Cytokine production and serum levels in systemic sclerosis.

Serum levels of various cytokines, tumor necrosis factor-alpha (TNF-alpha), interleukin 1-beta (IL1-beta), and interleukin 2 (IL2), and of soluble IL2 receptors (sIL2R) were determined in 30 patients with definite systemic sclerosis (SSc). Spontaneous and lipopolysaccharide-or mitogen-induced production of the cytokines, TNF-alpha, IL1-beta, and IFN-gamma, by peripheral blood mononuclear cells (PBMNC) of these SSc patients was measured by immunoassays. The patients were divided into three groups: 12 with limited cutaneous disease (lcSSc), 7 with diffuse cutaneous disease (dcSSc) < 3 years duration, and 11 with dcSSc > 3 years duration. None were treated with cytotoxic drugs or biologic response modifiers. Sera of patients with SSc had elevated sIL2R levels, and only low levels of IL2 (1-2 U/ml) were detected in 10/29 sera tested. Spontaneous production of TNF-alpha and IL1-beta by PBMNC of patients with SSc (829 pg/ml +/- 215 SEM and 728 pg/ml +/- 186, respectively) was significantly higher than that by normal PBMNC obtained from 30 volunteers (25 +/- 10 and 34 +/- 6 pg/ml, respectively) and tested at the same time as patients' PBMNC. The largest increases in spontaneous release of TNF-alpha or IL1-beta were seen in patients with early dcSSc. No significant difference in spontaneous IFN-gamma production by patient or control PBMNC was detected. On the other hand, the mean level of mitogen-induced IFN-gamma production by PBMNC was significantly depressed in patients with SSc (103 U/ml +/- 18 vs 255 +/- 33 U/ml in controls). In vitro-induced production of TNF-alpha or IL1-beta by patients' PBMNC was comparable to that of normal PBMNC. These data indicate that in vivo-activated PBMNC of patients with SSc spontaneously secrete excessive amounts of fibrogenic cytokines, which are involved in modulation of connective tissue synthesis.

Lymphokine-activated killer cell and natural killer cell activities in patients with systemic sclerosis.

OBJECTIVE: To determine the ability of T lymphocytes and natural killer (NK) cells from patients with systemic sclerosis (SSc) to respond to cytokines and to generate immune effector cells. METHODS: The numbers and percentages of peripheral blood T and NK cells were examined by 2-color flow cytometry, and NK and lymphokine-activated killer (LAK) cell function were measured in 4-hour 51Cr-release assays, in 34 patients with SSc. The patients were categorized into 3 subgroups: 10 had diffuse cutaneous disease of less than or equal to 3 years disease duration, 11 had diffuse cutaneous SSc of greater than 3 years duration, and 13 had limited cutaneous disease. RESULTS: Baseline and activated NK and T cell numbers and NK activity were normal in SSc patients. However, mean LAK activity was significantly depressed in all SSc subgroups. CONCLUSION: Decreased LAK cell function, despite normal numbers of circulating T and NK cells, indicates that SSc patients have poor ability to produce effector cells in response to interleukin-2.

Temporomandibular joint dysfunction syndrome: a close association with systemic joint laxity (the hypermobile joint syndrome)

Sixty-two patients admitted for elective reconstructive surgery of the temporomandibular joint (TMJ) and eight seen as outpatients with a chief complaint of TMJ dysfunction during the same time interval were evaluated for possible etiologic factors contributing to the disease. All hospitalized patients had severe, end-stage degenerative changes within the TMJ, whereas outpatients had less severe disease and did not require surgery. TMJ dysfunction in some patients was said to be a result of established causes including bruxism, malocclusion, and trauma. No patient in this series had evidence of a systemic inflammatory polyarthritis. Of the 70 patients, 38 (54%) met criteria, based on those of Carter and Wilkinson, as modified by Beighton et al., sufficient to warrant a diagnosis of the hypermobile joint syndrome. Five patients had classic Ehlers-Danlos syndrome and therefore were not patients with "benign hypermobility," and an additional two cases were described as "marfanoid" and as possible Ehlers-Danlos syndrome, respectively. Radiographs showed TMJ hyperextensibility in four hypermobile patients. Long-term surgical outcome was identical in the hypermobile and nonhypermobile groups. The incidence of hypermobility in this series is strikingly higher than the expected incidence in an otherwise population. Magnetic resonance images of the TMJs on separate groups of asymptomatic normal and hypermobile women identified excessive anterior movement in the hypermobile group, together with abnormal anterior disk position in some. We hypothesize that hypermobility within the TMJ may cause accelerated disk destruction and degenerative disease.

