Joseph H. Korn

Low-dose methotrexate treatment of rheumatoid arthritis. Long-term observations☆

Of 21 patients with rheumatoid arthritis who began to receive low-dose weekly methotrexate up to five years ago, 15 (71 percent) have continued to take this drug for a mean of 42 months and have received a mean total dose of 2,021 mg (range: 915 to 3,075). The clinical improvement noted at the first follow-up (11 months) was sustained throughout this follow-up period (42 months). Three patients (14 percent) have had complete clinical remission and nine others (43 percent) have had an excellent response. Methotrexate was discontinued in four patients between the first and second follow-up because of planned pregnancy (one), gastrointestinal toxicity (two), and fear of toxicity (one). Liver toxicity assessed in these 21 patients and four others receiving long-term methotrexate therapy revealed acute hepatitis in one and elevated transaminase levels in 12 (48 percent). Liver biopsy specimens in 17 patients after a mean of 1,950 mg of methotrexate (range: 915 to 3,125) revealed mild fibrosis in six and no cirrhosis. Methotrexate can continue to suppress rheumatoid synovitis over a prolonged period of time with minimal toxicity in most patients. Hepatic fibrosis and cirrhosis due to methotrexate may be less common in rheumatoid arthritis than has been reported in psoriasis.

Anti‐fas induces apoptosis and proliferation in human dermal fibroblasts: Differences between foreskin and adult fibroblasts

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 μg/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‐α (TNF‐α) 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. J. Cell. Physiol. 175:19–29, 1998.

In vitro growth and maintenance of two morphologically distinct populations of thymic epithelial cells

Cultures of thymic epithelial cells were generated and maintained in valine-free minimum essential medium (MEM) supplemented with 690 mg/liter of D-valine. These cultures have been maintained for 1 year through multiple passages by trypsinization of 60-70% confluent monolayers. Large and small epithelial cells were present in early cultures. They were separated into two stable subpopulations based on (1) their differential growth rates and (2) their differential adherence to the culture substratum. These morphologically distinct cell populations, TECS and TECL, were 100% keratin positive and contained cells with desmosomes and tonofilaments, all characteristics of epithelial cells. Esterase analysis of both cell populations revealed a 1 and 9% esterase-positive cell population in cultures of keratin-positive small (TECS) and large (TECL) cells, respectively. The percentages of esterase-positive cells corresponded to the 2 and 10% populations of TECS and TECL, respectively, that contained both desmosomes and phagolysosomes. These results establish conditions for the long-term propagation of pure thymic epithelial cells. Such cultures can be used to study the functional interactions between epithelial cells and lymphoid cells. Morphologic and histochemical analyses have identified subsets of these cells which may prove to have differential effects on thymocyte proliferative and developmental processes.

The production of regulatory cytokines for thymocyte proliferation by murine thymic epithelium in vitro.

Two separate cultures of pure, morphologically distinct thymic epithelial cells have been generated and maintained in culture for one year (A.C. Nieburgs et al., Cell. Immunol. 90, 439-450, 1985). Supernatants from one of these cell lines, TECs, were examined for functional activity on thymocytes in vitro. These supernatants contained three distinct intercellular mediators, each capable of modulating thymocyte responses to T-cell mitogens. Enhancement of thymocyte proliferation to suboptimal doses of mitogen was associated with a factor that eluted in the 97,000-Da region on molecular sieve chromatography and was functionally and physicochemically distinct from interleukin-1 and interleukin-2 (IL-1 and IL-2). Suppression of the thymocyte response to optimal doses of mitogen was mediated by a 1000- to 5000-Da factor. These two intercellular components have different susceptibilities to heat treatments and are trypsin insensitive. In addition, thymic epithelial cells produced significantly high levels of prostaglandin E2 (PGE2) which also suppressed thymocyte responses to mitogen, but only at high doses of supernatant. These epithelial cell-derived enhancing and inhibitory effects on thymocytes could play a role in regulating intrathymic events.

Synergy of interleukin 1 (IL-1) with arachidonic acid and A23187 in stimulating PGE synthesis in human fibroblasts: IL-1 stimulates fibroblast cyclooxygenase☆

The stimulation of prostaglandin E (PGE) synthesis by interleukin 1 (IL-1) has important physiologic effects in many target tissues. The mechanism(s) whereby IL-1 stimulates PGE synthesis is not well understood. Two alternative mechanisms have been postulated: hydrolysis of membrane phospholipid with release of arachidonic acid substrate and induction of cyclooxygenase activity. We examined these mechanisms in IL-1 stimulation of human dermal fibroblast PGE synthesis. IL-1 failed to induce release of previously incorporated radiolabeled arachidonic acid from fibroblasts under conditions where there was a 30-fold or greater stimulation of PGE synthesis. The calcium ionophore, A23187, gave much less stimulation of PGE synthesis while inducing 30-100% release of incorporated [14C]arachidonic acid. There was marked synergy between IL-1 and agents which increased availability of arachidonic acid. For example, the combination of IL-1 and A23187, used at concentrations where neither agent alone stimulated PGE synthesis, increased PGE synthesis from 3.1 to 22.5 ng/ml; similar synergy was seen between IL-1 and exogenous arachidonic acid. To examine a possible effect of IL-1 on cyclooxygenase synthesis, fibroblast cyclooxygenase was measured by radioimmunoassay. Following treatment with IL-1, fibroblasts demonstrated a two- to threefold increase in content of immunoreactive cyclooxygenase. These results suggest that IL-1 increases fibroblast PGE synthesis by a mechanism(s) other than making available increased membrane arachidonic acid, and that at least part of its effect may be mediated by induction of cyclooxygenase. Furthermore, the effect of IL-1 on fibroblast PGE synthesis is markedly potentiated by agents which increase available substrate.

