Monocytes control gamma/delta T-cell responses by a secreted product.

Gamma-irradiated ex vivo bovine monocytes induce proliferation of gamma/delta T cells in the autologous mixed lymphocyte reaction (AMLR), whereas when not irradiated they prevent this response. In contrast, non-irradiated autologous monocytes have no effect on bovine alpha/beta T-cell proliferation in the allogenic MLR suggesting that the regulation is specific for gamma/delta T-cell responses. Here, we showed that the inhibition was not mediated by inducing cell death and that the ability of ex vivo monocytes to prevent proliferation of gamma/delta T cells was not generalized in that gamma/delta T cells still responded to mitogenic stimulation. Inhibition of the AMLR by non-irradiated monocytes could not be overcome by addition of interleukin-2 to the cultures or by costimulation with antibodies to WC1, a gamma/delta T-cell-specific cell-surface differentiation antigen shown elsewhere by us to be involved in activation of gamma/delta T cells. Furthermore, we showed that monocytes inhibited gamma/delta T-cell responses via a soluble product since inhibition occurred even when monocytes and gamma/delta T cells were separated by membranes of transwells or when supernatants from monocyte cultures were added to AMLR cultures. Maximal secretion of the inhibitory product by the monocytes occurred during the first 6 hr of in vitro culture at 37 degrees, rapidly decreased thereafter, and did not occur when monocytes were incubated at 4 degrees. The inhibition was not attributable to nitric oxide, reactive oxygen intermediates, prostaglandin E2 or transforming growth factor-beta (TGF-beta) but the ability of monocyte supernatants to mediate inhibition was sensitive to heating at 65 degrees. Lipopolysaccharide and granulocyte-macrophage colony-stimulating factor activation of monocytes temporarily abrogated their ability to inhibit proliferation. In contrast, heat-shocking had no effect on their ability to inhibit. We hypothesize that non-irradiated monocytes produce the inhibitory material in vivo in order to regulate gamma/delta T-cell responses to self-derived monocyte membrane components, but that when monocytes are altered by infection, transformation, irradiation, or cytokine activation, production of the inhibitor is temporarily suspended allowing stimulation of gamma/delta T cells to occur.     

Role of interleukin-18 in intrahepatic inflammatory cell recruitment in acute liver injury

Although the innate immune system has been demonstrated to play important roles as the first line of defense against various infections, little is known about the interactions between intrahepatic inflammatory cells and the cytokine network in the liver. Here, we examined the role of IL-18 in IHL recruitment in acute liver injury. C57BL/6 mice were injected with an αCD40 mAb, and their serum IL-18 levels were observed to increase, with subsequent recruitment of IHLs into the liver. NKT cells were involved in this liver injury, as the serum ALT levels were reduced in NKT KO mice through the suppression of macrophage and monocyte migration and cytokine production. In contrast, depletion of neutrophils exacerbated the liver injury associated with high levels of TNF-α and IL-18 and increased numbers of macrophages and monocytes. Treatment with a neutralizing antibody against IL-18 reduced the serum ALT levels and inflammatory cell accumulation in the liver. Finally, additional administration of rIL-18 with αCD40 injection caused severe liver injury with increased IFN-γ production by NK cells. In conclusion, these findings demonstrate that IL-18 modulates liver inflammation by the recruitment of inflammatory cells, including NKT cells, macrophages, monocytes, and neutrophils.     

Engagement of CD30 shapes the secretion of cytokines by human gamma delta T cells

CD30 is a member of the TNF receptor superfamily, previously shown to be expressed on Hodgkin's lymphoma cells and on normal activated lymphocytes. We here show that CD30 is highly expressed on recently activated human gamma delta T cells. Elevated surface levels of this molecule persisted in long-term cultures of gamma delta cells, without further cell stimulation. CD30 acted as a co-stimulus in gamma delta T cells by potentiating the intracellular Ca(2+) fluxes induced by CD3 cross-linking. The engagement of CD30 enhanced the expression of several cytokines induced upon CD3 stimulation such as IL-4 and IFN-gamma but not IL-10. The CC chemokines RANTES and macrophage inflammatory protein-1beta were constitutively expressed and not affected by stimulation. The inducible expression of the neutrophil chemoattractant IL-8 was enhanced by CD30 co-stimulation, as well as that of the CC chemokines I-309 and MDC, whereas the secretion of the monocyte chemotactic protein-1 was not detected. Triggering of CD30 may therefore modulate the expression of several cytokines released by gamma delta cells; the expression of its physiologic ligand by APC and neutrophils at the site of infection may contribute to determine the outcome of an immune response.     

