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Previous studies have shown that supernatants from concanavalin A-stimulated human peripheral blood mononuclear cells and isolated Leu-2 suppressor/cytotoxic T cells are chemotactic for Leu-3 helper/inducer T cells. The current study shows that lymphocyte chemotactic factor (LCF) is also produced following antigen (tetanus toxoid) challenge of mononuclear cells obtained from recently immunized human donors. LCF was detected in 24-hr supernatants from mononuclear cells challenged with tetanus and was produced maximally at 48 hr. Tetanus toxoid challenge of mononuclear cells obtained from individuals whom had not received a tetanus immunization for 7 to 10 years prior to testing showed little or no production of LCF. Serial studies of these individuals following a tetanus booster immunization showed that LCF was produced by antigen-challenged mononuclear cells obtained 1-5 days postimmunization, was produced optimally 5-15 days postimmunization, and was still produced by antigen-challenged mononuclear cells obtained 6 weeks later. Fractionation of mononuclear cells from immunized donors into glass wool nonadherent lymphocytes, T lymphocytes, and non-T lymphocytes showed that tetanus-toxoid-induced LCF was produced by nonadherent lymphocytes and T cells but not non-T cells. Further fractionation of T lymphocytes into Leu-2 and Leu-3 T-cell subpopulations showed that LCF production by antigen-challenged isolated subpopulations was limited to the Leu-2 suppressor/cytotoxic T-cell subset. Characterization of both Con A and tetanus toxoid-induced LCF by gel filtration on Sephadex G-100 demonstrated the presence of two peaks of LCF corresponding to molecular weights of approximately 14,000-17,000 and 40,000-50,000.

The opioid neuropeptides have previously been shown to bind to and affect leukocyte function including lymphocyte proliferation, NK-cell activity, mononuclear cell chemotaxis, immunoglobulin synthesis, and lymphokine production. The effect of the opioid peptides beta-endorphin and Met-enkephalin on interferon gamma (IFN) production by concanavalin A-stimulated human mononuclear cells was examined. Both beta-endorphin and Met-enkephalin enhanced IFN production by the majority of donor mononuclear cells tested and did so at concentrations between 10(-14) and 10(-10) M. When 10(-12) M beta-endorphin or Met-enkephalin were included in concanavalin A-stimulated mononuclear cell cultures, IFN concentrations were significantly enhanced to 205 +/- 45 and 252 +/- 67% of control, respectively. Although the majority of cell preparations tested exhibited an enhanced production of IFN in response to these opioid peptides, some did not. When beta-endorphin or Met-enkephalin were utilized at 10(-11) M, 10 of 15 and 7 of 11 responded with IFN production greater than 20% above the control (untreated) level. There was not an absolute correlation between an enhanced response to beta-endorphin and Met-enkephalin, suggesting the presence of multiple receptor types on these cells for opioids. The opioid receptor antagonist, naloxone, did not significantly prevent the opiate effect. When 10(-8) M naloxone was included in cultures containing 10(-12) M beta-endorphin or Met-enkephalin no significant inhibition of the effect of either opioid on IFN production was observed.

Two T-cell chemotactic factors, lymphocyte chemotactic factor (LCF) and interleukin 2 (IL-2), were separated and characterized from culture supernatants of concanavalin A-stimulated human peripheral blood mononuclear cells. LCF was purified approximately 7800-fold to homogeneity from culture supernatant using gel filtration and high-performance liquid chromatography (HPLC). LCF was found to be distinct from both IL-2 and interleukin-1. Sephadex G-100 gel filtration of crude supernatants from concanavalin A-stimulated mononuclear cells showed two molecular weight regions of T lymphocyte chemotactic activity. A 10,000- to 25,000-Da region contained both IL-2 and LCF and a 45,000- to 75,000-Da region contained only a high molecular weight form of LCF. Both high and low molecular weight species of LCF eluted with 40-44% acetonitrile from a reversed-phase C18 HPLC column. IL-2 present only in the low molecular weight region eluted from the C18 column with 65-75% acetonitrile. The migration of T lymphocytes to IL-2 was totally inhibited by anti-interleukin 2 receptor antibody while the response of T cells to LCF was unaffected. LCF eluting off the C18 column was purified to homogeneity by two subsequent cycles of gel filtration HPLC. The resultant protein showed a single band by sodium dodecyl sulfate-polyacrylamide gel electrophoresis corresponding to a molecular weight of 10,500. The data presented here demonstrate that IL-2 and LCF are distinct lymphocyte chemotactic factors and although they are not readily separable from crude supernatants by molecular sieve chromatography, they can easily be distinguished by reversed-phase HPLC.