Interferon-gamma (IFN-gamma) is a pleiotrophic cytokine with immunomodulatory effects on a variety of immune cells. Mice with a targeted disruption of the IFN-gamma gene were generated. These mice developed normally and were healthy in the absence of pathogens. However, mice deficient in IFN-gamma had impaired production of macrophage antimicrobial products and reduced expression of macrophage major histocompatibility complex class II antigens. IFN-gamma-deficient mice were killed by a sublethal dose of the intracellular pathogen Mycobacterium bovis. Splenocytes exhibited uncontrolled proliferation in response to mitogen and alloantigen. After a mixed lymphocyte reaction, T cell cytolytic activity was enhanced against allogeneic target cells. Resting splenic natural killer cell activity was reduced in IFN-gamma-deficient mice. Thus, IFN-gamma is essential for the function of several cell types of the murine immune system.

Recombinant human tumor necrosis factor (rTNF alpha) injected intravenously into rabbits produces a rapid-onset, monophasic fever indistinguishable from the fever produced by rIL-1. On a weight basis (1 microgram/kg) rTNF alpha and rIL-1 produce the same amount of fever and induce comparable levels of PGE2 in rabbit hypothalamic cells in vitro; like IL-1, TNF fever is blocked by drugs that inhibit cyclooxygenase. At higher doses (10 micrograms/kg) rTNF alpha produces biphasic fevers. The first fever reaches peak elevation 45-55 min after bolus injection and likely represents a direct action on the thermoregulatory center. During the second fever peak (3 h later), a circulating endogenous pyrogen can be shown present using passive transfer of plasma into fresh rabbits. This likely represents the in vivo induction of IL-1. In vitro, rTNF alpha induces the release of IL-1 activity from human mononuclear cells with maximal production observed at 50-100 ng/ml of rTNF alpha. In addition, rTNF alpha and rIFN-gamma have a synergistic effect on IL-1 production. The biological activity of rTNF alpha could be distinguished from IL-1 in three ways: the monophasic pyrogenic activity of rIL-1 was destroyed at 70 degrees C, whereas rTNF alpha remained active; anti-IL-1 neutralized IL-1 but did recognize rTNF alpha or natural cachectin nor neutralize its cytotoxic effect; and unlike IL-1, rTNF alpha was not active in the mitogen-stimulated T cell proliferation assay. The possibility that endotoxin was responsible for rTNF alpha fever and/or the induction of IL-1 was ruled-out in several studies: rTNF alpha produced fever in the endotoxin-resistant C3H/HeJ mice; the IL-1-inducing property of rTNF alpha was destroyed either by heat (70 degrees C) or trypsinization, and was unaffected by polymyxin B; pyrogenic tolerance to daily injections of rTNF alpha did not occur; levels of endotoxin, as determined in the Limulus amebocyte lysate, were below the minimum rabbit pyrogen dose; and these levels of endotoxin were confirmed by gas chromatography/mass spectrometry analysis for the presence of beta-hydroxymyristic acid. Although rTNF alpha is not active in T cell proliferation assays, it may mimic IL-1 in a T cell assay, since high concentrations of rTNF alpha induced IL-1 from epithelial or macrophagic cells in the thymocyte preparations. These studies show that TNF (cachectin) is another endogenous pyrogen which, like IL-1 and IFN-alpha, directly stimulate hypothalamic PGE2 synthesis. In addition, rTNF alpha is an endogenous inducer of IL-1.(ABSTRACT TRUNCATED AT 400 WORDS)

