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Human recombinant IL-1 alpha stimulates in a dose-dependent fashion the production of 5-lipoxygenase-derived LTC4 and, to a lesser extent, of cyclooxygenase-derived eicosanoids (PGE2, 6-keto PGF1 alpha, TXB2) by murine resident peritoneal macrophages. Enhancement of eicosanoid production was evident on unstimulated macrophages, but only marginal on macrophages phagocytosing zymosan. The effect of IL-1 was similarly achieved at physiological (37 degrees C) as well as at higher (39 degrees C) temperatures. Human recombinant IL-1 beta, human natural IL-1, and partially purified murine IL-1-rich supernatant also stimulated eicosanoid production by macrophages, although IL-1 alpha appeared to be the most effective.

Monoclonal antibodies (MAbs) against two non-cross-reacting antigens of human IL-1 beta (Vhp20 and BRhC3) and human TNF alpha (B154.2 and B154.7) were applied to identify cytokine-containing cells in tissue sections and in cell suspensions. IL-1 beta- or TNF alpha-positive cells were not present in immunostained cytocentrifuge smears prepared from freshly isolated peripheral blood leukocytes, spleen, and lymph node cells. After 18 hours of culture with bacterial endotoxin (LPS), 80% to 90% of blood monocytes, 30% of spleen macrophages, and 2% to 28% of lymph node macrophages were strongly positive for IL-1 beta with either of the MAbs. Furthermore, 25% to 35% of blood monocytes and 6% to 60% of lymph node macrophages were stained for TNF alpha. Cells positive for IL-1 beta or TNF alpha were extremely rare in sections of normal thymus, spleen, and lymph nodes. Immunoreactivity for IL-1 beta or TNF alpha was frequently observed in sections of granulomatous lymphadenitis (N = 11). IL-1 beta or TNF alpha staining was confined to the epithelioid macrophages forming the granuloma, and the intensity of TNF alpha reactivity was generally stronger. The high frequency of cytokine-containing cells in this pathologic condition was confirmed in a cell suspension study showing that 20% of epithelioid macrophages were weakly positive for IL-1 beta and 80% were strongly positive for TNF alpha. The presence of cytokine-containing cells was investigated in cryostat sections of several nonlymphoid organs with normal histologic appearance. IL-1 beta reactivity was not observed in any of the tissues. TNF alpha reactivity was frequently demonstrated in isolated macrophages embedded in the interstitial connective tissue.

Resident mouse peritoneal macrophages (M phi) produced significant amounts of superoxide anion (O2-) in response to phagocytic stimuli. When M phi were exposed in vitro for 20 hr to fibroblast interferon (IFN-beta), their capacity to release O2- was significantly reduced, such reduction being more evident with increasing IFN-beta concentrations. In contrast, O2- production by M phi exposed for 20 hr to the lymphokine macrophage activating factor (MAF) or treated with either MAF or IFN-beta for 4 hr was not significantly different from that of control cells. This pattern of activity closely followed that of M phi-mediated suppression of lymphocyte proliferation, which was dramatically reduced by 20 hr exposure of M phi to IFN-beta, but unchanged by treatment with MAF. No correlation was however found between superoxide anion generation and enhancement of tumoricidal capacity in IFN-beta-treated M phi. We thus concluded that O2- does not play a relevant role in IFN-beta-induced M phi cytolysis, whereas the reduction of O2- production could be of major importance in the decrease of M phi suppression induced by IFN-beta.

Interleukin 1 (IL-1) has been shown to regulate several immunologic functions. Since tumor necrosis factor (TNF) shares many biologic properties with IL-1, we have investigated here the role of TNF in the modulation of the immune response. We have thus tested low doses of human recombinant TNF-alpha (hu rTNF-alpha) for its capacity to enhance the in vivo antibody responses evaluated at the cellular level in the hemolytic plaque assay. It was found that hu rTNF-alpha, like human IL-1 beta, is able to enhance the immune response to a T cell-dependent antigen (sheep red blood cells). Interestingly, at variance with human recombinant IL-1 beta, hu rTNF-alpha was not able to enhance the in vivo antibody response to a T cell-independent antigen (type III pneumococcal polysaccharide). These results suggest that low levels of TNF may have a role in the modulation of the immune response in vivo and shed new light on the biologic significance of this mediator.

