To monitor and better understand the immunoinflammatory sequelae in sepsis and septic shock, systemic and monocyte-related cytokine responses were evaluated in baboons with experimental peritonitis induced by an E. coli-laden fibrin clot. Despite similar bacterial inocula, considerable interindividual variability in clinical manifestation and outcome of infection was observed. Because monocytes and macrophages are a key component of innate immunity, we hypothesized that early polarization of distinct activation programs in circulating monocytes that culminates in the emergence of either classically (M1) or alternatively (M2) activated monocytes may underlie the observed susceptibility or resistance to infection. To test our hypothesis, we analyzed infection-induced expression of cytokine mRNAs in monocytes isolated from surviving and dead animals. Our data show that resistance to E. coli sepsis may well be associated with a mixed M1/M2 activation state of circulating monocytes, whereas M1 phenotype appeared to be prevailing in monocytes from animals that died. Together with data on systemic cytokine responses, the latter findings indicate that morbidity and mortality of animals with gram-negative sepsis may well result from an overwhelming proinflammatory response. Collectively, our data contribute to a better understanding of cytokine networking in the immunoinflammatory response to microbial infection and suggest M1/M2 immunophenotypic profiling of readily available circulatory monocytes for early prognosis of severe infections.

Macrophages are now well recognized to have a critical role in both innate and acquired immunity. The sentinel macrophage function is highly regulated and serves to allow for intrinsic plasticity of the innate immune responses to potential environmental signals. However, the mechanisms underlying the dynamic properties of the cellular arm of innate immunity are poorly understood. Therefore, we have conducted a series of in vitro studies to evaluate the contribution of immunoregulatory cytokines, such as IFN-γ, IL-10, and IL-12, in modulation of macrophage responses. We found that macrophages from IFN-γ knockout (IFN-γ−/−) mice exhibit only marginal LPS-induced TNF-α, IL-12, and NO responses, all of which can be fully restored in the presence of rIFN-γ. Pretreatment with substimulatory LPS concentrations led to reprogramming of IFN-γ−/− macrophage responses in a dose-dependent manner that manifested by an increased TNF-α and IL-12, but not NO, production upon the subsequent LPS challenge. These reprogramming effects were substantially attenuated and profoundly enhanced in macrophages from IL-12−/− and IL-10−/− mice, respectively, as compared with those modulated in macrophages from the congenic wild-type mice. LPS-dependent reprogramming was also fully reproduced in macrophages isolated from SCID mice after immunodepletion of NK cells. Our data strongly imply that cytokine (TNF-α and IL-12), but not NO, responses in macrophages may, at least in part, be governed by an autocrine IFN-γ-independent regulatory mechanism reciprocally controlled by IL-10 and IL-12. This mechanism may serve as an alternative/coherent pathway to the canonical IFN-γ-dependent induction of antimicrobial and tumoricidal activity in macrophages.

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We studied the potential role of a cytokine regulatory mechanism(s) in LPS-dependent reprogramming and modulation of TNF-α and nitric oxide (NO) responses in mouse peritoneal macrophages. Reciprocal regulation of TNF-α and NO production by LPS-primed and LPS-stimulated macrophages was found to be dependent on the presence of soluble secretory products released by the cells during the initial LPS priming interaction. Pretreatment of naïve macrophages with different mouse recombinant cytokines such as rIL-10, rIL-12, and rIFN-γ dose dependently and differentially regulated subsequent LPS-induced production of TNF-α, IL-6, and NO by cytokine-primed cells. Analysis of IL-12 and IL-10 levels present in culture supernatants of LPS-primed and LPS-stimulated macrophages revealed a high degree of correlation between the profiles of TNF-α and IL-12 as well as NO and IL-10. Furthermore, LPS priming of macrophages in the presence of anti-IL-12-neutralizing mAb attenuated TNF-α responses while at the same time up-regulated NO production. In contrast, neutralization of endogenous IL-10 with anti-IL-10 mAb resulted in considerable TNF-α response at LPS priming doses under conditions that would otherwise strongly inhibit TNF-α production. We also found that the initial LPS priming of naïve macrophages differentially and dose dependently regulates expression of mRNAs for IL-10, IL-12, and IFN-γ in LPS-primed macrophages. Collectively, our data provide experimental support for the hypothesis that a cytokine regulatory network, most probably autocrine, tightly controls the reciprocal modulation of TNF-α and NO responses in LPS-primed macrophages.

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