idiosyncratic responses of mesenchymal cells to TGFbeta.

Many effective treatments for scleroderma have emerged in recent years, including bosentan, an endothelin receptor antagonist, and epoprostenol, a prostacyclin, both of which target vasoconstriction. Cyclophosphamide may soon be proven effective against interstitial lung disease.

The pathogenesis of fibrosis in scleroderma involves a complex set of interactions between the fibroblast and its surroundings. Multiple fibrotic pathways are activated for reasons that are not completely clear, but involve immune activation, microvascular damage, and fibroblast transformation into the myofibroblast. Differential proliferation and apoptosis preserve the myofibroblast phenotype rather that leading to a selective depletion of activated fibroblasts after an acute injury has healed. Disproportionate fibroblast activity could result from a combination of possible cellular and matrix defects that include fibrillin protein abnormalities, autoantibody formation, type II immune response, excessive endothelial reaction to injury, and excessive fibroblast response to TGF-beta. Development of therapies that are targeted to correcting these abnormalities will eventually lead to effective treatment for the fibrotic complications of scleroderma.

The hallmark of scleroderma is cutaneous and visceral fibrosis characterized and by increased biosynthesis of multiple matrix proteins by interstitial fibroblasts. Studies over recent years have delineated pathways involved in promoting matrix synthesis and elucidated the molecular pathways of regulation. Central to the regulation of fibrosis are extracellular mediators, called cytokines, which are elaborated by a variety of cells, including those in the immune system, vascular cells, and fibroblasts themselves. The concept that inhibiting or promoting the action of these naturally occurring profibrotic or antifibrotic molecules, respectively, is a rational therapeutic approach to treating scleroderma and other fibrotic diseases finds support in animal studies and anticytokine therapy conducted in relation to rheumatoid arthritis and other disorders. This review looks at cytokines known or thought to play a role in scleroderma and/or other fibrotic states and at potential therapy directed at these mediators. Potential targets for therapy include transforming growth factor beta (TGF-beta), connective tissue growth factor (CTGF), IL-4, IL-13, MCP-1, and endothelin, among others.

Objective: Fibrillin, an extracellular matrix protein implicated in dermal fibrosis, is increased in the reticular dermis of systemic sclerosis (SSc) skin. We undertook this study to investigate the hypothesis that transforming growth factor beta (TGFbeta) or other cytokines regulate fibrillin matrix formation by normal and SSc fibroblasts. We further investigated the mechanism of TGFbeta-induced fibrillin fibrillogenesis and its relationship to myofibroblasts., Methods: Fibrillin and fibronectin matrix deposition and alpha-smooth muscle actin expression by fibroblast cultures from normal and SSc skin treated with TGFbeta or other cytokines were analyzed by immunofluorescence. Supernatant and extracellular matrix from normal and SSc fibroblasts treated with or without TGFbeta were evaluated by Western blot and Northern blot for fibrillin protein and messenger RNA (mRNA) expression, respectively., Results: Immunofluorescence demonstrated increased fibrillin matrix formation by normal and scleroderma fibroblasts after TGFbeta treatment. Other cytokines, including tumor necrosis factor alpha, interleukin-1beta (IL-1beta), IL-4, granulocyte-macrophage colony-stimulating factor, and platelet-derived growth factor, did not affect fibrillin fibrillogenesis. Fibrillin matrix formed in proximity to myofibroblasts and independently of up-regulation of fibronectin matrix or cell number. Western blot analysis of extracellular matrix confirmed increased fibrillin after TGFbeta stimulation of normal or scleroderma fibroblasts. However, TGFbeta did not alter the expression of either soluble fibrillin protein or fibrillin mRNA., Conclusion: Our data show that TGFbeta induces fibrillin protein incorporation into the extracellular matrix without affecting fibrillin gene expression or protein synthesis, suggesting that fibrillin matrix assembly is regulated extracellularly. TGFbeta might increase fibrillin matrix by activating myofibroblasts. Such TGFbeta-mediated effects could account for the increased fibrillin matrix observed in SSc skin.