The individual roles of the murine type 1 and type 2 tumor necrosis factor (TNF) receptors (TNF-R1 and TNF-R2) were investigated utilizing (i) the strong species specificity of TNF-R2 for murine TNF compared to human TNF and (it) agonistic rabbit polyclonal antibodies directed against the individual TNF receptors. Proliferation of mouse thymocytes and the murine cytotoxic T-cell line CT-6 is stimulated by murine TNF but not by human TNF. Consistent with this observation, polyclonal antibodies directed against TNF-R2 induced proliferation in both of these cell types, whereas polyclonal antibodies directed against TNF-R1 had no effect. In contrast, cytotoxicity in murine LM cells (which are sensitive to murine and human TNF) was induced by antibodies against TNF-R1 but not by antibodies against TNF-R2. Also, the steady-state level of manganous superoxide dismutase mRNA in the murine NIH 3T3 cell line was induced by murine TNF, human TNF, and anti-TNF-R1 but not by anti-TNF-R2. These results suggest that TNF-R2 initiates signals for the proliferation of thymocytes and cytotoxic T cells, whereas TNF-R1 initiates signals for cytotoxicity and the induction of the protective activity, manganous superoxide dismutase. The nonredundant signaling observed for the two TNF receptors cannot be explained simply by the differential expression of the two TNF receptors in the various cell types, because LM cells express on their surface higher levels of TNF-R2 than TNF-R1, and LM cells, NIH 3T3 cells, and thymus cells all express mRNA corresponding to both receptor types. It is therefore likely that the two receptors initiate distinct signaling pathways that result in the induction of different cellular responses. (17)(18)(19)(20).A number of recent reports have described initial studies investigating the individual roles of the two human TNF receptors. Polyclonal and monoclonal antibodies directed against human TNF-R1 have been shown to behave as receptor agonists and elicit several TNF activities, such as cytotoxicity, fibroblast proliferation, resistance to chlamidiae, and synthesis of prostaglandin E2 (15,21,22). Monoclonal antibodies against human TNF-R1 that block the binding of TNF to TNF-R1 and antagonize several TNF effects have also been described (21-23). Although no direct signaling role for TNF-R2 has yet been identified with either receptor agonists or transfection studies, several reports have described monoclonal antibodies directed against TNF-R2 that can partially antagonize TNF responses (such as cytotoxicity and activation of NF-KB) and enhance the antagonistic effects of anti-TNF-R1 monoclonal antibodies (22)(23)(24). These reports suggested that both TNF receptors are active in signal transduction and that there is redundancy in the function of the two receptors. However, the reported effects of the TNF-R2 antagonists have been quite small and were observed exclusively at very low TNF concentrations. It is therefore possible that TNF-R2 is only participating as a minor accessory component to TNF-R1 ...

The HER2 protooncogene encodes a receptor tyrosine kinase, p185HER2. The overexpression of p185HER2 has been associated with a worsened prognosis in certain human cancers. In the present work we have screened a variety of different tumor cell lines for p185HER2 expression using both enzyme-linked immunosorbent and fluorescence-activated cell sorting assays employing murine monoclonal antibodies directed against the extracellular domain of the receptor. Increased levels of p185HER2 were found in breast (5/9), ovarian (1/6), stomach (2/3) and colorectal (5/16) carcinomas, whereas all kidney and submaxillary adenocarcinoma cell lines tested were negative. Some monoclonal antibodies directed against the extracellular domain of p185HER2 inhibited growth in monolayer culture of breast and ovarian tumor cell lines overexpressing p185HER2, but had no effect on the growth of colon or gastric adenocarcinomas expressing increased levels of this receptor. The most potent growth-inhibitory anti-p185HER2 monoclonal antibody in monolayer culture, designated mumAb 4D5 (a murine IgG1 kappa antibody), was also tested in soft-agar growth assays for activity against p185HER2-overexpressing tumor cell lines of each type, with similar results. In order to increase the spectrum of tumor types potentially susceptible to monoclonal antibody-mediated anti-p185HER2 therapies, to decrease potential immunogenicity issues with the use of murine monoclonal antibodies for human therapy, and to provide the potential for antibody-mediated cytotoxic activity, a mouse/human chimeric 4D5 (chmAb 4D5) and a "humanized" 4D5 (rhu)mAb 4D5 HER2 antibody were constructed. Both engineered antibodies, in combination with human peripheral blood mononuclear cells, elicited antibody-dependent cytotoxic responses in accordance with the level of p185HER2 expression. Since this cytotoxic activity is independent of sensitivity to mumAb 4D5, the engineered monoclonal antibodies expand the potential target population for antibody-mediated therapy of human cancers characterized by the overexpression of p185HER2.

