Amelioration of antigen-induced arthritis in rabbits treated with monoclonal antibodies to leukocyte adhesion molecules.

OBJECTIVE: To examine the effects of treatment of antigen-induced arthritis in rabbits with a monoclonal antibody against CD18, the common beta chain of the leukocyte adhesion molecules. Intraarticular injection of antigen into primed rabbits elicits an acute inflammatory response followed by chronic arthritis in this model. METHODS: Anti-CD18 was given at the time of intraarticular antigen administration, and effects on the acute and chronic arthritis were investigated. Twenty-four rabbits were examined (11 controls, 3 receiving normal mouse IgG, and 10 receiving anti-CD18). RESULTS: Flow cytometry of blood leukocytes at anti-CD18 administration showed saturating amounts of mouse Ig coating all the circulating cells. Treatment effects on the acute arthritis (measured by quantitating the synovial cell exudate 24 hours after arthritis induction) were a profound reduction in the number of inflammatory cells and a striking decrease in the proportion of polymorphonuclear leukocytes recovered from the synovial cavity, indicating a decrease in acute inflammation. Treatment effects on the chronic synovitis (2 and 4 weeks later) compared with controls showed a significant decrease in synovial fluid cell counts at 2 weeks (1.7 versus 21.0 x 10(6)/joint, P less than 0.03) and at 4 weeks (7.4 versus 22.6 x 10(6)/joint, P less than 0.05). Histologic evaluation of the synovium (0-3+ scale, scored "blindly") of anti-CD18-treated rabbits and controls showed marked decreases in subsynovial cell infiltration and lymphoid follicle formation both at 2 weeks (1.0 versus 2.25, P less than 0.005; and 0 versus 1.75, P less than 0.001) and at 4 weeks (1.48 versus 2.17, P less than 0.01; and 0.75 versus 2.08, P less than 0.02). Quantitation of cartilage-bound immune complexes, and of synovial synthesis of Ig and specific antibody showed no differences between groups. CONCLUSION: These findings indicate that treatment with monoclonal antibody to CD18 not only modifies the initial acute arthritis, but also results in significant amelioration of the subsequent chronic inflammation in this animal model of rheumatoid arthritis.

Detection of major group rhinoviruses by soluble intercellular adhesion molecule-1 (sICAM-1).

Soluble intercellular adhesion molecule-1 (sICAM-1) was shown to be the receptor for the major subgroup of rhinoviruses. It was demonstrated that this molecule can inhibit the binding and subsequent infection of target cells by rhinoviruses belonging to the major but not the minor subgroup. The data reported now describe an ELISA-based system utilizing biotinylated sICAM-1 as a means of detecting rhinoviruses belonging to the major subgroup.

Role of adherence vs. spreading in the induction of membrane-associated interleukin-1 on mouse peritoneal macrophages.

Adherence of peritoneal exudate cells (PEC) to plastic has been shown to activate several PEC functions, such as tumor cell lysis and membrane-associated interleukin-1 (mIL-1) expression. Several studies have demonstrated that leukocyte adherence is dependent on divalent cations. In this study, ethylenediaminetetraacetic acid (EDTA), a known chelator of divalent cations, was used to evaluate the role of cell attachment vs. spreading in adherence-induced mIL-1 activity on resident C57BL/6 mouse PEC. Significant inhibition of PEC spreading on plastic and mIL-1 expression was noted when PEC were cultured in the presence of 10 mM EDTA. However, PEC remained adherent in the presence of EDTA and were able to express mIL-1 activity in response to a soluble stimulus lipopolysaccharide (LPS) 1 microgram/ml. These results suggest that the divalent cation-dependent spreading of PEC on plastic initiates or enhances the expression of mIL-1 activity. Additionally, adhesion and LPS stimulate mIL-1 expression by independent mechanisms.

Synergistic and overlapping activities of tumor necrosis factor-alpha and IL-1.

TNF-alpha and IL-1 induce the production of PGE2 from human chondrosarcoma, fibrosarcoma, and carcinoma cell lines. When combined at sub-optimal concentrations, TNF-alpha and IL-1 synergistically stimulate PGE2 production. The synergy of TNF-alpha and IL-1 on the induction of PGE2 is partially neutralized by specific antibodies. In vitro, human rTNF-alpha is directly cytotoxic to several human transplantable tumor cell lines. These include a human carcinoma, human chondrosarcoma, and a human transformed fibroblast cell line. The cytotoxic effect of TNF-alpha was abrogated by a specific, neutralizing, polyclonal antibody. IL-1 had no direct cytotoxic effect on these cell lines; however, IL-1 enhanced the cytotoxic effects of TNF-alpha. The synergy of these two cytokines in the cytotoxic assay was neutralized by the addition of specific neutralizing antibodies.

