Lymphocyte-Derived Cytokines Augment Macrophage Tumor Necrosis Factor-α and Interleukin-6 Secretion during Experimental Gram-Negative Bacterial Sepsis

Abstract

Although lymphocyte-derived cytokines are known to augment macrophage cytokine production in vitro, their effect on macrophage tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) secretion during gram-negative bacterial sepsis has not been characterized. The purpose of this study was to examine the effect of lymphocyte-derived cytokines on macrophage TNFα and IL-6 secretion during gram-negative bacterial peritonitis. To examine this problem, uninfected and infected mice were studied. Mice were infected with Escherichia coli 0111:B4 and two subgroups were examined consisting of those pretreated iv 1 hr prior to bacterial challenge with either (1) saline or (2) anti- E. coli 0111:B4 LPS mAb 2A3, the latter administered to abrogate the effects of LPS in vivo. Thus, three groups of mice were studied in relation to pretreatment and infectious challenges: (1) saline/saline (control); (2) saline/ E. coli (saline); and (3) mAb 2A3/ E. coli (mAb 2A3). Nonadherent splenocytes (>95% lymphocytes by histologic staining criteria) harvested 16 hr later from mice in each group were incubated in culture ex vivo for 3 hr to obtain supernatants containing lymphocyte-derived cytokines. These supernatants containing lymphocyte-derived cytokines then were incubated in vitro with naive splenic macrophages with or without E. coli 0111:B4 LPS. Macrophage TNF-α and IL-6 levels were determined using L929 and B9 bioassays. Lymphocyte-derived cytokines obtained 16 hr after infection from mice pretreated with saline significantly stimulated TNF-α and IL-6 secretion compared to those obtained from uninfected mice (TNF-α, 525 ± 67 pg/ml versus 16 ± 03 pg/ml, P < 0.001; IL-6, 19.2 ± 7.7 ng/ml versus 1.5 ± 0.0 ng/ml, P < 0.05) and synergistically enhanced macrophage TNF-α secretion in combination with LPS compared to that in medium (1601 α 378 pg/ml versus 850 ± 146 pg/ml, P < 0.05). Pretreatment of infected animals with anti-LPS mAb 2A3 blocked this effect (525 ± 67 pg/ml versus 183 ± 73 pg/ml, P < 0.01; 1601 ± 378 pg/ml versus 791 ± 46 pg/ml, P < 0.05). Interferon-γ (IFN-γ) concentrations in the lymphocyte-derived cytokines of all groups were <35 pg/ml. Thus, LPS released during infection induces lymphocytes to secrete cytokines other than IFN-γ that may act to amplify the host cytokine response. Characterization of the cytokines responsible for this phenomenon may prove important in further delineating the pathophysiology of the host septic response.