Cyclosporin A (CsA) has proven effective as an inhibitor of a variety of T cell responses in vitro, including antigen-specific proliferation, macrophage-mediated antigen presentation, and antigen-and lectin-induced cytokine production, including IL-1, IL-2, IL-3, and IFN-y, but not IFN-a/# (1) . CsA appears to function in the prevention of graft rejection by affecting the early activation of immunocompetent cells, possibly by blocking antigen-specific receptor binding, class I and II expression, inhibition of phospholipase A2 activity, and/or inhibition of synthesis of cytokine mRNA (1-4). The selective suppressive activity of CsA for cell-mediated immunity prompted its use in human organ transplantation (5) .Transforming growth factors (TGFs) are a family of peptides that, under certain conditions, can induce normal cells to express a transformed phenotype (6). At least two types of TGFs have been described : TGF-a, which is homologous to epidermal growth factor (EGF), and TGF-f3. Mature TGF-a is a 25 kD homodimer held together by disulfide bonds that binds to a specific receptor(s) distinct from the TGF-a or EGF receptor (6) . TGF-0 inhibits IL-2-induced T cell proliferation, IL-2-R induction, IL-1-induced thymocyte proliferation, IFNa but not IL-2-induced NK activity, B cell proliferation to growth factors and IFN-y-induced class II antigen expression on Hs294T melanoma cells (7)(8)(9)(10)(11) .In this report, we describe the effects of CsA and TGF-(3 on the production of TNF-a (also referred to as cachectin), TNF-# (lymphotoxin), and IFN-y by human PBMC and murine peritoneal macrophages . Our results demonstrate that CsA and TGF-0 have suppressive effects on cytokine production that are dependent on their selective activity on specific cell types . Materials and MethodsCytokines. Recombinant human IFN-y (rHuIFN-,y) and murine IFN-,y (rMuIFN-y), cloned and expressed in Escherichia coli (12, 13), were purified to >99% purity~Rinder-knecht, E., unpublished results) .

The HER2 protooncogene encodes a 185-kDa transmembrane protein (p185HER2) with extensive homology to the epidermal growth factor (EGF) receptor. Clinical and experimental evidence supports a role for overexpression of the HER2 protooncogene in the progression of human breast, ovarian, and non-small cell lung carcinoma. These data also support the hypothesis that p185HER2 present on the surface of overexpressing tumor cells may be a good target for receptor-targeted therapeutics. The anti-p185HER2 murine monoclonal antibody (muMAb) 4D5 is one of over 100 monoclonals that was derived following immunization of mice with cells overexpressing p185HER2. The monoclonal antibody is directed at the extracellular (ligand binding) domain of this receptor tyrosine kinase and presumably has its effect as a result of modulating receptor function. In vitro assays have shown that muMAb 4D5 can specifically inhibit the growth of tumor cells only when they overexpress the HER2 protooncogene. MuMAb 4D5 has also been shown to enhance the TNF-alpha sensitivity of breast tumor cells that overexpress this protooncogene. Relevant to its clinical application, muMAb 4D5 may enhance the sensitivity of p185HER2-overexpressing tumor cells to cisplatin, a chemotherapeutic drug often used in the treatment of ovarian cancer. In vivo assays with a nude mouse model have shown that the monoclonal antibody can localize at the tumor site and can inhibit the growth of human tumor xenografts which overexpress p185HER2. Modulation of p185HER2 activity by muMAb 4D5 can therefore reverse many of the properties associated with tumor progression mediated by this putative growth factor receptor. Together with the demonstrated activity of muMAb 4D5 in nude mouse models, these results support the clinical application of muMAb 4D5 for therapy of human cancers characterized by the overexpression of p185HER2.