Interactions between cells of the immune system and hyaluronate synthesis by human dermal fibroblasts.

Mitogen- or alloantigen-activated human peripheral blood mononuclear cells (MNC) produce a soluble factor which selectively stimulates up to twenty-fold the synthesis of glycosaminoglycan (GAG) by cultured normal human fibroblasts. Confluent fibroblast monolayers were incubated with active MNC supernatants and newly synthesized GAG was measured by the incorporation of [3H]glucosamine into cetylpyridinium chloride-precipitable material. The GAG-stimulatory factor (GAG-SF) was a product of T lymphocytes. Alloreactive T cell clones obtained from the peripheral blood produced the factor after reactivation with the irradiated stimulators, and its production was dependent on HLA-DR-mediated recognition. The CD3+CD4+ clones derived from the skin-infiltrating lymphocytes in patients with early scleroderma also produced the GAG-SF upon in vitro activation with a mitogen. The GAG-SF was purified to apparent homogeneity from supernatants of concanavalin A-activated MNC by Sephadex gel filtration, ion-exchange chromatography and reverse-phase HPLC. The GAG-SF is a 67,000 Da glycoprotein with pI of 5.6. It is not mitogenic to fibroblasts and does not modulate collagen synthesis. Its purification and characterization are important, because of a possible involvement of activated lymphocytes and their products in the immunopathogenesis of human diseases characterized by fibrosis, stromal reactions and local lymphocytic infiltrates.

Heterogeneous synthetic phenotype of cloned scleroderma fibroblasts may be due to aberrant regulation in the synthesis of connective tissues.

Clones of dermal fibroblasts from the skin of 4 normal subjects and 5 patients with progressive systemic sclerosis (PSS; scleroderma) were established, and their synthetic and proliferative characteristics were compared. A limiting-dilution assay was used to determine frequencies of cloning in the microcultures of dermal fibroblasts plated. The clones derived from single cells were expanded in vitro and examined (in passages C-H) for growth and synthesis of glycosaminoglycan (GAG) and collagenase-sensitive protein (CSP). The clonogenicity of PSS fibroblasts was not significantly different from that of normal fibroblasts. Normal fibroblast clones were characterized by low levels of GAG and CSP synthesis, and there was a correlation between the GAG and CSP phenotypes. In contrast, clones of PSS fibroblasts were often, but not always, high producers of GAG and CSP, but there was no correlation between the levels of GAG and CSP synthesis. It appears that scleroderma skin is composed of fibroblast clones that are unable to regulate the synthesis of connective tissue components and often synthesize large amounts of connective tissue macromolecules.

Systemic joint laxity (the hypermobile joint syndrome) is associated with temporomandibular joint dysfunction.

A consecutive series of 37 individuals admitted to the hospital for elective temporomandibular joint (TMJ) reconstructive surgery and 3 seen as outpatients with TMJ disease were evaluated for rheumatic disease or for another etiologic factor that might account for this problem. These 40 patients were screened by history, physical examination, and laboratory study. We soon noticed that many patients had generalized joint laxity. Eighteen of the first 40 individuals satisfied established criteria for the hypermobile joint syndrome. An additional 3 were found to have Ehlers-Danlos syndrome or a forme fruste of this disorder. Many were markedly hypermobile and could perform a number of flexible maneuvers. Although excessive joint laxity is known to be associated with a variety of rheumatic conditions, TMJ disease has not been recognized as one of them. No patient in this series had a systemic inflammatory disorder or any other apparent etiologic factor for TMJ disease. We suggest that there is a cause-and-effect relationship between generalized joint laxity and TMJ disease.

Persistence of scleroderma-like phenotype in normal fibroblasts after prolonged exposure to soluble mediators from mononuclear cells.