Membrane-associated interleukin 1 activity on human U937 tumor cells: stimulation of PGE2 production by human chondrosarcoma cells.

Membrane-associated interleukin 1 (IL 1) activity was induced on the human macrophage tumor cell line, U937, by pretreatment with phorbol myristic acid (PMA). Incubation of PMA-treated, paraformaldehyde-fixed U937 cells with the murine cell line D10.G4.1 in the presence of concanavalin A caused an increase in DNA synthesis as measured by the uptake of tritiated thymidine. Paraformaldehyde-fixed U937, not pretreated with PMA, showed little or no activity. A rabbit polyclonal antibody directed against human IL 1 neutralized all membrane-associated IL 1-like activity, as measured by the inhibition of D10.G4.1 cell proliferation. PMA-treated U937 caused a pronounced enhancement of PGE2 production from a human chondrosarcoma cell line, SW-1353. Membrane-associated IL 1 induced a more potent PGE2 response than did a maximal concentration of soluble IL 1. Rabbit antihuman IL 1 neutralized membrane-bound IL 1 induction of PGE2. The data presented here raise the possibility that membrane-bound IL 1 may play a primary role in the pathophysiology of the inflammatory disease process.

Effect of UV irradiation on expression of membrane IL 1 by rat macrophages.

The effect of UV-B irradiation on the expression of membrane-associated IL 1 (mIL 1) by rat pulmonary alveolar macrophages (PAM) was studied. We found that although there was an increase in secreted IL 1 by PAM exposed to UV-B, the expression of mIL 1 was inhibited in a dose-dependent manner. Furthermore, PAM that were allowed to express mIL 1 before UV-B irradiation had a faster decay of mIL 1 activity than unirradiated cells. These data suggested that mIL 1 expression is inhibited by UV-B irradiation, and that under normal circumstances, mIL 1 synthesis and degradation is at a steady state, with the half-life of mIL 1 activity being 24 hr when assayed in an IL 1-dependent cell line proliferation assay. These data indicate that secreted forms of IL 1 and mIL 1 are differentially regulated and that the therapeutic effects of UV irradiation may be due to its inhibition of mIL 1 activity.

Regulatory mechanisms in cytotoxic T lymphocyte development. I. A suppressor T cell subset that regulates the proliferative stage of CTL development.

The alloantigen-induced suppressor function of cells from 3-day mixed leukocyte culture (MLC) was studied. These cells, when co-cultured with normal syngeneic lymphocytes and cells of the same haplotype as the original inducing alloantigen, inhibited the generation of cytotoxic T lymphocytes (CTL). Suppression was mediated by a radiation-resistant Lyt-2+ T cell. The suppressor T cells appeared to act by inhibiting the clonal expansion of CTL precursors in the responder cell population, determined by limiting dilution analysis. Levels of endogenous interleukin 2 (IL 2) in co-cultures with suppressor T cells were diminished, and the addition of exogenous IL 2 to co-cultures cancelled the suppressor T cell effects. The suppressor cell population was shown to be capable of absorbing IL 2 from lymphokine preparations, and in contrast to mitogen-induced suppressor T cells, after exposure to IL 2 the allostimulated suppressor T cell remains active. The results are discussed in terms of possible modes of action of the suppressor T cell.

Recovery of humoral and cellular immunity by soluble mediators after 5-fluorouracil-induced immunosuppression.

5-Fluorouracil (5-FU) administered in daily injections to mice (15-60 mg/kg; subcutaneous) was differentially toxic to helper T cells. Precursors for both antibody forming cells and cytotoxic T lymphocytes (CTL) were spared. 5-FU suppressed the in vitro T cell-dependent antibody response to sheep red blood cells (SRBC). This low response was restored to normal levels by the addition of T cell replacing factor (TRF) or mixed lymphocyte culture (MLC) supernatants to the culture system. T cell-independent antibody responses to TNP-Ficoll or TNP-LPS were not eliminated by 5-FU but, in contrast, were elevated two-four-fold. These results indicate that precursors for antibody forming cells for T cell-dependent and -independent antibody responses were not eliminated by 5-FU, 5-FU administered in the same regimen did not reduce the number of CTL precursors as shown by limiting dilution analysis, but did cause a reduction in the capacity of lymphocytes from pre-treated mice to generate a CTL response in vitro. This low CTL response was restored to control levels by adding Lyt 1+2- T cells or sources of interleukin 2 (IL2) to the culture system, indicating that 5-FU similarly eliminated helper cells for CTL precursor differentiation as well as helper cells for antibody synthesis.