Objective: To document disease activity and functional status in patients with scleroderma (systemic sclerosis [SSc]) and Raynaud's phenomenon (RP) and to determine the sensitivity to change, reliability, ease of use, and validity of various outcome measures in these patients., Methods: Patients with SSc and moderate-to-severe RP participating in a multicenter RP treatment trial completed daily diaries documenting the frequency and duration of RP attacks and recorded a daily Raynaud's Condition Score (RCS). Mean scores for the 2-week periods prior to baseline (week 0), end of trial (week 6), and posttrial followup (week 12) were calculated. At weeks 0, 6, and 12, physicians completed 3 global assessment scales and performed clinical assessments of digital ulcers and infarcts; patients completed the Health Assessment Questionnaire (HAQ), the Arthritis Impact Measurement Scales 2 (AIMS2) mood and tension subscales, 5 specific SSc/RP-related visual analog scales (VAS), and 3 other VAS global assessments. We used these measures to document baseline disease activity and to assess their construct validity, sensitivity to change, and reliability in trial data., Results: Two hundred eighty-one patients (248 women, 33 men; mean age 50.4 years [range 18-82 years]) from 14 centers participated. Forty-eight percent had limited cutaneous SSc; 52% had diffuse cutaneous SSc. Fifty-nine patients (21%) had digital ulcers at baseline. Patients had 3.89 +/- 2.33 (mean +/- SD) daily RP attacks (range 0.8-14.6), with a duration of 82.1 +/- 91.6 minutes/attack. RCS for RP activity (possible range 0-10) was 4.30 +/- 1.92. HAQ scores (0-3 scale) indicated substantial disability at baseline (total disability 0.86, pain 1.19), especially among the subscales pertaining to hand function (grip, eating, dressing). AIMS2 mood and tension scores were fairly high, as were many of the VAS scores. Patients with digital ulcers had worse RCS, pain, HAQ disability (overall, grip, eating, and dressing), physician's global assessment, and tension, but no significant difference in the frequency of RP, duration of RP, patient's global assessment, or mood, compared with patients without digital ulcers. VAS scores for digital ulcers as rated by the patients were not consistent with the physician's ratings. Factor analysis of the 18 measures showed strong associations among variables in 4 distinct domains: disease activity, RP measures, digital ulcer measures, and mood/tension. Reliability of the RCS, HAQ pain and disability scales, and AIMS2 mood and tension subscales was high. The RP measures demonstrated good sensitivity to change (effect sizes 0.33-0.76)., Conclusion: Our findings demonstrate that the significant activity, disability, pain, and psychological impact of RP and digital ulcers in SSc can be measured by a small set of valid and reliable outcome measures. These outcome measures provide information beyond the quantitative metrics of RP attacks. We propose a core set of measures for use in clinical trials of RP in SSc patients that includes the RCS, patient and physician VAS ratings of RP activity, a digital ulcer/infarct measure, measures of disability and pain (HAQ), and measures of psychological function (AIMS2).

Tissue fibrosis is the result of a complex series of events focusing on regulation of fibroblast proliferation, synthesis of extracellular matrix, and apoptosis. Transforming growth factor-beta is important for the stimulation of the fibrotic response by promoting the production of extracellular matrix proteins, by promoting the differentiation of the myofibroblast cell morphology, and by protecting these cells against apoptotic stimuli. Other cytokines such as interleukin-1 may have stimulatory and counter-regulatory effects on fibrosis. The effects of these signaling molecules depend on cellular environment and are organ specific. Furthermore, intercellular interactions and cell-matrix interactions can stimulate or inhibit the apoptotic pathway. Through selective inhibition of apoptosis in myofibroblasts, fibrosis can become dysregulated and lead to diseases such as systemic sclerosis.

