The immunoregulatory effects of transforming growth factor beta (TGF-beta) and recombinant murine tumor necrosis factor alpha (rMuTNF-alpha) on CTL generation and activity were examined. The results demonstrate that TGF-beta, in a dose-dependent manner, inhibited CTL generation but not CTL activity. The inhibitory effects were detected only when TGF-beta was added within the first 48 h of the MLC. Little activity was seen when it was added thereafter, including the addition of TGF-beta to the cytotoxicity assay. The production of TNF-alpha, which occurs during early phases of the MLC and which is inhibited in the presence of TGF-beta, appears to have an important regulatory role, as altering the levels of TNF-alpha in an MLC can significantly influence CTL development. The inhibitory effects of TGF-beta on the MLC can be significantly reversed by the addition of rMuTNF-alpha to the cultures. These results demonstrate that TGF-beta can inhibit MLC and subsequent CTL generation at early stages of the reaction, and such inhibition may involve the suppression of TNF-alpha production.

Type II collagen-induced arthritis (CIA) in mice and rats is an inflammatory polyarthritis that displays several characteristics similar to human rheumatoid arthritis (1, 2). The onset of CIA is associated with the development of both a cellular and humoral response to type II collagen (3) and is restricted in mice to animals of the H-2 q or H-2 r haplotype (4). The humoral response to type II collagen appears to be an essential element in the development of the disease although the role of cellular immunity is less clear.The L3T4 antigen, defined by a rat anti-mouse monoclonal antibody GK1.5, is limited to T cells responsible for class II major histocompatibility complex (MHC)-restricted functions and is analogous to the OKT4/Leu-3 antigen on human T cells (5, 6). Recent reports (7, 8) have indicated that treatment of murine experimental autoimmune diseases with monoclonal anti-L3T4 antibody under either preventive or therapeutic protocols dramatically decreases disease expression. In this paper we demonstrate the prevention of murine CIA by in vivo treatment with monoclonai antibody to the L3T4 antigen. Materials and MethodsAnimals. DBA/1 male mice, 8-10 wk old, were obtained from The Jackson Laboratory, Bar Harbor, ME.Type H Collagen. Bovine articular cartilage was processed through four 24-h extractions with 5 vol of 4 M guanidine HCI, 50 mM Tris, pH 7.0, at 4°C. The slices were washed twice for 2 h in 1 M NaCI, 50 mM Tris, pH 7.5, once for 2 h in distilled H20, and twice for 3 h with 0.5 M HAc, and then digested overnight with 10 vol of 200 ttg/ml pepsin in 0.5 M HAc. The digest was centrifuged for 1 h at 10,000 g; solid NaCI was added to the supernatant to a concentration of 0.9 M and held overnight. After centrifugation, the pellet was resuspended in 5 vol of 1 M NaCI, 50 mM Tris, pH 7.5. The solution was adjusted to pH 8 by addition of 3 N NaOH and, after centrifugation as above, the supernatant was reprecipitated by the addition of solid NaC! to a final concentration of 4 M. After centrifugation, the pellet was suspended in 5 vol of 0.5 M HAc, dialyzed against 0.5 M HAc, and centrifugated at 27,000 g for 1 h. The purity of the type II collagen in the supernatant preparation was determined by sodium dodecyl sulphate gel electrophoresis, which showed a single band identical to a sample of pure bovine type II collagen (kindly provided by Dr.

