Evaluation of chemokine- and phlogistin-mediated leukocyte chemotaxis using an in vivo sponge model.

We have directly compared the in vivo activity of a number of chemokines and phlogistins using a modified murine in vivo sponge model in which gelatin sponges are soaked with chemoattractant and implanted in the peritoneal cavity. Sponges soaked with murine JE/MCP-1 (monocyte chemoattractant protein-1) or zymosan promoted the chemotaxis of specific leukocyte populations in a time-dependent manner, as judged by multiparameter flow cytometry, with granulocytes predominating in zymosan-soaked sponges and granulocytes and macrophages present in JE/MCP-1-soaked sponges. Smaller numbers of B, T and dendritic cells were identified as well. Eotaxin selectively chemoattracted eosinophils in this model, while MIG induced significant T cell migration relative to other chemokines. Cell migration was inhibited by administration of methotrexate, piroxicam or dexamethasone, and JE/MCP-1-mediated trafficking was impaired by treatment with anti-JE antibody or with IL-10, suggesting a role for pro-inflammatory factors in amplifying the JE/MCP-1-induced response. This amplification phase involves the production of the chemokine KC, since anti-KC antibody significantly attenuated JE/MCP-1-induced chemotaxis. These results indicate that intraperitoneally implanted chemoattractant-soaked gelatin sponges are capable of inducing a pronounced inflammatory response characterized by the selective migration of leukocyte populations, and suggest that this model may be useful for delineating the activity of novel inhibitors of leukocyte chemotaxis.

Regulatory effects of interleukin-4 and interleukin-10 on human neutrophil function ex vivo and on neutrophil influx in a rat model of arthritis.

OBJECTIVE: To assess the capacity of interleukin-4 (IL-4) and IL-10 to block polymorphonuclear neutrophil (PMN) activation in an ex vivo human model system, and to confirm their effect on neutrophil function in an animal model of arthritis. METHODS: The ex vivo phagocytic capacity of cytokine-activated human PMNs was assessed by use of assays for measuring the ingestion of heat-killed yeast and by subsequent hexose-monophosphate shunt activation using nitroblue tetrazolium reduction. The in vivo activity of IL-4 and IL-10 was measured using a rat adjuvant arthritis model in which the mycobacterial antigen concentration was titrated to modify disease intensity. RESULTS: IL-4 and IL-10 suppressed the ex vivo activation state of interferon-gamma- and tumor necrosis factor alpha-activated human neutrophils. In the rat adjuvant arthritis model, treatment with systemic murine IL-10 (mIL-10) effectively suppressed all disease parameters in rats that received the lower concentrations of mycobacteria, whereas systemic mIL-4 was effective against even the most severe disease. Both cytokines were effective in lowering the absolute PMN cell number recovered and the PMN activation state in the joint synovia. We also observed lower levels of the messenger RNA transcript for CINC protein (cytokine-induced neutrophil chemoattractant; a rat homolog for human IL-8) in the synovia. CONCLUSION: IL-10 is an effective antiarthritic agent and has a major effect on the presence and function of PMNs in the joint synovia when disease intensity is not severe. IL-4 has an inhibitory profile that is similar to that of IL-10, but is effective in modifying even the most severe disease. Both cytokines reduced the phagocytic activation of human PMNs in response to proinflammatory cytokines. These data demonstrate that IL-4 and IL-10 can exert powerful regulatory effects on neutrophil function that translate into a therapeutic response in a disease model of arthritis. Treatment with these cytokines alone or in combination may therefore be very useful in the management of patients with rheumatoid arthritis.

Interleukins 6 and 11 protect mice from mortality in a staphylococcal enterotoxin-induced toxic shock model.

BALB/By mice given doses of D-galactosamine plus Staphylococcus aureus enterotoxin B die within 48 h of administration. The cause of death is a syndrome much like toxic shock syndrome in humans. We used this model to investigate the role of two cytokines, interleukin 6 and interleukin 11, which share the signal transducing subunit, gp130, of their respective receptors. We observed that pretreatment of mice with antibody to interleukin 6 increased mortality from 55% to nearly 90% (P < 0.001), while pretreatment with either cytokine reduced death. The protection was dose dependent; however, interleukin 6 was about 10-fold more potent that interleukin 11. These data indicate that endogenous interleukin 6 plays a protective role in attenuating acute inflammatory responses; furthermore, interleukin 6 and interleukin 11 can abrogate T-cell activation due to triggering by superantigen. A possible clinical role for these cytokines in the treatment of toxic shock merits further investigation.

Oncostatin M stimulates proliferation in B9 hybridoma cells: potential role of oncostatin M in plasmacytoma development.