Supernatants of mononuclear cells (MNC-SN) were shown to increase synthesis of glycosaminoglycan (GAG) by cultured normal dermal fibroblasts. Fibroblasts from the skin of patients with progressive systemic sclerosis (PSS, scleroderma) were hyporesponsive. We exposed fibroblasts outgrowing from explants of normal adult skin to MNC-SN for up to 30 generations in culture. MNC-SN were obtained by incubating normal MNC with concanavalin A. Four experimental, 4 normal control, and 3 PSS control lines were passaged by trypsinizing and splitting the cultures 1:2 every 7 days. At the third and fifth passages, portions of the experimental fibroblasts were removed from MNC-SN, then passaged in medium alone. Cell counts, assays for GAG, and electron microscopy were performed and increases in GAG after brief reexposure to MNC-SN were determined at the third, fifth, and eighth passages. In normal dermal fibroblasts, baseline GAG production, measured by 3H-glucosamine uptake, was low and increased as much as 15 times after reexposure to MNC-SN. In contrast, production was high in both experimental and PSS lines, and increases after reexposure to MNC-SN were consistently small. This PSS-like behavior persisted in experimental fibroblasts removed from MNC-SN at the third and fifth passages. Growth of experimental and scleroderma fibroblasts was slower than that of control fibroblasts. Ultrastructurally, both scleroderma and experimental dermal fibroblasts differed from normal fibroblasts by their oval cellular shape, indentations in nuclear membrane, numerous organelles and bundles of microfilaments, prominent Golgi, and intranuclear inclusions. These experiments indicate that normal adult dermal fibroblasts subjected to MNC-SN in vitro acquire a scleroderma-like phenotype that persists for many generations.

Soluble mediators from mononuclear cells increase the synthesis of glycosaminoglycan by dermal fibroblast cultures derived from normal subjects and progressive systemic sclerosis patients.

Dermal fibroblast cultures from patients with progressive systemic sclerosis (PSS) synthesize up to 5 times more glycosaminoglycan (GAG) than normal cultures. In an in vitro model of fibroblast-lymphocyte interactions, we show that the supernatants of activated mononuclear cells (MNC) modulate GAG synthesis, as measured by the incorporation of 3H-glucosamine into GAG following incubation of the confluent fibroblast monolayers with active supernatant preparations. GAG accumulation was selectively increased up to 18 times in normal dermal fibroblast cultures. Cell viability was not affected, and 3H-thymidine uptake and cell numbers were depressed in cultures treated with the supernatants. In contrast to normal dermal fibroblast cultures, PSS fibroblasts responded to MNC supernatants by only a 1-2-fold increase in GAG. Supernatants of concanavalin A-activated PSS MNC had higher stimulatory activity than those of normal MNC. Supernatants made with MNC that had been depleted of monocytes on Sephadex G-10 columns were only minimally stimulatory. The GAG-stimulatory supernatants modulated the synthesis, but not the degradation of GAG. Gel filtration on a calibrated Sephadex G-100 column indicated the presence of stimulatory activity in both the 50,000 and 15,000 molecular weight fractions. These activities were trypsin-sensitive, but had different susceptibilities to heat. The active column fractions also contained interleukin-1 activity, as shown in an assay measuring proliferation of mouse thymocytes. Like our factors, interleukin-1 preparations increased GAG in normal and PSS dermal fibroblasts. Products of activated MNC may modulate normal and pathologic processes in human skin.

Products of activated mononuclear cells modulate accumulation of collagen by normal dermal and scleroderma fibroblasts in culture.

Supernatants of human mononuclear cells activated with concanavalin A contain soluble factors that modulate accumulation of collagen in human dermal fibroblasts in culture. The supernatants decreased collagen accumulation by 67% in normal fibroblast lines and by greater than 80% in lines established from skin of patients with scleroderma. Collagen was measured by incorporation of 3H-proline into collagenase-sensitive protein. The inhibitory activity of collagen was optimal after a 24-hour incubation of confluent fibroblast monolayers with unfractionated mononuclear cell supernatants. Kinetic studies of this response showed a delay in accumulation of collagen-sensitive protein in supernatant-treated cultures. The 3H-thymidine uptake and fibroblast cell count and viability were only minimally altered. Noncollagen protein was inhibited by 30% to 40%. The presence of serum in fibroblast cultures did not affect this activity. The inhibitory factors were produced by purified T-lymphocyte subpopulations. Supernatant inhibitory activity was present after removal of monocytes from mononuclear cell cultures. By gel filtration on Sephadex G-100, active supernatants fractionated into at least three peaks of activity with molecular weight of 50,000, 30,000, and 15,000 daltons. Interleukin 1 might be one of the factors in mononuclear cell supernatants that modulates production of collagenase-sensitive protein in human dermal fibroblasts. Factors modulating collagen-sensitive protein are important in processes leading to excessive accumulation of the connective tissue and fibrosis in certain diseases.

