Human monocytes produce IL-1 and an inhibitor of IL-1 in response to two different signals.

Recently, IL-1 inhibitors from urine, monocytes, or monocyte lines have been described. The relationship of these inhibitors to the production, release, and immunological effects of IL-1 is unclear. The present studies were initiated to describe and quantitate the production of IL-1 and a 23 to 45-kDa IL-1 inhibitor from human monocytes in response to certain stimuli using a mouse thymocyte system responsive to IL-1. Zymosan stimulated monocytes to produce IL-1 but not IL-1 inhibitor. Adherent immune complexes, human IgG1-4, and Fc fragments, but not F(ab')2, stimulated monocyte production of IL-1 inhibitor and little if any IL-1. Fibronectin and three of its fragments had neither effect. These observations suggest that monocytes produce IL-1 or IL-1 inhibitor in response to two different signals, through "endotoxin or beta-glucan" and Fc receptors, respectively. The inhibitor decreases IL-1-induced CD-1, C3H/HeJ, and D10 G4.1 cells but not IL-2-induced CD-1, C3H/HeJ, or CTLL-2 proliferation. The inhibitor competitively blocked binding of radiolabeled rIL-1 to the IL-1 receptor on murine thymoma cells. Preincubation of thymocytes with the inhibitor prevented IL-1-induced proliferation; however, this effect was reversed by washing thymocytes and inhibitor activity was markedly reduced when added 24 hr after stimulation with IL-1. These observations suggest that the inhibitor acts on IL-1 receptors to prevent thymocyte proliferation. Monocytes from patients with systemic lupus erythematosus produced less IL-1 inhibitor than cells from normal volunteers. The decrease in IL-1 inhibitor production may play a role in disease states.

Selective immunosuppression with anti-interleukin 2 receptor-targeted therapy: helper and suppressor cell activity in rat recipients of cardiac allografts.

(LEW X BN)F1 cardiac allografts are rejected within 8 days in unmodified LEW rats. ART18, a mouse anti-rat IgG1 monoclonal antibody which binds specifically in vitro to the interleukin 2 receptor (IL 2R) molecule expressed primarily on activated T cells, prolongs allograft survival in a dose-dependent fashion to ca. 3 weeks (p less than 0.001) after being administered for 10 days after transplantation. This effect was related to the specificity of the antibody for IL 2R, as therapy with ART62 (a monoclonal antibody recognizing MHC class I antigen but not binding the rat IL 2R) was ineffectual. Suppressor activity was detected in spleen cells of ART18-treated grafted hosts: in vivo, splenic T suppressor/cytotoxic fraction adoptively transferred into normal LEW improved donor-specific but not third-party test graft survival (17 days, vs. 8 days, respectively, p less than 0.001); in vitro, mixed lymphocyte reaction was profoundly but nonspecifically inhibited (less than 5% of test mixed lymphocyte reaction, p less than 0.001 as compared to acutely rejecting controls). In contrast, splenic T helper (Th) cells from ART18-treated hosts were functionally depressed, as noted by their passive transfer into immunologically anergic B recipients of cardiac allografts (rejection in ca. 40 days, vs. ca. 13 days after transfer of Th from specifically sensitized rats). ART18 treatment also resulted in diminished elaboration of IL 2 as compared to normal (p less than 0.005) or acutely rejecting hosts (p less than 0.001); however, a remarkable increase in the production of IL 3 occurred (p less than 0.001). These results demonstrate that IL 2R-targeted therapy of immunocompetent graft recipients produces a selective immune defect in which donor-specific T suppressor cells are spared, but Th cells attenuated or destroyed. Decreased elaboration of IL2 concomitantly augments the release of IL 3, a lymphokine which might play a role in suppressor effect in vivo. In addition, IL 2R-targeted therapy of the immunodeficient graft recipients abrogates the capacity of alloactivated T cells to re-establish acute immune responsiveness.

Characterization of the Ia antigens involved in suppressor T cell generation in the rat.