The transcription factor cKrox was originally identified as a protein that bound to a negative transcription regulatory element in the murine alpha1(I) collagen promoter. We recently reported the cloning and characterization of human cKrox (hcKrox). Overexpression of hcKrox in NIH3T3 fibroblasts efficiently repressed the promoters of the fibronectin and alpha1(I) collagen genes (70-90%) in transient transfection assays and suppressed the endogenous genes in hcKrox expressing permanent cell lines. We have now isolated genomic clones and cDNAs encoding two novel transcription factors related to hcKrox termed hcKrox-beta and hcKrox-gamma (the original clone is now referred to as hcKrox-alpha). Both contain three kruppel-like zinc-finger DNA binding motifs that are 71-78% identical to those of hcKrox-alpha. The NH(2)-terminus of all three proteins contains a POZ domain, a conserved 120 amino acid motif involved in transcriptional repression and protein dimerization. RT-PCR experiments demonstrate that all three hcKrox family members are expressed in foreskin and dermal fibroblasts. Transient transfection studies in NIH3T3 fibroblasts demonstrate that hcKrox-alpha -beta and -gamma, as well as the murine cKrox-beta homologue, LRF, suppress transcription driven by promoters for the alpha1(I) and alpha2(I) collagen, fibronectin and elastin genes. Electrophoretic mobility shift assays and coimmunoprecipitation studies suggest that homo- and heterodimerization occurs between cKrox family members. Dimer formation is influenced by amino acids in the NH(2)-terminal POZ domain and the Zn(+2)-finger region. Immunoprecipitation studies indicate that cKrox can form heterodimers in solution in the absence of DNA. Thus, a multi-gene family exists that can coordinately regulate several extracellular matrix genes and has the potential to form many heterodimeric transcription factors.

Objective: We hypothesized that pathophysiologic events during the development of systemic sclerosis (SSc) may lead to selection and propagation of certain apoptosis-resistant fibroblast subpopulations. The aim of this study was to examine a possible role for apoptosis in fibroblast selection in SSc and the role of transforming growth factor beta1 (TGFbeta1)., Methods: We compared SSc and normal fibroblasts for their susceptibility to anti-Fas-induced apoptosis and analyzed 2 models that might lead to fibroblast resistance to apoptosis in this process: long-term exposure to either anti-Fas or TGFbeta1., Results: SSc-derived fibroblasts were resistant to anti-Fas-induced apoptosis, showing 5.5 +/- 17.2% (mean +/- SD) apoptosis, compared with 32.1 +/- 14.0% among normal fibroblasts (P < 0.05). Anti-Fas-selected normal fibroblasts showed 9.0 +/- 3.7% apoptosis, compared with 21.6 +/- 5.9% for sham-treated cells, which is consistent with the elimination of apoptosis-susceptible subpopulations. Normal fibroblasts subjected to 6 weeks of TGFbeta1 treatment showed not only resistance to apoptosis, but also proliferation (118.5 +/- 35.4%), after anti-Fas treatment, compared with sham-treated cells (35.1 +/- 11.1% apoptotic cell death). TGFbeta1 treatment also increased the proportion of myofibroblasts (47% versus 28% in controls). Cultured SSc fibroblasts had a greater proportion of myofibroblasts (32-83%) than did normal fibroblasts (4-25%). We also examined the relationship between collagen gene expression and the myofibroblast phenotype in normal and SSc skin sections. Only 2 of 7 normal sections had alpha-smooth muscle actin (a-SMA)-positive cells (mean +/- SD score 0.29 +/- 0.49 on a scale of 0-3), but all SSc sections were positive for alpha-SMA, with a mean score of 1.90 +/- 0.88 for lesional and 1.50 +/- 0.71 for nonlesional sections. Scores for alpha1(I) procollagen messenger RNA (mRNA) in lesional skin (mean +/- SD 3.30 +/- 0.82 on a scale of 1-4) were significantly higher than in normal (1.43 +/- 0.79) or nonlesional (1.40 +/- 0.52) skin, but scores varied, and there was no correlation between collagen mRNA and alpha-SMA levels., Conclusion: Our results show that resistance to apoptosis is an important part of the SSc phenotype. TGFbeta1 may play a role by inducing apoptosis-resistant fibroblast populations, and also by inducing myofibroblasts and by enhancing extracellular matrix synthesis.

