The macrophage has a major role in normal wound healing and the reparative process around implants. Murine macrophage-like cells RAW 264.7 were used to investigate the effect of titanium surfaces on macrophage activation and secretion of proinflammatory cytokines [interleukin (IL)-1 beta, IL-6, and tumor necrosis factor (TNF)-alpha] and chemokines (monocyte chemoattractant protein-1 and macrophage inflammatory protein-1 alpha). Four topographies were used: those produced by mechanically polishing, coarse sand blasting, acid etching, and sandblasting and acid etching (SLA). Macrophages were plated on the four titanium surfaces at a population density of 5 x 10(5) cells/mL/well. Tissue culture plastic and tissue culture plastic plus lipopolysaccharide (LPS) served as negative and positive control, respectively. In addition, all surfaces were tested for their effects on macrophages in the presence of LPS. Supernatants were collected for assays after 6, 24, and 48 h and the numbers of macrophages attached to the surfaces were quantified using the DAPI (4,6-di-amidino-2-phenylindole) assay. Cytokine and chemokine levels were measured with sandwich enzyme-linked immunosorbent assays. Statistical comparison between the surfaces and the controls was determined by using the two-way analysis of variance including interaction effect (two tailed and p < or = 0.05). Unstimulated macrophages increased their secretion of the proinflammatory cytokine (TNF-alpha) when attached to rough surfaces (acid etching and SLA, p < or = 0.05). In macrophages stimulated with LPS, the roughest surface SLA produced higher levels of IL-1 beta, IL-6, and TNF-alpha at 24 and 48 h than all other surfaces (p < or = 0.05). Surface topography also modulated the secretion of the chemokines monocyte chemoattractant protein-1 and macrophage inflammatory protein-1 alpha by macrophages. Unstimulated macrophages attached to the SLA surface down-regulated their production of chemokines (p < or = 0.05) whereas LPS-stimulated macrophages attached to the SLA surface up-regulated their production (p < or = 0.05). Moreover, the SLA surface was found to act synergistically with LPS as well as the combination of blasting and etching features of the SLA surface resulted in significant release of proinflammatory cytokines and chemokines by stimulated macrophages at 24 and 48 h (p < or = 0.05). This in vitro study has demonstrated that surface topography, in particular the SLA surface, modulated expression of proinflammatory cytokines and chemokines by macrophages in a time-dependent manner.

An antagonist of human monocyte chemoattractant protein (MCP)-1, which consists of MCP-1(9-76), had previously been characterized and shown to inhibit MCP-1 activity in vitro. To test the hypothesis that, by inhibiting endogenous MCP-1, the antagonist has antiinflammatory activity in vivo, we examined its effect in the MRL-lpr mouse model of arthritis. This strain spontaneously develops a chronic inflammatory arthritis that is similar to human rheumatoid arthritis. Daily injection of the antagonist, MCP-1(9-76), prevented the onset of arthritis as monitored by measuring joint swelling and by histopathological evaluation of the joints. In contrast, controls treated with native MCP-1 had enhanced arthritis symptoms, indicating that the inhibitory effect is specific to the antagonist. In experiments where the antagonist was given only after the disease had already developed, there was a marked reduction in symptoms and histopathology, although individuals varied in the magnitude of the response. The mechanism of inhibition of disease is not known, although the results suggest that it could be more complex than the competitive inhibition of ligand binding that is observed in vitro. The demonstration of the beneficial effects of an MCP-1 antagonist in arthritis suggests that chemokine receptor antagonists could have therapeutic application in inflammatory diseases.

Monocyte-derived cells, including macrophages and foreign body giant cells, can determine the performance of implanted devices. Upon contact with biomaterials, macrophages can be activated into a classic inflammatory (M1) or wound-healing (M2) phenotype. Previously, we showed that high macrophage density on rough SLA implants was associated with early bone formation. This study examined a possible mechanism, namely, surface roughness activation of macrophages to the M2 phenotype to enhance bone formation on the SLA surface. RAW 264.7 macrophages were seeded on SLA or smooth (Po) epoxy substrates and the expression of the M1 and M2 specific markers, NOS2 and Arg-1 measured by qPCR on days 1, 3, and 5. Additionally, secretion of inflammation-associated cytokines and chemokines was studied by antibody arrays and ELISAs. Controls included RAW 264.7 macrophages primed into the M1 or M2 phenotypes by LPS/IFN-γ and IL-4, respectively. Rough SLA surfaces did not activate Arg-1 and NOS2 expression, but relative to Po surfaces MCP-1 and MIP-1α were upregulated after 5 days, whereas the secretion of the M1-associated chemokine IP-10 was lowered. RAW 264.7 macrophages on the SLA surface thus adopted elements of an M2-like phenotype, suggesting that when implanted the SLA surfaces may enhance wound repair.

Pulpal disease is intimately associated with the immune system's response to bacteria products. Clinical pathology is mediated in part by the production of pyrogenic cytokines, especially interleukin (IL)-1, tumor necrosis factor (TNF)-alpha, and IL-6. Methyl mercaptan (CH3SH), a volatile sulfur compound produced by anaerobic Gram-negative bacteria, has been shown to contribute to the production of IL-1 by human mononuclear cells. In this report, we investigated the production of IL-1, TNF-alpha, and IL-6 by human pulp fibroblasts when stimulated for various periods of time with lipopolysaccharide (LPS) with or without the presence of CH3SH. We found that LPS and CH3SH had no effect on the production of IL-1 or TNF-alpha. However, LPS stimulated IL-6 production, and this production was augmented when CH3SH was present. We conclude that the volatile sulfur compound CH3SH plays a role in activation and modulation of the immune response through its role in production of IL-6.

The therapeutic mAb rituximab induced the expression of the CCL3 and CCL4 chemokines in the human lymphoma line BJAB following binding to the CD20 Ag. Induction of CCL3/4 in vitro was specific, was observed in several cell lines and freshly isolated lymphoma samples and also took place at the protein level in vitro and in vivo. To investigate the role of these β-chemokines in the mechanism of action of rituximab, we synthesized a N-terminally truncated CCL3 molecule CCL3(11–70), which had antagonist activity on chemotaxis mediated by either CCL3 or BJAB supernatant. We also set up an established s.c. BJAB tumor model in athymic mice. Rituximab, given weekly after tumors had reached 250 mm2, led to complete disappearance of the lymphoma within 2–3 wk. Treatment of mice with cobra venom factor showed that complement was required for rituximab therapeutic activity. Treatment of BJAB tumor bearing mice every 2 days with the CCL3(11–70) antagonist, starting 1 wk before rituximab treatment, had no effect on tumor growth by itself, but completely inhibited the therapeutic activity of the Ab. To determine whether CCL3 acts through recruitment/activation of immune cells, we specifically depleted NK cells, polymorphonuclear cells, and macrophages using mAbs, clodronate treatment, or Rag2−/−cγ−/− mice. The data demonstrated that these different cell populations are involved in BJAB tumor eradication. We propose that rituximab rapidly activates complement and induces β-chemokines in vivo, which in turn activate the innate immunity network required for efficient eradication of the bulky BJAB tumor.