The WRC rat, an intra-class II recombinant strain (RT1.B beta nB alpha aD alpha, beta a), was used to study the relative roles of the two class II loci in mixed lymphocyte reaction (MLR) proliferation and suppressor T cell (Ts) generation. Both MLR proliferation and Ts generation were noted in cultures of WRC with DA (RT1a) stimulator cells. In contrast, cultures of WRC with BN (RT1n) stimulator cells proliferate but do not generate significant amounts of Ts. The data suggest that RT1.B beta incompatibility is important in the generation of Ts in the WRC rat. Suppressor cells generated in cultures of WF (RT1u) with WRC stimulator cells potently suppressed a WF + WRCx test MLR, with less suppression when tested against either the WF + DAx or WF + BNx MLRs. The latter experiments suggest that Ts clones may be produced to either class II subregion, and therefore that MLR proliferation and Ts induction are not necessarily linked, but vary with particular genotypes. The current lack of other rat intra-class II recombinant strains precludes assignment of suppressor induction/activation to a single locus.

Systemic natural killer activity following cardiac engraftment in the rat: lack of correlation with graft survival.

The systemic NK activity was studied both in untreated rats which acutely reject allogeneic heterotopic heart grafts and in cyclosporine-treated rats which tolerate their transplants. The trend and magnitude of changes in NK activity were similar at all time points for the two animal groups. Compared to naive rats, peak NK activity was noted 7-8 days after engraftment in untreated rats and 7-12 days after engraftment in cyclosporine-treated hosts. In both groups, NK activity returned to normal levels by 3 weeks. No evidence could be found for inactivation of NK cells or their precursors in vivo in ungrafted rats undergoing cyclosporine treatment alone. These data are consistent with prior studies and suggest that non-specific cytotoxic activity does not represent a crucial force contributing to acute rejection of vascularized organ grafts.

Cyclosporine-induced transplantation unresponsiveness in rat cardiac allograft recipients: in vitro determination of helper and suppressor activity.

Lymphocytes from spleen, peripheral blood, thymus, and lymph node of naive rats, nonimmunosuppressed recipients of MHC-incompatible heart grafts, and cyclosporine-treated recipients of MHC-incompatible heart grafts were tested for their ability to augment or suppress proliferation of naive cells in an in vitro MLR co-culture assay. Rats treated with cyclosporine for only 7 days maintained their grafts indefinitely. Potent suppressor activity was found in the peripheral blood and spleen of adult naive rats. In untreated engrafted rats, increased suppressor activity was found 1 wk after transplantation and increased helper activity 2 wk after transplantation. In contrast, subnormal helper and suppressor activity was found in cyclosporine-treated rats 1 wk after transplantation. Subsequently, suppressor activity peaked at 2 to 3 wk and helper activity at 4 wk after transplantation. Beyond 5 wk, the cyclosporine-treated rat was indistinguishable from naive ungrafted rats. Two types of suppressor activity were identified that differed in buoyant density and cyclophosphamide sensitivity. Neither suppressor activity demonstrated antigen specificity. These data suggest that one role of cyclosporine in this rat model is to delay the initial helper mechanisms until generalized suppressor activity is operable. The increased antigen-nonspecific activity is only transient, presumably until the final antigen-specific mechanisms become operative.

The modification of tryptophan in bovine thrombin.

2-Hydroxy-5-nitrobenzyl bromide, at a 100-fold molar excess, was observed to react withthrombin at pH 4.0 to give a modified enzyme which possessed 20% of the fibrinogen clotting activity and 80% of the esterase activity compared to a control preparation. Spectrophotometric analysis of the modified protein indicated that this effect on catalytic activity was associated with the incorporation of 1 mol of reagent per mol of thrombin. Amino acid analysis showed no loss of amino acids other than tryptophan. The reaction of N-bromosuccinimide with thrombin at 2-fold molar excess resulted in the modification of one tryptophan per mol of enzyme with the loss of 80% of the fibrinogen clotting activity with, as above, a considerably smaller loss of esterase activity. Oxidation of thrombin with N-bromosuccinimide decreased the extent of subsequent tryptophan modification with 2-hydroxy-5-nitrobenzyl bromide. Thrombin modified with 2-hydroxy-5-nitrobenzyl bromide showed a 3-4 fold increase in Km and a decrease in V for the ester substrate. The reaction of thrombin with 2-acetoxy-5-nitrobenzyl bromide, a substrate analogue, also resulted in the inactivation of the enzyme. The data are interpreted to show the presence of a tryptophan residue at or near the enzyme's substrate binding site.