Background: Relaxin is a pregnancy-related hormone that has tissue remodeling and antifibrotic effects. Systemic sclerosis (scleroderma) is characterized by fibrosis of the skin, vasculature, and internal organs., Objective: To assess the efficacy, safety, and dose-response effect of recombinant human relaxin in patients with scleroderma., Design: Multicenter, parallel-group, randomized, double-blind, placebo-controlled trial., Setting: Academic referral centers., Patients: 68 patients who had had stable, diffuse scleroderma (moderate to severe) for less than 5 years., Intervention: Recombinant human relaxin, 25 or 100 microg/kg of body weight per day, or placebo administered by continuous subcutaneous infusion over 24 weeks., Measurements: Modified Rodnan skin score was the primary efficacy measure. Secondary measurements were pulmonary function, the Health Assessment Questionnaire, and other measures of scleroderma that reflected fibrosis., Results: Patients who received 25 microg/kg of recombinant human relaxin per day had significantly lower skin scores than those who received placebo (mean change, -3.6 at 4 weeks [P = 0.021], -7.5 at 12 weeks [P < 0.001], and -8.7 at 24 weeks [P = 0.040]). Similar trends were noted in other outcome measures, including forced vital capacity, measures of oral aperture and hand extension, functional status, and global assessment. Patients who received 100 microg/kg of relaxin per day did not differ from those who received placebo. Drug-related adverse events included menometrorrhagia, reversible anemia, and complications of the subcutaneous drug administration system (site irritation and local infection)., Conclusions: Twenty-four weeks of recombinant human relaxin, 25 microg/kg per day, is associated with reduced skin thickening, improved mobility, and improved function in patients with moderate to severe diffuse scleroderma.

Heme oxygenase-1 (HO-1) catalyzes the rate-limiting step in heme catabolism and presumably is involved in cellular iron homeostasis. It is induced by a variety of cellular stresses, including oxygen deprivation and free radical-mediated stress. We examined induction of HO-1 mRNA in skin fibroblasts and investigated the mechanism by which it occurs. Hypoxia did not appear to act via induction of oxygen free radicals: induction of HO-1 was not sensitive to the free radical scavenger GSH or other antioxidants. Moreover, hypoxia did not increase steady-state levels of free radicals generated by fibroblasts. In contrast, HO-1 induction by the oxidants, H(2)O(2) and carbonyl cyanide m-chlorophenylhydrazone (CCCP) was significantly attenuated in the presence of free radical scavengers. This correlated with increased levels of free radical production in fibroblasts treated with these oxidants. Iron depletion by desferrioxamine mesylate, a specific iron complexon, completely inhibited hypoxic stimulation of HO-1 but did not attenuate the effect of H(2)O(2) and CCCP on HO-1 mRNA. Addition of Fe(2+), Fe(3+), or holo-transferrin to fibroblasts increased levels of HO-1 mRNA. Treatment of cells with hypoxia, but not H(2)O(2) or an exogenous source of iron, significantly increased the half-life of HO-1 mRNA. The data suggest hypoxia regulates HO-1 gene expression by a specific posttranscriptional mechanism: stabilization of mRNA. Hypoxia has previously been shown to increase fibroblast collagen synthesis and is thought to play a role in pathogenesis of systemic sclerosis (SSc). Skin fibroblasts isolated from patients with SSc demonstrated significantly stronger induction of HO-1 by hypoxia than did fibroblasts from normal controls. We hypothesize that exposure of SSc fibroblasts to hypoxic conditions leads to in vivo selective proliferation of cells that adapt to hypoxia.

Objective: To evaluate the short- and long-term effects of intravenous epoprostenol in patients with pulmonary hypertension (PH) associated with systemic sclerosis (SSc)., Methods: Sixteen patients with SSc-associated PH and New York Heart Association (NYHA) class III or IV symptomatology underwent right heart catheterization for determination of baseline hemodynamic values. Vasoreactivity was assessed with either inhaled nitric oxide or intravenous adenosine. After a medication washout period, all patients received intravenous epoprostenol in incrementally increasing doses; tolerance was assessed according to symptoms and hemodynamic findings at each dose increment and at the conclusion of the medication trial. Once a stable medication regimen was established, patients were discharged and followed up as outpatients for assessment of symptoms and exercise tolerance as measured by change in the NYHA class. Repeat hemodynamic testing was performed in 4 patients at 1 year and in 2 patients at 2 years of treatment., Results: Therapeutic response to epoprostenol, defined by a reduction in the pulmonary vascular resistance of > or =25%, was achieved in the short-term treatment period in 13 of 16 patients (81.3%). Improvement in symptoms and exercise tolerance occurred in all patients, and a significant short-term hemodynamic response was observed. Followup hemodynamic tests revealed persistent favorable responses in all 4 of the patients studied., Conclusion: Most patients with PH secondary to SSc manifest favorable hemodynamic responses to epoprostenol in the short term. Long-term epoprostenol was generally well tolerated and provides a potential therapeutic option for patients with PH secondary to SSc.