The interleukin 1 receptors (IL-1R) on the human B lymphoma RAJI and on the murine thymoma EL4-6.1 have been characterized. Equilibrium binding analysis using both 125I-labeled IL-1 alpha and IL-1 beta showed that RAJI cells have a higher number of binding sites/cell for IL-1 beta (2400, Kd 2.2 nM) than for IL-1 alpha (316, Kd 0.13 nM). On the other hand, EL4-6.1 cells have more receptors/cell for IL-1 alpha (22 656, Kd 1 nM) than for IL-1 beta (2988, Kd 0.36 nM). Dexamethasone (DXM) induced on RAJI cells a time-dependent increase in binding sites for both IL-1 beta and IL-1 alpha without affecting their binding affinities. However, while receptor-bound 125I-IL-1 alpha was displaced with equal efficiency by both IL-1 forms, only unlabeled IL-1 beta could effectively displace 125I-IL-1 beta. Cross-linking experiments indicated that RAJI cells have a predominant IL-1R of about 68 kDa, while EL4-6.1 cells have an IL-1-binding polypeptide of 80 kDa. These results suggest that B and T cells possess structurally different IL-1R with distinct binding properties for IL-1 alpha and IL-1 beta.

Resident peritoneal macrophages (M phi) from untreated mice and inflammatory M phi induced by a sterile irritant were able to exert significant suppressive activity on lymphocyte functions. M phi-mediated suppression was evident in lymphocyte proliferation and in a proliferation-independent lymphocyte response (i.e. lymphokine production). The lymphokine macrophage-activating factor (MAF), which enhances tumoricidal capacity of inflammatory but not of resident M phi in vitro, was ineffective in modulating suppressive activity of both resident and inflammatory M phi. In contrast, a significant effect on M phi-mediated suppression was observed upon treatment with IFN-beta. In fact, suppression of lymphoproliferation and of lymphokine production by either resident or inflammatory M phi was significantly decreased or abolished by IFN-beta. Like MAF, IFN-beta was able to increase M phi cytotoxicity against tumor cells. Such as effect, however, was evident in both resident and inflammatory M phi, thus confirming different M phi activation mechanisms for MAF and IFN-beta. These data indicate that IFN-beta acts mainly on mature M phi, which thus appear as the major M phi type involved in suppression. The contrasting effects of IFN-beta on M phi suppression and on M phi-mediated cytotoxicity strongly suggest a dissociation between the 2 induction mechanisms of suppression and cytotoxicity. The in vivo relevance of these two IFN activities was demonstrated by treating mice with the potent IFN inducer polyinosinic polycytidylic acid (poly(I).poly(C). M phi from poly(I).poly(C)-primed mice simultaneously showed enhanced tumoricidal activity and complete abolishment of suppressive capacity.

The pleiotropic activities of IL-1 have fostered a series of studies on the structure-function relationship in these proteins. In fact, the attempt to dissociate different biological functions of IL-1 should simplify its therapeutic use. About human IL-1 beta, which has been more extensively studied in this respect, enzymatic cleavage of the precursor protein to generate the mature polypeptide appears necessary for its full biological activity. The almost complete integrity of the mature IL-1 beta protein is also required for its ability to bind to the receptor and trigger cellular functions. However, by the use of monoclonal antibodies and recombinant or synthetic peptides, it has been possible to map some IL-1 beta regions important for different activities. Both N-terminal and C-terminal fragments are important for receptor binding. A domain around amino acids 187-204 is apparently involved in the hyperalgesic effects of IL-1 beta. Finally, the fragment in position 163-171 appears to be responsible for a restricted series of the IL-1 beta activities, mainly directed to the immune system, although irrelevant for inflammation-related effects and unable of binding to the IL-1R. It is thus possible, within the sequence of a cytokine, to isolate selectively active domains. This will give us new tools for new therapeutic approaches. Thus, IL-1 might be the prototype of a new generation of cytokines developed with the goal of stimulating specific biological activities without activating the cascade effects which are typical for many cytokines.

Mouse peritoneal macrophages (M phi) expressed enhanced tumoricidal activity upon in vitro stimulation either with the lymphokine M phi-activating factor (MAF) or with fibroblast interferon (IFN-beta). In contrast, M phi suppressive activity on lymphoproliferation was not affected by MAF pretreatment, but was drastically reduced or abolished by IFN-beta. Catalase, the enzyme involved in the destruction of hydrogen peroxide (H2O2), did significantly decrease M phi suppressive capacity but had no effect on M phi tumoricidal activity. Analysis of the phagocytosis-dependent H2O2 production by IFN-beta-treated M phi demonstrated a strong impairment of the oxygen metabolite release, which strictly paralleled the decreased M phi suppressive capacity. On the other hand, MAF did not modify H2O2 release by M phi. Studies on M phi antibacterial activity against Salmonella typhimurium, a function thought to depend upon H2O2 production, showed that exposure of M phi to IFN-beta significantly impaired their bactericidal and bacteriostatic capacity, again in close correlation with the decrease in H2O2 production. Thus, IFN-beta appears as modulating both suppressive and antibacterial capacities of M phi through reduction of their oxygen metabolism, whereas regulation of M phi anti-tumour activity is possibly controlled by different mechanisms.