Thymic epithelium in vitro: III. Cytokine production by a thymic epithelial subset

We have previously shown that two populations of thymic epithelium can be separated in culture on the basis of their differential growth rates and their adherence to the culture substratum, and maintained as long-term, morphologically distinct cell cultures, TECs and TECL. We have also described the effects of supernatants from the small epithelial cell (TESs) on the proliferative responses of thymocytes cocultured with mitogen and TESs over a 72-hr period. We now describe the effects in thymic epithelial supernatants (TESL) of soluble factors produced by TECL (the large epithelial cell) on thymocytes costimulated with mitogen and compare their effects to those derived from TECs. Both TESL and TESs suppress optimally stimulated thymocytes and enhance the proliferative responses of suboptimally stimulated thymocytes over a 72-hr period. The suppressive activities produced by TECL and TECs appear distinct, based upon markedly different molecular weights, but have similar sensitivities to heat treatment. The enhancing activities are of similar molecular weight, but have different sensitivities to heat treatment. In addition, TECL synthesize four- to fivefold less PGE2 than TECs. These results provide additional distinctions between the two cell types, and taken in conjunction with data on the anatomic distribution of similar cells, suggest that although they have similar functional effects in vitro, they may prove to have separable roles in vivo.

Factor XI deficiency, juvenile rheumatoid arthritis and systemic lupus erythematosus: Report of the first case☆

Abstract A number of inborn errors have recently been associated with a diathesis for collagen vascular disorders. For example, hereditary deficiencies of C1r or of the second component of complement (C2) have been associated with certain features of systemic lupus erythematosus (SLE). We describe the association of juvenile rheumatoid arthritis and SLE with a familial deficiency of factor XI (plasma thromboplastin antecedent). The propositus, a 26 year old Ashkenazi woman, presented at age four with symmetrical destructive polyarthritis, fever and rash. Progressive renal disease began at age seven, and nine criteria for SLE became manifest beginning at age 15. Three maternal relatives have arthritis, two maternal relatives had mild bleeding after surgery. Physical findings included a deforming arthritis and rash. Low levels of C3 and C4, antinuclear antibodies, high titers of native DNA antibodies, positive lupus erythematosus cell preparations and C1q precipitins were present. A skin biopsy specimen showed deposits of immunoglobulin G, immunoglobulin M, C1q and C3 at the dermal-epidermal junction. Factor XI levels were 5 per cent in the propositus and mother, 25 per cent in the maternal grandfather and within normal limits in her sister, father, and a maternal and paternal aunt, implying a dominant mode of inheritance. An HL-A identical sister whose lymphocytes showed no stimulation with those of the patient in unidirectional mixed lymphocyte culture manifested neither collagen vascular disease nor factor XI deficiency. There was no evidence for a circulating anticoagulant, and other clotting factors were normal. The present case represents a third and new association of an inborn error in the clotting system (factor XI deficiency) and collagen vascular disease with immune complex deposition.

Inhibition of thymocyte mitogenic responses by coculture with thymic epithelial cells

We have previously reported the isolation and culture of two morphologically distinct types of murine thymic epithelial cells (TECL and TECs) and have studied their capacity to produce soluble factors as well as to bind thymocytes in vitro. In the present study, we examined whether direct interactions of thymic epithelial cells with thymocytes regulate thymocyte function by studying the effect of these thymic epithelial cell lines on thymocyte responses to mitogen. We found that direct contact between thymocytes and epithelial cells markedly suppresses both thymocyte and splenocyte responses to mitogen. This effect is irreversible after a 24-hr incubation on thymic epithelial cells, is not H-2 restricted, and appears to be epithelial cell specific. The inhibitory effect is mediated by a molecule or molecules present constitutively on the plasma membrane of epithelial cells. One mechanism of inhibition involves the generation of suppressor cells on contact of thymocytes with thymic epithelial cells or cell membranes. However, it is also possible that molecules with direct immunosuppressive activity may be present on these cell membranes.

Allogeneic fibroblasts and endothelial cells support mitogen but not antigen responses of tetanus-responsive T-cell lines

Recent studies suggest a role for endothelial cells (EC) and fibroblasts (FB) in performing certain accessory functions of monocytes in immune responses. We examined the ability of allogeneic adherent cells (AC), umbilical vein EC, and foreskin FB to support antigen and mitogen responses of tetanus toxoid-responsive human T-cell lines (TCTet). Syngeneic AC supported antigen and mitogen (phytohemagglutinin and pokeweed mitogen, PHA and PWM) responses of TCTet. Allogeneic AC, EC, and FB supported mitogen but not antigen responses of TCTet, in a dose-dependent manner. PHA-activated mononuclear cell supernates or EC or FB supernates could not replace accessory cells in mitogen responses. We provide further evidence that EC and FB can function as fully competent accessory cells, a function that may be of significance in in vivo initiation of immune responses.