Epoprostenol (prostacyclin) is currently approved for treatment of primary pulmonary hypertension; however, it is being evaluated in other forms of pulmonary hypertension, particularly scleroderma. Side effects associated with this medication are usually minor; serious complications are most often due to the delivery system required for continuous infusion. We describe a life threatening side effect of acute epoprostenol infusion (pulmonary edema) in a patient with pulmonary hypertension associated with limited scleroderma and discuss its management and potential etiology. This is the first case where epoprostenol has been successfully reinstituted.

Scleroderma currently affects approximately 75,000-100,000 individuals in the United States. Fibroblasts isolated from lesional skin of scleroderma patients overexpress collagens and other matrix components, and this abnormality is maintained for multiple passages in culture. To understand the molecular basis for matrix gene overexpression, we performed a differential display comparison of fibroblasts from clinically lesional and nonlesional scleroderma skin. The results suggested that protease nexin 1 (PN1), a protease inhibitor, is overexpressed in scleroderma fibroblasts. Northern blot verification showed that lesional and nonlesional scleroderma fibroblasts had three- to five-fold increased levels of PN1 mRNA compared with healthy fibroblasts. Western analysis showed that scleroderma fibroblasts also secreted more PN1. In situ hybridization of skin biopsy specimens demonstrated PN1 expression in the dermis of four out of six scleroderma patients but no PN1 expression in the dermis of six healthy volunteers. Transient or stable overexpression of PN1 in mouse 3T3 fibroblasts increased collagen promoter activity or endogenous collagen transcript levels, respectively. PN1 mutagenized at its active site and antisense PN1 both failed to increase collagen promoter activity. These results suggest that overexpression of enzymatically active PN1 may play a pathogenic role in the development of the scleroderma phenotype.

Inflammation and tissue injury are strong stimuli for fibroblast activation and initiation of reparative processes. In certain disease states, pathological fibrosis occurs. Fibroblasts isolated from these diseased tissues often display a persistently abnormal phenotype characterized by increased synthesis of matrix components such as collagen. This metabolic abnormality is apparently independent of continued exposure to any pathological stimulus that may have initiated the process. Since fibroblasts are heterogeneous in proliferative capacity, in synthesis of collagen and other matrix proteins and in response to immune mediators and growth factors, clonal selection, i.e. selective increase in fibroblast subpopulations, may explain the long-term effects of acute in vivo activation on fibroblast behavior. Studies of SSc fibroblasts are consistent with clonal selection and/or clonal activation, processes that may play an important role in fibrosis in this and other disorders.

Abnormalities of matrix biosynthesis by systemic sclerosis fibroblasts underlie the cutaneous and visceral fibrosis seen in this disease. The fundamental basis of the abnormal fibroblast phenotype has not been determined but primary metabolic abnormalities, responses to abnormal environmental signals, and clonal selection have all been hypothesized to play a role. In the past year, evidence supporting each of these explanations was forthcoming. In a study of Choctaw Indians with a high incidence of scleroderma, it was shown that genetic linkage to fibrillin may play a role. This should be considered in light of demonstrated abnormalities of fibrillin structure in the tight skin mouse. Other studies have shown that abnormalities of transforming growth factor beta receptor levels, abnormalities of production and response to tissue inhibitor of matrix metal-loproteinase, and abnormalities of response to endothelial cell factors are all present in scleroderma fibroblasts. In addition to these studies in scleroderma fibroblasts, work elucidating the role of transcription factors Sp1, Sp3, and cKrox in regulating collagen metabolism provided important data upon which future studies may build.

