Effect of cytokines on polymorphonuclear neutrophil infiltration in the mouse. Prostaglandin- and leukotriene-independent induction of infiltration by IL-1 and tumor necrosis factor.

The i.p. injection of mice with highly purified recombinant human rIL-1 alpha or beta resulted in the rapid influx of a large number of polymorphonuclear neutrophils (PMN) into the peritoneal cavity. Significant increases in the number of PMN were induced by doses of IL-1 which ranged from 0.005 to 5 ng/injection. Interestingly the dose response for PMN influx was bell-shaped because 50 ng of IL-1 did not result in a significant increase in peritoneal PMN. IL-1 induced PMN infiltration was detectable by 1 h with peak levels of PMN obtained by about 2 h, followed by a subsequent decline by 24 h. Other cytokines, IL-2, IFN-gamma, IFN alpha beta, granulocyte-CSF, granulocyte-macrophage-CSF, IL-3, TNF-alpha, and TNF-beta were compared to IL-1 for their ability to induce a PMN influx into the peritoneum. Only TNF-alpha or TNF-beta (lymphotoxin) were able to induce a significant influx of PMN within 2 h. However, based on total protein administered, about 100 times more TNF than IL-1 was required to produce a comparable PMN infiltration. Intraperitoneal injection of inhibitors of the cyclooxygenase or lipoxygenase pathways did not inhibit the IL-1-induced influx of PMN. Also, neither IL-1 nor TNF triggered an increase in PG or leukotriene release from peritoneal cells in vitro. Furthermore, direct peritoneal injection of leukotriene B4, a potent PMN chemoattractant in vitro, did not induce any significant increase in PMN in the peritoneal cavity indicating that chemotactic activity alone is insufficient for inducing peritoneal infiltration. These results suggest that the local production of very low levels of IL-1 in vivo would be sufficient to initiate a sequence of events that results in a rapid accumulation of PMN. Because IL-1 was not chemotactic for PMN in vitro, our data suggest that IL-1 induces production of factors that are chemotactic for PMN. Alternatively, IL-1 may act on other stages of the complex sequence of events that regulates the emigration of PMN into tissue sites in vivo. The synergy apparent in PMN influx when suboptimal concentrations of IL-1 and TNF were injected suggests that the local production of very low concentrations of these cytokines in situ could play a critical role in the emigration of PMN during infection.

Nickel--magnesium interactions in carcinogenesis: dose effects and involvement of natural killer cells.

The effects of magnesium carbonate (MgCarb) on carcinogenesis and natural killer (NK) cell modulation by nickel subsulfide (Ni3S2) were studied. Male Fischer F344/NCr rats, 50-90 g body wt, 20 rats per group, received single i.m. injections into both thigh muscles of 2.5 mg Ni3S2 alone or combined with different proportions of MgCarb; the Mg/Ni molar ratio ranged from 0.25 to 4.0. Control rats received i.m. injections of normal saline or magnesium acetate (MgAcet), or s.c. MgCarb at a site distant from Ni3S2. The animals were observed over 79 weeks for the development of tumors. The NK cell activity was determined over the first 3 weeks of the experiment in separate groups of rats treated as above, with the use of the 51Cr/YAC-1 release assay for blood and spleen cells and the peroxidase localization of Ox-8-immunoreactive lymphocytes at the injection site. I.m. administration of MgCarb mixed with Ni3S2 up to the Mg/Ni molar ratio of 1.0 inhibited the carcinogenicity of Ni3S2 in a dose-related manner; final incidence of sarcomas decreased from 100 to 55% and the appearance of first tumors was delayed from 25 to 39 weeks. Higher doses of MgCarb did not exert further effect. Distant s.c. injection of MgCarb or local i.m. application of MgAcet did not change the carcinogenic potency of i.m. Ni3S2. MgCarb or saline alone did not produce any tumors. I.m. Ni3S2 had no significant influence on the activity of NK cells in blood and spleen, while i.m. MgCarb alone did not affect the NK activity in blood but doubled it transiently in the spleen 24 h after injection. In the injected muscle, Ox-8-positive cells became abundant around MgCarb but could not be found close to Ni3S2. This inhibitory effect of Ni3S2 was partially reversed by MgCarb. Also, numerous multinucleated giant cells infiltrated the sites of injection of MgCarb alone and MgCarb + Ni3S2 but not Ni3S2 alone. The results indicate a dose-dependent and strictly local character of the inhibition by MgCarb of Ni3S2 carcinogenesis, as well as a possible involvement of NK and phagocytic cells in this inhibition.

Analysis of a cytostatic lymphokine produced by incubation of lymphocytes with tumor cells: relationship to leukoregulin and distinction from recombinant lymphotoxin, recombinant tumor necrosis factor, and natural killer cytotoxic factor.

Supernatants from the coculture of peripheral blood lymphocytes and the NK-susceptible cell line K562 were highly growth inhibitory for a variety of tumor cell lines. No correlation was observed between the susceptibility of the target cell lines to growth inhibition and to lysis by NK cells. Rather, the spectrum of cytostatic activity and the characteristics of the soluble factor were similar to those of leukoregulin, a recently described lymphokine. The supernatants of tumor-lymphocyte cultures contained only low levels of IFN-alpha and IFN-gamma, and antibodies to interferons did not affect the observed growth inhibition. The pattern of target cell susceptibility to growth inhibition by this factor was also quite distinct from that seen with purified recombinant LT or TNF. Furthermore, monoclonal antibodies to these cytokines also had no effect on the cytostasis, arguing against a requirement for, or synergistic interaction with, low levels of these cytokines. Some of the targets susceptible to the factor were only growth inhibited but not lysed, thereby distinguishing it from NKCF. Furthermore, the cytostasis was not inhibited by mannose-6-PO4 or rabbit antibodies to granule cytolysin, both of which have been reported to block NKCF. Therefore, the results show that a cytostatic factor is released in tumor-lymphocyte incubation that is quite distinct from interferons, LT, and TNF but has characteristics that resemble those of leukoregulin.

Natural killer activity in the rat. IV. Distribution of large granular lymphocytes (LGL) following intravenous and intraperitoneal transfer.

Highly enriched populations of rat large granular lymphocytes (LGL) and T lymphocytes were prepared on discontinuous density gradients of Percoll, labeled with either 111In-oxine or 51Cr and injected either intravenously (iv) or intraperitoneally (ip) into normal syngeneic recipients. Following iv inoculation of labeled LGL or T cells into normal recipients, a large proportion of radioactivity (18 to 33%) was recovered within minutes in the lungs. By 2 to 4 hr following transfer, significantly more LGL (13.5%) than T cells (6.4%) remained in the lungs. This difference persisted through 48 hr (5.4 vs 0.8%). Decreasing levels of radioactivity in the lungs were accompanied by corresponding increases in counts in the spleen and liver. At early time points, a significantly higher proportion of T cells was found to distribute to the spleen, while labeled LGL persisted for longer periods in the blood as well as in the lungs. Following ip inoculation into normal recipients, there was a slow clearance of radiolabeled LGL and T cells from the peritoneal cavity, with less than 20% of the radiolabel found in peripheral organs by 24 hr. These results demonstrate a distribution pattern for LGL and T cells that resembles the previously reported proportions of these cells in various organs. In addition, these studies provide a firm basis for the formulation of further experiments to examine the usefulness of adoptive immunotherapy with LGL or immune T cells.