The clinical and immunological responses to typhoid vaccination with parenteral (TAB) and oral (Ty21a) vaccines in two groups of 30 adult male subjects were studied. Parameters monitored included specific anti-Salmonella typhi cell-mediated immunity and total and specific antilipopolysaccharide fecal immunoglobulin A (IgA) titers in Ty21a-vaccinated subjects. Peripheral blood lymphocytes antibacterial activity was significantly increased only in Ty21a-vaccinated subjects. Serum arming activity and results of human F(ab')2 anti-IgG and -IgA inhibition tests suggest antibody-dependent cellular cytotoxicity mediated by IgA in those vaccinated with Ty21a. Interestingly enough, the cells of TAB-vaccinated subjects were able to mediate IgG-dependent cellular cytotoxicity, as was observable from the results of blocking experiments. Moreover, total and specific antilipopolysaccharide fecal IgA levels were observed to be significantly increased with Ty21a, up to 8 months post-vaccination schedule. An early-onset, transitory increase in serum IgM rheumatoid factor was also found, exclusively in subjects treated with TAB, and was no longer detectable on day 240. Ty21a was well tolerated and free of side effects, whereas 65% of subjects administered TAB reported fever, headache, malaise, and local tenderness at the injection site. Our data show that the two typhoid vaccines induce different cell-mediated specific immune responses. The role of these responses in protection against Salmonella infection, however, requires further investigation.

Large granular lymphocytes (LGL) were observed in the peripheral blood, spleen, lung and, to a lesser extent, bone marrow and lymph nodes, but not in the thymus of C3H/HeN mice 8 weeks old. The organ distribution of natural-killer (NK) cytotoxicity closely followed that of LGL. Nude mice had higher LGL percentages and NK activity than normal mice. In addition, the age distribution of LGL from the peripheral blood followed that of NK activity. Employing discontinuous Percoll density gradients the percentage of LGL and the NK cytotoxicity of the low density fractions could be enriched in comparison with the original populations of lymphocytes from peripheral blood and spleen, but not from thymus. These results suggest that, as recently shown for humans and rats, in mice too LGL are associated with NK activity.

Mice exposed to a sublethal dose of X-rays were immunized with alum-precipitated DNP-KLH (dinitrophenyl-keyhole limpet haemocyanin) and B. pertussis either before or after irradiation. The primary anti-DNP antibody response was evaluated during 8 weeks after immunization by the equilibrium dialysis technique using ammonium sulphate- precipitated serum globulins and the ligand 3H-labelled xi-DNP-L-Lysine. The serum concentrations of antibody sites in mice immunized 1-5 days before or 2 h-8 weeks after 450 rad were below the values in unirradiated controls at all bleeding times. Antibody affinity, however, was found to be up to 20 fold higher in irradiated mice than in control mice when antigen was injected before, or 3-8 weeks after, irradiation. Spleen cells from mice exposed to 450 rad 1-9 weeks before killing were stimulated in vitro with PHA, ConA, or LPS. Recovery profiles of mitotic responsiveness suggest that enhancement of antibody affinity in irradiated mice could result from relative lack of suppressor T Cells.

The synthetic nonapeptide VQGEESNDK, corresponding to the fragment 163-171 of human IL-1 beta, showed in vivo immunomodulatory capacities qualitatively and quantitatively comparable to those of the mature human IL-1 beta protein. In fact, both IL-1 beta and the 163-171 fragment stimulated the immune response of normal mice and restored immune reactivities of immunocompromised animals. In addition, the synthetic IL-1 peptide was as efficient as the entire protein in inducing tumor rejection and radioprotection. On the other hand, the 163-171 fragment did not cause any of several inflammation-associated metabolic changes inducible by the whole IL-1 beta molecule in vivo: hypoferremia, hypoglycemia, hyperinsulinemia, increase in circulating corticosterone, SAA and fibrinogen, decrease in hepatic drug-metabolizing enzymes. Furthermore, at variance with IL-1 beta, the 163-171 peptide did not show the toxic effects causing shock and death in adrenalectomized mice. Thus, these results confirm our previous in vitro observations that functional domains are identifiable within the multipotent cytokine IL-1 beta, and demonstrate the biological relevance of this finding in a variety of in vivo systems. The identification of a selectively active fragment of a cytokine may thus represent a significant step towards a better directed and more rational immunotherapeutic approach.