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Human activated T cells adhere to synovial fibroblast-like cells in vitro. The present study was conducted to investigate the consequences of T cell-synovial fibroblast interactions with regard to induction of adhesion molecules and proinflammatory cytokines. A sensitive Western blot technique, polymerase chain reaction (PCR) amplification, and fluorescence-activated cell sorter (FACS) analysis were used to analyze the induction of VCAM-1 and ICAM-1 expression in T cell-synovial fibroblast cocultures. VCAM-1 and ICAM-1 expression could be induced in synovial fibroblast-like cells by 2 h. PCR amplification showed that both forms of VCAM-1 mRNA are found after the interaction of synovial fibroblasts with T cells. Up-regulation of VCAM-1 and ICAM-1 was confined to synovial fibroblasts; T cells did not express VCAM-1 or increased ICAM-1. In contrast to the T cell-synoviocyte interaction, the interaction between T cells and dermal fibroblasts resulted in the up-regulation of ICAM-1 but not VCAM-1, suggesting tissue-specific regulation of VCAM-1. The T cell-synovial fibroblast interaction also resulted in increased levels of tumor necrosis factor (TNF), interferon-gamma, and interleukin-6 in coculture supernatant. Of the neutralizing antibodies used against these cytokines, only anti-TNF could significantly inhibit VCAM-1 and ICAM-1 expression. When T cells were separated from synoviocytes by a chamber that allowed medium exchange but no cell contact, VCAM-1 and ICAM-1 failed to be up-regulated and cytokine accumulation in cocultures was drastically reduced. Our results demonstrate mutual cell activation of T cells and synoviocytes upon cell contact as shown by the release of T cell- and synoviocyte-specific cytokines and suggest a cell contact-mediated and T cell-initiated mechanism for the chronic accumulation and retention of mononuclear cells via VCAM-1/ICAM-1 by synovial fibroblasts in the rheumatoid synovium.

The role for the two tumor necrosis factor (TNF) receptors in discriminating TNF and lymphotoxin ␣ (LT␣) effects has been studied. TNF and LT␣ were equally mitogenic in Fs4 fibroblasts, which express a high amount of the p55 compared to the p75 TNF receptors (TNFRs). In contrast, TNF was more potent than LT␣ in mediating gene regulation and cytotoxicity in SW480-␤Gal cells and KYM-1 cells, which have a high p75/p55 TNFR ratio. Both TNF and LT␣ showed comparable affinities for the two TNFRs. However, in contrast to LT␣, TNF dissociated rapidly from the p75 TNFR, whereas both cytokines dissociated slowly from the p55 TNFR. Soluble p55 TNFR was much more potent than soluble p75 TNFR in inhibiting TNF cytotoxicity, whereas both soluble receptors moderately decreased LT␣-mediated cytotoxicity with comparable efficacy. Antagonistic monoclonal antibodies against either TNFR types markedly inhibited TNF effects. However, only the p55 TNFR antagonistic antibody significantly decreased LT␣-mediated cytotoxicity and cytomegalovirus promoter activation, whereas blocking of the p75 TNFR enhanced the LT␣ effects. These data suggest that whereas the p75 TNFR can both directly propagate TNF signals and "pass" TNF to the p55 TNFR, it attenuates LT␣ and may serve as a decoy receptor for this cytokine. Tumor necrosis factor ␣ (TNF)1 and lymphotoxin ␣ (LT␣, TNF␤) are pleiotropic cytokines which mediate a large variety of inflammatory, immunostimulatory, and antiviral responses (1). They are both members of the TNF ligand and receptor family, which now contains at least 12 ligand-receptor pairs (2). TNF and LT␣ are unique among this family in sharing the two TNF receptors, the p55 TNFR (CD120a) and the p75 TNFR (CD120b), both of which are expressed on most mammalian cells (3). Both TNF and LT␣ exist as homotrimeric molecules, each with the capability of complexing with three receptors (4, 5), and both cytokines are believed to elicit their effects by the cross-linking of cell surface receptors (6 -8). LT␣ has also been shown to form a cell surface heterocomplex with LT␤ (9). While the LT␣ homotrimer binds to the p55 and p75 TNFRs, the heteromeric LT␣⅐LT␤ complex interacts with a TNFR-related protein (LT␤-R) (10). Gene knock-out experiments in mice suggest that the LT␣-LT␤ heteromer is involved in the development of the peripheral lymphoid tissues (11), while the two TNFRs are responsible for the resistance for certain types of bacterial pathogens (12, 13).The ratio of the two receptors on the cell surface varies among cells, i.e. some cell types have a high proportion of the p75 TNFR versus the p55 TNFR, and vice versa. Furthermore, the cell surface expression of the two TNFRs is independently regulated in many cell types. It has been shown, for instance, that activation of B cells results in a marked up-regulation of the p75 but not the p55 TNFR (14). Also, the generation of soluble receptors differs for the two TNFRs, because in vitro stimulation of monocytes with lipopolysacharide results in the specific secretion of the p75 TNFR...