In the present studies we found that incubation of human lung fibroblasts with transforming growth factor-beta 1 (TGF-beta 1) potentiated the interleukin-1 beta (IL-1 beta) and/or tumor necrosis factor-alpha (TNF-alpha)-stimulated production of prostaglandin E2 (PGE2). Analysis of fibroblast proteins showed the induction of cyclooxygenase-1 (Cox-1) by TGF-beta 1 and the induction of Cox-2 by IL-1 beta and TNF-alpha. The levels of transcripts for Cox-1 were minimally modified by IL-1 beta or TNF-alpha, however, they were increased by 12-fold by TGF-beta 1. Transcripts for Cox-2 were induced by IL-1 beta or TNF-alpha and their induction was potentiated by TGF-beta 1. TGF-beta 1 alone did not induce Cox-2 transcripts. In vitro transcription assays showed that IL-1 beta and TNF-alpha increased the transcription of the Cox-2 gene, whereas TGF-beta 1 had no effect. Addition of TGF-beta did not increase further the transcription of Cox-2 in IL-1 beta-treated cells, but increased the stability of the corresponding transcripts. The transcription rate of the Cox-1 gene was not increased by any of the cytokines studied. In summary, we demonstrate that the potentiation of PGE2 production by TGF-beta 1 in IL-1 beta and TNF-alpha-treated fibroblasts is the result of transcriptional stimulation of the Cox-2 gene by IL-1 beta and TNF-alpha and the stabilization of the resulting transcripts by TGF-beta 1.

Objective: To evaluate the efficacy and tolerability of an oral preparation of iloprost, a prostacyclin analog, in patients with Raynaud's phenomenon (RP) secondary to systemic sclerosis (scleroderma)., Methods: A multicenter, randomized, parallel-group, placebo-controlled double-blind study was performed at university and community-based medical centers. Patients were randomly assigned to receive either 50 microg of iloprost orally twice daily or an identical gelatin-coated capsule containing placebo for 6 weeks. Outcome measures included average total daily duration of RP attacks, average number of RP attacks, and RP condition scored via a standardized daily diary., Results: Three hundred eight patients with scleroderma (272 women, 36 men, mean age 49 years [range 18-80]) were enrolled. One hundred fifty seven were assigned to receive iloprost and 151 to receive placebo. One hundred forty-three patients in the iloprost group (91.1%) and 144 in the placebo group (95.4%) completed the 6-week treatment phase. Fifteen of these treated patients (8 iloprost, 7 placebo) failed to complete all of the followup visits. The mean reduction in the average duration of attacks from baseline to week 5-6 was 24.32 minutes in the iloprost group and 34.34 minutes in the placebo group (P = 0.569). Likewise, the mean reduction from baseline to week 5-6 in the daily frequency of attacks was 1.02 in the iloprost group and 0.83 in the placebo group (P = 0.459). The Raynaud's condition score, a patient-completed assessment of the severity of RP attacks, was reduced by 1.32 in the iloprost group and 1.00 in the placebo group (P = 0.323). The lack of significant difference between treatment groups did not change when a variety of factors, including use of other vasodilators, duration of disease, classification of scleroderma (limited versus diffuse), or number of baseline digital ulcers were taken into account. Premature withdrawal from the study due to adverse events occurred in 10 patients (6.4%) in the iloprost group and 3 (2.0%) in the placebo group (P = 0.058)., Conclusion: Oral iloprost at a dosage of 50 microg twice daily is no better than placebo for management of RP secondary to scleroderma, either during 6 weeks of treatment or during 6 weeks of posttreatment followup.

Apoptosis, or programmed cell death, is a naturally occurring process mediated by extracellular signals. We studied anti-Fas (CD95/Apo-1) antibody-induced apoptosis in cultured human foreskin and adult dermal fibroblasts. Induction of apoptosis was identified by fluorescence in situ DNA end-labeling. Anti-Fas antibody induced apoptosis in fibroblasts in a dose- and time-dependent manner. Adult dermal skin fibroblasts were more susceptible to anti-Fas antibody-induced apoptosis than foreskin fibroblasts, with 21-52% dead cells in different strains. In foreskin fibroblasts, anti-Fas antibody (1.0 microg/ml) predominantly induced proliferation ([3H]thymidine incorporation increased to 115-165% of control level) and only low levels of apoptotic cell death after 48 hours of treatment. No induction of proliferation by anti-Fas was found in the adult fibroblasts. Addition of tumor necrosis factor-alpha (TNF-alpha) slightly augmented the anti-Fas antibody-induced apoptosis in both cell types. When we examined the levels of Fas expression using flow cytometry, we found two- to threefold higher Fas expression in adult fibroblasts. C6-ceramide treatment, which induces Fas-independent apoptosis, gave similar levels of cell death in both foreskin and adult fibroblasts. No proliferation was observed in C6-ceramide-treated fibroblasts. Thus, this difference in apoptosis between adult dermal and foreskin fibroblasts appears to be related to the level of Fas expression. When clones of foreskin fibroblasts were examined, there was heterogeneity of anti-Fas antibody-induced apoptosis and proliferation in the cloned fibroblast subpopulations, but this was not correlated with differences in Fas expression. Alterations in fibroblast populations during the process of differentiation and aging may result from selective loss of apoptosis-susceptible populations.

