Previous studies have shown that engagement of Toll-like receptors (TLR) 2 and 4 can induce macrophages to express a variety of proinflammatory cytokines. We have recently demonstrated that TLR2 agonists poorly induce a subset of TLR4-inducible proinflammatory genes (e.g., inducible protein (IP)-10, inducible NO synthase (iNOS), monocyte chemoattractant protein-5, IL-12p40), due in part to differential activation of IFN-β production and phosphorylation of the transcription factor STAT1. TLR4, but not TLR2, agonists can induce IFN-β expression via a mechanism that requires the adapter protein Toll-IL-1R domain-containing adapter protein (TIRAP)/myeloid differentiation protein 88 (MyD88) adapter-like (Mal), but not the adapter protein MyD88. Thus, the failure of TLR2 agonists to induce STAT1-dependent genes results, in part, from their failure to induce the expression of IFN-β. In this study, we show that IL-6 expression is also preferentially induced by activation of TLR4. TLR4-dependent induction of IL-6 expression did require Toll-IL-1R domain-containing adapter protein (TIRAP)/MyD88 adapter-like (Mal), but unlike iNOS and IP-10, it did not require the expression of IFN-β. Although exogenous IFN-β and IFN-γ could synergize with TLR2 agonists to restore high levels of iNOS expression and NO production, these IFNs could not synergize with TLR2 agonists to induce high levels of IL-6. Similarly, neutralizing anti-IFN Abs could block iNOS gene expression in LPS-stimulated murine macrophages, whereas these Abs had little effect on IL-6 gene expression in these cells. Together, these studies demonstrate that IL-6, like iNOS and IP-10, is differentially expressed in macrophages stimulated via TLR2 vs TLR4, although these differences appear to arise from distinct signaling mechanisms.

Outer membrane proteins extracted from isolated cell walls of Proteus mirabilis were able to combine with cell wall phospholipids in a model membrane system. The presence of outer membrane proteins in vesicular model membranes mediated the release of previously entrapped [14C]sucrose while [3H]inulin was retained. Incorporation of lipopolysaccharide from the same cell walls was not required for the formation of such selectively permeable membranes.

The immunoglobulin M (IgM) and the IgG1, IgG2ab, and IgG3 subclasses of plaque-forming cells (PFC) specific for lipopolysaccharide (LPS) were measured after immunization of mice with LPS alone and compared with the responses to LPS in combination with nonbacterial proteins and with bacterial membrane phospholipid vesicles or two major outer membrane proteins from Proteus mirabilis. The relative numbers of IgG PFC belonging to the IgG1, IgG2, or IgG3 subclasses induced by immunization with LPS alone depended upon the type of LPS administered. Phospholipids and the proteins effected characteristic alterations in not only the strength but also the subclass of the IgG responses to LPS. The results suggest that the hydrophobic-hydrophilic nature or state of aggregation of the preparations plays a role in the induction of IgG1 and IgG2 subclasses of PFC specific for LPS. Complex formation with LPS and adjuvant was apparently necessary to obtain these effects.

Macrophages are potent elicitors of inflammatory reactions that can play both positive and negative roles in radiotherapy. While several studies have investigated the effects of X-rays or gamma-rays on macrophages, virtually no work has been done on the responses of these cells to irradiation with carbon ions. Investigations into the effects of carbon ion irradiation are of particular interest in light of the fact that this type of radiation is being used increasingly for cancer therapy. In the present investigation we compared the effects of 250 kV X-rays with those of 9.8 MeV/u carbon ions on RAW 264.7 macrophages over a wide range of radiation doses. Macrophage functions including vitality, phagocytic activity, production of the proinflammatory cytokines IL-1beta and TNFalpha and production of nitric oxide (NO) were measured. In comparison to lymphocytes and fibroblasts, macrophages showed only a small decrease in vitality after irradiation with either X-rays or carbon ions. Proinflammatory cytokines and NO were induced in macrophages by LPS but not by irradiation alone. X-rays or carbon ions had little modulating effect on LPS-induced TNFalpha production. However, LPS-induced NO increased in a dose dependent manner up to 6-fold after carbon ion irradiation, while X-ray irradiation did not have this effect. Carbon ion irradiation mediated a concomitant decrease in IL-1beta production. Carbon ions also had a greater effect than X-rays in enhancing the phagocytic activity of macrophages. These results underscore the greater potential of carbon ion irradiation with regard to radiobiological effectiveness.

Antibody-producing cell responses of mice to a protein isolated from the outer membrane of Proteus mirabilis were typical of the responses to a thymus-dependent antigen. The immunoglobulin G antibody-producing cell responses to the protein were increased after administration of the antigen complexed with either lipopolysaccharide or with vesicles of phospholipids extracted from P. mirabilis. The protein in turn significantly increased the immune response to lipopolysaccharide and also converted this response from predominantly immunoglobulin M to predominantly immunoglobulin G.