Oncostatin M was found to stimulate the IL-6-addicted hybridoma line B9. Leukaemia inhibitory factor did not stimulate proliferation of this line. Both of these factors bind to the gp130 of the IL-6 receptor. In another cell line that is stimulated by LIF (DA.1), neither IL-6 nor oncostatin M stimulated proliferation. Previously it had been thought that the gp130 alone is sufficient to bind ligand and transduce signal and that oncostatin M could bind to and activate the LIF receptor, but these data show this is not always the case. Mice primed with Pristane were found to have IL-6 in their sera and peritoneal fluid only at a few time points following Pristane treatment; this was determined by IL-6-specific ELISA. When the same samples were analysed on IL-6-addicted B9 cells, stimulatory activity was found at all time points. When the Pristane-primed samples were assayed for oncostatin M activity in the A375 melanoma assay, there was oncostatin M activity at various time points. IL-6 did not have activity on the A375 cells. These data indicate that oncostatin M play a role in the generation of plasmacytoma in vivo.

Protective role of interleukin 6 in the lipopolysaccharide-galactosamine septic shock model.

C57BL/6J mice given low doses of lipopolysaccharide (LPS) (100 ng per mouse) plus D-galactosamine (8 mg per mouse) die within 24 h following LPS administration. We used this septic shock model to confirm the role of tumor necrosis factor in mortality using a monoclonal antibody to tumor necrosis factor to prevent lethality. Furthermore, we demonstrated that interleukin 6, rather than playing a lethal role, protected mice against death in this septic shock model. Antibody to interleukin 6 did not affect the fatal outcome in this low-LPS-dose model. However, pretreatment with antibody to tumor necrosis factor did protect the mice against death, in a dose-dependent manner. Moreover, mortality was enhanced by pretreatment with antibody to interleukin 6 when tumor necrosis factor was partly limited by anti-tumor necrosis factor treatment. Mortality was significantly reduced by pretreatment with both recombinant interleukin 6 and low doses of antibody to tumor necrosis factor; in the absence of the low dose of antibody to tumor necrosis factor, interleukin 6 alone did not confer any protection. These data demonstrate in vivo antagonistic activities of tumor necrosis factor and interleukin 6 and show that interleukin 6 can play a protective role against death from septic shock.

Two pathways of signal transduction are activated in the same cell by different cytokines.

NFS60, a murine leukemia cell line, responds to both interleukin 3 and 6 by proliferating, apparently by different signal transduction pathways. Although stimulation by both cytokines increases the uptake of 3H-arachidonic acid, the response to IL-6 was much faster. Furthermore, the effect of various arachidonic acid metabolites on the response to cytokine was different. PGE2 inhibited IL-6-induced proliferation and potentiated the response to IL-3. Additionally the G proteins which coupled the IL-3 and IL-6 receptor to the proliferative response are probably different, based on the ability of cholera toxin to inhibit the IL-3 but not the IL-6 response. These data are evidence of two pathways of signal transduction.

An immunotoxin composed of a monoclonal antitransferrin receptor antibody linked by a disulfide bond to the ribosome-inactivating protein gelonin: potent in vitro and in vivo effects against human tumors.

An immunoconjugate was prepared containing a disulfide linker between a murine monoclonal antibody (5E9), which recognized the human transferrin receptor, and the ribosome-inactivating protein gelonin. This immunoconjugate was found to consist of two major species, 5E9-gelonin2 and 5E9-gelonin1, and a minor species of 5E9-gelonin3 and less than 10% of either free antibody or gelonin. 5E9-gelonin was extremely toxic in vitro to human tumor cell lines expressing the 5E9 antigen, including a Burkitt's lymphoma, an adult T-cell acute lymphocytic leukemia, an acute myelogenous leukemia, a promyelocytic leukemia, and a cervical carcinoma line. A 24-hour exposure to 10(-9) M immunoconjugate killed 90-99.9% of tumor cells, depending on the cell line. A 5E9-negative murine leukemia was not sensitive to this conjugate. Pharmacokinetic analysis of the disappearance of this immunoconjugate from the murine circulation revealed that it had a biphasic clearance, with an initial rapid phase with a half-life (t1/2) of 3 hours and a later, slower phase with a t1/2 of about 1 day. Analysis of blood samples by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis revealed that a substantial degree of disulfide-linker breakdown occurred in vivo and that the 5E9-gelonin2 species was cleared more rapidly than the 5E9-gelonin1. With use of the same clonogenic assays used to measure in vitro toxicity, biologically active immunoconjugate could be detected in murine plasma for up to 24 hours after iv administration, but the concentration of immunoconjugate by this measure was considerably less than that predicted by SDS-gel electrophoresis. The ability to deliver immunoconjugate to tumor cells in vivo was studied with use of the Burkitt's lymphoma Namalwa as a xenograft in nude mice. It was possible to deliver substantial amounts of immunoconjugate to Namalwa cells in xenografted ascites with direct ip inoculation; lower but significant amounts of immunoconjugate could be delivered to this xenograft after systemic iv administration, provided the tumor burden was low. The 5E9-gelonin conjugate, when administered iv at the time of ip tumor inoculation, prolonged survival of nude mice bearing Namalwa or other human tumors as ascites xenografts and delayed or prevented the growth of subcutaneous nodules of Namalwa in an antigen-specific fashion after a single iv injection. Direct intratumoral administration also inhibited the growth of visible subcutaneous nodules of Namalwa. This immunoconjugate may be useful in the treatment of human cancer.