cKrox is a novel zinc finger-containing transcription factor that binds to the alpha1(I) and alpha2(I) collagen gene promoters. The gene coding for cKrox is a new member of a family of early growth response genes, that play important roles in development. In the mouse, cKrox is expressed, beginning at 9.5 days of gestation and at 10.5 days in regions destined to become skin. In adult animals, expression is predominantly in skin, one of the two major organs where type I collagen is expressed. We have isolated cDNA clones for human cKrox. Theoretical translation of the nucleic acid sequence reveals 90% conservation of amino acids between the mouse and human proteins; however, the human gene product contains a 117-amino-acid N-terminal extension. The amino acid sequences of the zinc-finger DNA binding domains of mouse and human cKrox are identical. RT-PCR analysis of human fibroblasts indicates constitutive low-level expression of cKrox which can be transiently elevated by treatment with retinoic acid. Transient transfection assays indicate that hcKrox represses transcription of the alpha1(I) procollagen promoter, and electrophoretic mobility shift assays demonstrate that hcKrox binds to both the human and murine promoter DNA. Deletion derivatives of hcKrox demonstrate transcription-activating potential that is promoter-dependent. NIH3T3 cells permanently expressing hcKrox demonstrate a threefold and 10-fold decrease in alpha1(I) procollagen and fibronectin mRNA levels, respectively, compared to control cells. Consistent with this finding, a fibronectin promoter reporter construct is repressed more than 80% by hcKrox. These data suggest that hcKrox represses collagen transcription directly, and it may function as a repressor of fibronectin and possibly other matrix genes.

The effect of human mast cells on fibroblast activity was studied using an organotypic skin-equivalent culture system. Human mast cell-1 (HMC-1) cells were embedded in a collagen gel with neonatal dermal fibroblasts at a ratio of 1:4; keratinocytes then were allowed to stratify above this composite culture. Analysis of type a1(I) procollagen mRNA synthesis by in situ hybridization revealed a substantial increase in mRNA levels in the presence of mast cells and especially following degranulation, induced by calcium ionophore A23187. Tryptase, a major product of human mast cells, could substitute for mast cells in this culture system, up-regulating procollagen mRNA synthesis. Tryptase pretreated with the specific protease inhibitor bis(5-amidino-2-benzimidazo-lyl)methane (BABIM) markedly attenuated the collagen mRNA up-regulation. Further studies revealed HMC-1 cell sonicates stimulated fibroblast chemotaxis and procollagen mRNA synthesis. Inhibition of HMC-1 sonicates with either BABIM or a neutralizing mAb against tryptase resulted in significant reduction of fibroblast chemotaxis and procollagen mRNA, implying that tryptase accounted for the majority of HMC-1 sonicate activity. Tryptase directly stimulated fibroblast chemotaxis with optimal concentrations between 10 pM and 1 nM. The maximal response of optimal concentrations of tryptase was comparable with the known fibrogenic factor, TGF-beta. Inhibition of tryptase with BABIM resulted in approximately 50% reduction in chemotactic activity. Additional studies revealed that tryptase (0.3-3 nM) stimulated procollagen mRNA synthesis in confluent monolayers of dermal fibroblasts.

The frequency of the use of deception in American psychological research was studied by reviewing articles from journals in personality and social psychology from 1921 to 1994. Deception was used rarely during the developmental years of social psychology into the 1930s, then grew gradually and irregularly until the 1950s. Between the 1950s and 1970s the use of deception increased significantly. This increase is attributed to changes in experimental methods, the popularity of realistic impact experiments, and the influence of cognitive dissonance theory. Since 1980 there appears to have been a decrease in the use of deception as compared to previous decades which is related to changes in theory, methods, ethical standards, and federal regulation of research.