The structure-function relationship in the human IL-1 beta protein has been analyzed by means of computer prediction, synthesis of peptides and use of monoclonal antibodies of predetermined specificity. A nine amino acid-long fragment (VQGEESNDK), corresponding to the human IL-1 beta sequence in position 163-171, was shown to possess only some of the IL-1 activities, i.e. those directed to the immune system, while being devoid of IL-1-like inflammatory effects. It is thus proposed that discrete domains within the IL-1 beta protein might be responsible for the different biological activities of the molecule and that the 163-171 fragment may represent one of the immunostimulatory sites of IL-1 beta.

This report initially reviews the progressive steps of research designed to build up a new, well-defined helper system triggering both the non-specific and the tumor-specific immune reactivity of a host bearing a tumor, in order to impair tumor growth. Tumor-specific T-helper lymphocytes were first generated in vitro from the spleen of mice with evident tumors. As these lymphocytes inhibit tumor growth by recruiting host reactivity through the release of lymphokines, the peri-tumoral injection of interleukin-2 (IL-2) was then experimented. Repeated injections of 10 units of IL-2 are only weakly effective, but its triggering of an efficient anti-tumor reactivity is markedly enhanced when non-reactive lymphocytes directly obtained from tumor-bearing mice are artificially admixed with the challenge tumor cells. Lastly, in order to ascertain whether lymphocytes themselves could be dispensed with, lymphokines were injected around tumor-draining lymph nodes. Ten daily injections of 1 pg of the 163-171 synthetic nonapeptide of human IL-1 beta appeared very efficient in activating tumor inhibition, particularly when combined with IL-4 or (to a lesser extent) IL-2.

A monoclonal antibody (mAb) specific for human recombinant IL-1 beta (hu rIL-1 beta) was produced by immunizing BALB/c mice with hu rIL-1 beta purified with classical methods. This mAb recognizes an epitope within the highly hydrophylic fragment spanning amino acid 133-147. The affinity constant of this mAb towards IL-1 beta was determined by RIA. An affinity column was prepared by covalent binding of the mAb to Sepharose CL-4B. The column was capable of selectively binding hu rIL-1 beta produced in Escherichia coli directly from crude homogenates. The IL-1 beta protein yield was higher than 90% with a very good recovery of IL-1 beta biological activity. Moreover, the immunosorbent retained at least two thirds of its IL-1 beta-binding capacity after 20 cycles of purification.

In vitro natural anti-bacterial activity against Salmonella typhimurium by lymphocytes from Peyer's patches and spleens was assessed in several mouse strains. C3H/HeN and CBA/J mice, which are resistant to S. typhimurium infections, showed a natural anti-bacterial activity significantly higher than BALB/c, C57BL/10, C57BL/6 and C3H/HeJ mice, i.e. strains susceptible to the in vivo bacterial infection. In these susceptible strains and also in A/J mice, a significantly higher natural activity was observed in females compared to males. The sex control of natural anti-bacterial activity was further stressed by the fact that orchidectomy could induce a strong activity in low responder C57BL/10 male mice. With the exception of Beige mice, a low natural killer (NK) strain also with no natural activity against S. typhimurium in both sexes, the genetic distribution of natural anti-bacterial activity was extremely different from that of the NK activity. Thus, these results further stress the difference between natural anti-bacterial activity and NK cytotoxicity. Furthermore, our data establish a possible link, although with some exceptions, between in vivo susceptibility to S. typhimurium infections and in vitro natural activity against these bacteria.

The effect of IFN-beta on the development of the inflammatory reaction was studied in an experimental animal model, carrageenan-induced pleurisy in the mouse. Intrapleural inoculation of IFN-beta at the same time as carrageenan administration inhibited both migration of inflammatory cells and exudate formation in the pleural cavity in a dose-dependent fashion. Similarly, IFN-beta decreased the presence of the arachidonate metabolites PGI2, TXA2 and PGE2 (highly active molecules involved in the regulation of the inflammatory reaction) in inflammatory exudates. A marked inhibition of the inflammatory response to carrageenan was also evident when IFN-beta was administered several hours after the inflammatory challenge. In contrast, administration of IFN-gamma did not modify significantly any of the inflammatory parameters considered.