Immunoenhancing activity of NPT 15392: a potential immune response modifier.

NPT 15392, a new immunomodulating compound related to inosine in structure and isoprinosine in action, enhances T-cell dependent immune responses. Antibody responses to sheep red blood cells are augmented two to threefold in mice receiving NPT 15392 while T-cell independent antibody responses to TNP-LPS are unaffected. NPT 15392 does not enhance or alter the number of clonable B cells. This drug also increases cytotoxic T-lymphocyte responses to allogeneic tumor cells but does not alter the number of cytotoxic precursor cells. Immature hematopoietic cell classes (clonable progenitor cells) were also monitored and found not to be influenced by NPT 15392.

Effects of gamma-irradiation on lymphocyte subpopulations participating in the development of the cytotoxic T lymphocyte response.

Differences in radiosensitivity among lymphocyte subpopulations involved in generating allogeneic cytotoxic T lymphocyte (CTL) responses were studied. Gamma irradiated splenic lymphocytes from C57BL/6 mice (B6, H-2b) were stimulated in vitro with DBA/2 (H-2d) spleen cells or the DBA/2 mastocytoma, P815. Limiting dilution analysis indicated that the cytotoxic T lymphocyte effector cells (CTLe) for either antigen arose from the same pool of cytotoxic T lymphocyte precursors (CTLp), and that the D37 value of the B6 anti-H-2d CTLp was approximately 165 R. In mixed lymphocyte cultures, however, differential effects of irradiation were seen between the 2 antigens in the ability of irradiated responder B6 spleen cells to generate CTLe. For induction of cytotoxic activity against DBA/2 splenocytes, the CTLp was the most radiosensitive component of the response, whereas the generation of a cytotoxic response against P815 was limited by the radiosensitivity of an Lyt-1 T helper cell (D37 approximately 85 R). The radiosensitive Lyt-1 cell could be replaced by mixed lymphocyte reaction supernatants or semipurified interleukin 2 (IL-2) from Con A-stimulated murine spleen cells. The requiremet of B6 anti-H-2d CTLp for a radiosensitive accessory cell in the CTL response against P815, in contrast with their activation by DBA/2 splenocytes, may center on the fact that the tumor cells do not possess Ia antigens.

Recovery of the in vivo cytotoxic T-cell response in cyclophosphamide-treated mice by injection of mixed-lymphocyte-culture supernatants.

Mice given injections of high antileukemic doses of cyclophosphamide lost the capacity to generate cytotoxic T-cells in vivo to allogeneic tumor cells. These low responses were not due to the elimination of cytotoxic T-lymphocyte precursors because normal cytotoxic responses were obtained in vivo after cyclophosphamide treatment by injection of helper factor derived from mixed-lymphocyte-culture supernatants.

Inhibition of cytotoxic T-cell clonal expansion by cyclophosphamide and the recovery of cytotoxic T-lymphocyte precursors by supernatants from mixed-lymphocyte cultures.

Spleen cells from mice treated with cyclophosphamide (150 mg/kg) and cultured at suboptimal concentrations do not generate a cytotoxic T-lymphocyte (CTL) response to allogeneic tumor cells. The reduced response of spleen cells from cyclophosphamide-treated mice is not due to the elimination of CTL precursors because normal responses are obtained by the addition of a helper factor(s) derived from mixed lymphocyte culture supernatants. The results indicate that helper cells, required for development of CTL responses to tumor alloantigens, are eliminated by cyclophosphamide in the absence of evident toxicity to CTL precursors.

Recovery of the capacity for cytotoxic T cell generation in cyclophosphamide-treated mice by the addition of LYT-1/2- helper cells.

Spleen cells from mice treated with cyclophosphamide (150 mg/kg) and cultured at suboptimal concentrations do not generate cytotoxic T lymphocytes to allogeneic tumor cells. The reduced response of spleen cells from cyclophosphamide-treated mice is not due to the elimination of cytotoxic T cell precursors because normal responses are obtained by the addition of Lyt-1+2- T helper cells to the culture system. These results indicate that helper cells, required for the development of cytotoxic T cell responses to tumor alloantigens, are eliminated by cyclophosphamide in the absence of evident toxicity to cytotoxic T cell precursors.