Objective: The goal of this study was to quantitatively analyze the distribution of collagen synthesis in normal and systemic sclerosis (SSc) fibroblast populations in order to determine the extent of activation in SSc populations., Methods: We used quantitative in situ hybridization to assess the population distribution of type I collagen synthesis. Fibroblast cultures were derived from both clinically involved and uninvolved skin regions of patients with SSc, and from healthy adults, and assessed for levels of alpha 1(I) procollagen messenger RNA (mRNA)., Results: Dermal fibroblasts from both patients with SSc and normal adults were heterogeneous for distribution of alpha 1(I) procollagen mRNA when assessed by in situ hybridization, with a wide range of grains per cell. In contrast, clones of neonatal fibroblasts showed a relatively homogeneous distribution of grain counts. Involved SSc skin fibroblasts had a larger proportion of cells in the high collagen-producing mRNA subpopulation (mean +/- SEM 28.4 +/- 6.85%), compared with normal fibroblasts (1.75 +/- 1.44%) and uninvolved fibroblasts (9.6 +/- 6.73%). Conversely, within the low collagen-producing mRNA subpopulation, involved fibroblasts had a smaller proportion of cells (mean +/- SEM 14.0 +/- 5.63%) than did uninvolved fibroblasts (37.8 +/- 13.69%), while normal fibroblasts had a majority of the cells in this subpopulation (53.5 +/- 8.68%)., Conclusion: These results suggest that only a specific subset of fibroblasts are activated in SSc, as evidence by an increased proportion of cells with high levels of alpha 1(I) procollagen mRNA. Differences between the SSc and normal fibroblast populations appeared to be quantitative rather than qualitative. This may be a result of either clonal selection or selective activation in the pathogenesis of SSc.

The capacity of mast cell products to mediate T-cell adhesion to fibroblasts was explored using heterotypic coculture systems or by exposing fibroblasts to mast-cell-conditioned media (MCCM), prepared by degranulating mast cells with calcium ionophore. Experimental results indicated that fibroblasts exposed to MCCM for 24 h bound fivefold more T cells than control fibroblasts. Binding was inhibited with intercellular adhesion molecule-1 (ICAM-1) or vascular cell adhesion molecule-1 (VCAM-1) neutralizing antibodies. Enzyme-linked immunosorbent assay and fluorescence-activated cell sorter analysis revealed that fibroblasts exposed to MCCM markedly increased ICAM-1 and VCAM-1 surface expression by 4 h, with levels maximal at 16 h and returning toward baseline by 48 h. A dose-dependent response of ICAM-1 and VCAM-1 expression was noted using serial dilutions of MCCM or by altering the ratio of degranulated mast cells cocultured with fibroblasts. Similar results were obtained using human fibroblasts derived from the dermis, synovium, and lung, although lung fibroblasts were generally less responsive. Northern analysis confirmed that MCCM regulated ICAM-1 and VCAM-1 expression at the mRNA level. In summary, mast cell products stimulated fibroblast surface expression, steady-state mRNA levels, and functional expression of ICAM-1 and VCAM-1. Experimental data suggest that mast-cell-derived tumor necrosis factor-alpha may be in large part responsible for these observations, although further studies using human mast cells will be required. Using a skin-equivalent organotypic coculture model with fibroblasts admixed with mast cells, we observed increased ICAM-1 expression in both keratinocytes and fibroblasts after activation of the mast cells.

Monoclonal antibodies were developed against human dermal fibroblast cell-surface membranes. Our goal was to find evidence of differential expression of antigens on fibroblast clones derived by limiting dilution. Antibodies were then used to isolate and identify membrane proteins. By sequence analysis of membrane immunoprecipitates, one monoclonal antibody, BR2, was subsequently shown to recognize the enzyme aminopeptidase N (hAPN; EC 3.4.11.2), originally described as a marker for certain hematopoetic cells. We have used biochemical techniques, electron microscopy, flow cytometry, and ELISA to characterize aminopeptidase N expression by human dermal fibroblasts. The presence of abundant aminopeptidase N confers enzymatic properties to human dermal fibroblasts which heretofore have been largely unexplored and suggest that the cellular distribution of aminopeptidase N is wider than originally appreciated.