Surface proteins of scleroderma fibroblasts in culture.

Scleroderma skin fibroblasts, as examined by a lactoperoxidase-iodine labeling technique, lack a surface associated protein of apparent molecular weight 120,000 daltons present in normal skin fibroblasts and contain 2 proteins 74,000 and 59,500 daltons, which are absent in normal-fibroblasts. Relative concentrations of several other cell surface proteins also appear to be different in these fibroblasts.

Scleredema adultorum of Buschke: a clinical, pathologic, and cell culture study of two patients.

Two patients with scleredema of Buschke are presented. The second patient developed scleredema after a febrile drug reaction. Biopsies of both involved and uninvolved skin were obtained for histologic examination and cell culture studies. Immunofluorescent studies of the biopsy specimens revealed staining with IgG, IgM and C3 at the dermal-epidermal junction in the involved skin of the first patient. This has not been previously reported. Cell culture studies revealed that fibroblasts from the involved skin produced more glycosaminoglycans (GAG) than uninvolved skin and most of the GAG produced was hyaluronic acid. Possible pathogenic mechanisms for this unusual condition are discussed.

Progressive systemic sclerosis (PSS, scleroderma) dermal fibroblasts synthesize increased amounts of glycosaminoglycan.

Fibroblast monolayers were established by explant culture of dermis from seven patients with early, rapidly advancing PSS (scleroderma) and from six normal individuals. Net GAG synthesis was measured as [3H]glucosamine incorporation into the polymer and also as total uronic acid accumulation in the cultures. Cultures from patients with PSS accumulated up to fivefold more GAG than did normal cultures. Evidence suggests that this is a result of increased synthesis rather than decreased degradation. PSS cultures simultaneously accumulated increased amounts of CSP as well as GAG. Data obtained from studies of several monolayers, followed for as many as 10 generations, show that the difference between PSS and normal cultures can be propagated. The total quantity of GAGs was increased in PSS cultures, and there were differences in the distribution of individual GAGs on a percentage basis in PSS and normal cell cultures. GAG, as well as collagen, may be important in the development of connective tissue thickening and induration in scleroderma.

D-penicillamine treatment of progressive systemic sclerosis (scleroderma): a comparison of clinical and in vitro effects.

Punch biopsies of skin were obtained from the forearms of 3 patients with progressive systemic sclerosis with diffuse scleroderma before and after treatment for 1 year or more with D-penicillamine. While on therapy, each patient studied had demonstrated a marked reduction in skin thickening. Collagenase-sensitive protein and glycosaminoglycan accumulation were measured in fibroblast cultures derived from these biopsies. No differences were observed pre- and post-treatment. We conclude that although D-penicillamine may exert its effect in vivo on connective tissue synthesis, maturation and/or turnover, fibroblasts remaining in the thinned dermis retain their potential for increased synthesis of connective tissue.

Clinical and serologic study of Sjögren's syndrome in patients with progressive systemic sclerosis.

Fifty-eight patients with progressive systemic sclerosis (PSS) were evaluated clinically and by biopsy of the minor salivary glands of the lips for the presence of Sjögren's syndrome. Clinical findings included dry eyes in 38%, dry mouth in 32%, parotid enlargement in 4%, and an abnormal Schirmer's test in 34%. Histologic changes in lip biopsies included lymphocytic infiltrates characteristic of Sjögren's syndrome in 17 individuals (29%). In 19 (33%) there was periglandular and intraglandular fibrosis (PSS-fibrosis) without significant inflammation, an alteration characteristic of PSS per se. In the remaining 22 patients (38%) with PSS, no abnormality was found. Of those with PSS and Sjögren's syndrome, 53% had serum antibodies to SS-A and/or SS-B, while only 1 patient with a normal biopsy had either of these antibodies. Anti-SS-A and anti-SS-B were not detected in patients with glandular fibrosis alone. The mortality rate of the PSS-fibrosis group was higher due to a variety of severe internal manifestations related to PSS. Antibodies to SS-A and SS-B are useful serologic markers of the presence of Sjögren's syndrome in patients with PSS.