Induced autoantibodies to fetal erythroid antigens in the chicken.

Cornell Obese (OS) (B13B13), Special C (Sp.C) (B13B13) and K-strain White Leghorn chickens (B15B15) were examined for both naturally occurring and induced autoantibodies directed against erythroid fetal antigens. Obese chickens have been reported to produce high levels of autoantibodies to thyroglobulin and to occasionally produce antinuclear autoantibodies specific for erythrocytes. Despite producing high levels of antithyroglobulin autoantibodies and antibodies cross-reactive with erythrocytes, no OS chickens were found to possess naturally occurring autoantibodies to fetal-specific antigens on erythrocytes. However, such antibodies were induced following immunization with fetal erythrocytes derived from a strain allogeneic for the major histocompatibility complex (MHC), the B-complex. Challenge with both intact erythrocytes and erythroid membranes in Freund's complete adjuvant resulted in production of antibodies specific for fetal antigens. The ability to induce autoantibodies to fetal erythrocytes was not restricted to the OS-strain; similar antibodies were detected in the Cornell K-strain and Special C-strain chickens even when intrastrain immunizations were employed. These results suggest that antibodies can be induced to fetal antigens on chicken erythrocytes in both autoimmune and normal chickens given the appropriate antigen presentation.

Autoanti-thyroglobulin production in obese chickens: influence of age and sex as measured by ELISA.

The kinetics of serum autoantibody production against thyroglobulin (Tg) was examined in two strains of chicken using an enzyme-linked immunosorbant assay (ELISA). Both strains are homozygous for the B13 haplotype. The OS strain develops spontaneous autoimmune thyroiditis at several weeks of age while the Sp. C strain, which serves as the control for the OS strain, has virtually undetectable autoantibody levels as determined by hemagglutination assay (HA). Serum autoantibody levels in both strains were monitored bi-weekly from 6 through 14 weeks of age post-hatching, using both the ELISA and HA techniques. With the more sensitive ELISA, absolute serum autoantibody concentrations were determined and both the Sp. C and OS strains were found to have readily detectable serum autoantibodies against Tg; however, the OS did have significantly higher autoantibody levels than the Sp. C strain. In the latter strain, autoantibodies increased significantly with age while the pattern was somewhat reversed in the OS strain. The ELISA revealed that the decline of Tg autoantibodies with age in the OS strain was restricted primarily to males, with females maintaining constant levels of autoantibodies. In contrast, the HA detected only the differences in autoantibody levels between the OS and Sp. C strains.

Quail fetal antigen (QFA): identification and comparison with chicken fetal antigen.

Quail fetal antigen (QFA), an analogous hematopoietic antigen to chicken fetal antigen (CFA), was identified and shown to be a developmental antigen on the blood cells of Japanese quail and other avian species. Rabbit antiserum against 14-day embryonic quail red blood cells (RBCs) was specifically adsorbed to achieve fetal specificity and to eliminate any cross reactivity with the CFA system. Complement-mediated microcytotoxicity and hemagglutination assays were used to detect the presence of QFA on hematopoietic cells. QFA was found on 100% of the peripheral RBCs from 10-day quail embryos but incidence of the antigen gradually declined with increasing development. Complete loss of QFA from RBCs occurred just prior to sexual maturation between 31 and 42 days of age. No qualitative differences in erythroid expression of QFA were observed during development; however, RBCs from both embryonic duck and interspecific quail- chicken hybrids reacted with R-anti QFA. Like CFA, quail fetal antigen was associated with lymphocytes, particularly those from primary lymphoid organs. Similarities in the developmentally controlled tissue distribution of QFA and CFA suggest that developmental hematopoietic antigens are a common feature of avian species and are useful cell surface markers for studies of development and differentiation.