Tumor necrosis factors (TNF) a and (i are structurally related cytokines that mediate a wide range of immunological, inflammatory, and cytotoxic effects. During bacterial infection of the bloodstream (sepsis), TNF-a induction by bacterial endotoxin is thought to be a major factor contributing to the cardiovascular collapse and critical organ failure that can develop. Despite antibiotic therapy, these consequences of sepsis continue to have a high mortality rate in humans. Here we describe a potent TNF antagonist, a TNF receptor (TNFR) immunoadhesin, constructed by gene fusion of the extracellular portion of human type 1 TNFR with the constant domains of human IgG heavy chain (TNFR-IgG). When expressed in transfected human cells, TNFR-IgG is secreted as a disulfide-bonded homodimer. Purified TNFR-IgG binds to both TNF-a and TNF-,B and exhibits 6-to 8-fold higher affinity for TNF-a than cell surface or soluble TNF receptors. In vitro, TNFR-IgG blocks completely the cytolytic effect of TNF-a or TNF-( on actinomycin D-treated cells and is markedly more efficient than soluble TNFR (24-fold) or monoclonal anti-TNF-a antibodies (4-fold) in inhibiting TNF-a. In vitro, TNFR-IgG prevents endotoxin-induced lethality in mice when given 0.5 hr prior to endotoxin and provides significant protection when given up to 1 hr after endotoxin challenge. These results confim the importance of TNF-a in the pathogenesis of septic shock and suggest a clinical potential for TNFR-IgG as a preventive and therapeutic treatment in sepsis.Tumor necrosis factors a (TNF-a; cachectin) and ( (TNF-f3; lymphotoxin) are related proteins, secreted by activated macrophages and lymphocytes, respectively (1-3). These cytokines have been implicated in diverse biological processes including immunoregulation, inflammation, antiviral defense, cachexia, angiogenesis, and septic shock. The biological effects of TNF-a and TNF-p8 are mediated through specific receptors. Molecular cloning has demonstrated the existence of two distinct types of TNF receptor (TNFR), each of which binds to both TNF-a and TNF-,B (4-8). The extracellular portions of both receptors are found naturally also as soluble TNF binding proteins (7,8).Several lines of evidence indicate that TNF-a is a principal mediator in the pathogenesis of septic shock. First, neutralizing anti-TNF-a antibodies can prevent the pulmonary failure and death associated with administration of endotoxin or Escherichia coli in mice (9) or baboons (10). Second, intravenous infusion of TNF-a leads to a toxic syndrome indistinguishable from that caused by endotoxemia and gramnegative sepsis (11,12). In addition, the levels of TNF-a increase substantially in the circulation of animals and humans who have received endotoxin or have septic shock (13,14) and correlate with mortality in severe sepsis (15)(16)(17).To create a TNF antagonist that might block the lethal effect of TNF in endotoxic shock, we constructed an immunoadhesin (18) containing the extracellular portion of human type 1 TNFR and the hinge and F...

We compared the ability of recombinant human tumor necrosis factor- alpha (rHuTNF-alpha) and tumor necrosis factor-beta (rHuTNF-beta) to stimulate polymorphonuclear neutrophil (PMN) migration and superoxide production. Significant PMN migration occurred across polycarbonate filters after stimulation with rHuTNF-alpha at concentrations ranging from 10(-7) to 10(-10) mol/L and at 10(-7) to 10(-8) mol/L for rHuTNF- beta and N-formylmethionyl-leucyl phenylalanine (FMLP), whereas recombinant human interferon-gamma was only minimally active at 10(-7) mol/L and recombinant human interleukin-1 alpha was inactive at the doses tested. In addition, antibodies to rHuTNF-alpha completely inhibited rHuTNF-alpha but not rHuTNF-beta or FMLP-induced PMN migration. Combinations of rHuTNF-alpha and rHuTNF-beta (at similar molar concentrations) stimulated PMN migration levels comparable to that obtained with rHuTNF-alpha alone. Checkerboard analyses performed by placing different concentrations of rHuTNF-alpha and rHuTNF-beta above and below polycarbonate filters of microchemotaxis chambers demonstrated that rHuTNF-alpha and rHuTNF-beta stimulated both chemotactic and chemokinetic responses by PMN. Additional studies demonstrated that 1 X 10(-8) mol/L rHuTNF-alpha and 3 X 10(-9) mol/L rHuTNF-beta (which represents 10(4) U/mL of each cytokine) were similar in their ability to induce superoxide production by PMNs; however, at ten- to 100-fold lower molar concentrations (10(3) and 10(2) units), rHuTNF-alpha was significantly more active than rHuTNF-beta. At the doses tested, both cytokines were less active than phorbol myristate acetate at stimulating O2- release. The results demonstrate that rHuTNF- alpha and rHuTNF-beta differ quantitatively but not qualitatively in their effects on PMN functions in vitro and suggest that rHuTNF-beta may be less toxic than rHuTNF-alpha in vivo.