Monocyte/macrophage lineage commitment and distribution are affected by the lack of regulatory T cells in scurfy mice

Foxp3⁺ regulatory T (Treg) cells play a pivotal role in maintaining immunological tolerance. Loss‐of‐function mutations in the Foxp3 gene result in multiorgan inflammation known as immunodysregulation, polyendocrinopathy, enteropathy, X‐linked syndrome in humans and scurfy (Sf) disease in mice. While the impact of missing Treg cells on adaptive immune cells is well documented, their role in regulation of myeloid cells remains unclear. Here we report that Sf mice exhibit an altered composition of stem and progenitor cells, characterized by increased numbers of myeloid precursors and higher efficiency of macrophage generation ex vivo. The proportion of monocytes/macrophages in the bone marrow, blood, and spleen was significantly elevated in Sf mice, which was accompanied with tissue‐specific monocyte expression of homing receptor and phagocytic activity. Sf mice displayed high levels of M‐CSF and other inflammatory cytokines, including monocyte‐recruiting chemokines. Adoptive transfer of WT CD4⁺ cells and in vivo neutralization of M‐CSF normalized frequencies of monocyte subsets and their progenitors and reduced high levels of monocyte‐related cytokines in Sf mice, while Treg cell transfer to RAG2^?/? mice had no effect on myelopoiesis and monocyte/macrophage counts. Our findings illustrate that deregulated myelopoiesis in Sf mice is mainly caused by the inflammatory reaction resulting from the lack of Treg cells.     

Murine eosinophil granulocytes bind the murine macrophage-monocyte specific monoclonal antibody F4/80.

Studies designed to identify a panel of monoclonal antibodies useful for the separation of murine eosinophil myeloid progenitors revealed that F4/80, an antigen heretofore thought to be expressed only by murine monocyte/macrophage lineage cells, was expressed by eosinophil granulocytes. Eosinophils from several strains of mice stained positively with specific antibody for the epitope. A novel pathway for myeloid differentiation is proposed in which neutrophil progenitors exit a common lineage prior to a common progenitor of monocytes and eosinophils. Moreover, the results demonstrate that binding of anti-F4/80 can no longer be viewed as exclusive for mononuclear phagocytes.     

Skewing of cytotoxic activity and chemokine production,but not of chemokine receptor expression,in human type-1/-2 gamma delta T lymphocytes

Human Vgamma9/Vdelta2(+) T lymphocytes participate in the immune response against intracellular pathogens through the secretion of type-1 cytokines and chemokines and by killing of infected cells. Little is known of the effects by type-2 differentiation of gamma delta cells on these functions. Here, we report that bona fide naive cord blood-derived gamma delta lymphocytes expanded in vitro with the mycobacterial antigen isopentenyl pyrophosphate (IPP) can be differentiated as either type-1 or type-2 cells, in the presence of an appropriate cytokine milieu. Instead, peripheral gamma delta cells from PPD-negative healthy adults displayed a type-1 cytokine profile, i.e. IPP-stimulated secretion of IFN-gamma, but not of IL-4 and IL-10. Moreover, they released the macrophage inflammatory protein (MIP)-1beta, but not IL-8 nor the Th2 chemoattractants I-309 and TARC (thymus and activation-regulated chemokine). This cytokine profile was not significantly affected by in vitro culture in Th2 polarizing conditions. Only in one case out of seven were peripheral gamma delta cells fully differentiated to type-2 lymphocytes, characterized by sustained IL-4 and IL-10 production, along with secretion of substantial amounts of IL-8, I-309 and TARC. Type-2 gamma delta T lymphocytes preferentially expressed the co-stimulatory molecule CD30; conversely, no skewing in chemokine receptor expression was observed. Both polarized populations displayed high levels of CXCR3 in the absence of CCR3, CCR4 and CCR5. Finally, type-1, but not type-2, gamma delta T lymphocytes killed IPP-pulsed U937 cells and displayed elevated perforin content. Overall, our data suggest that type-2 differentiation of gamma delta T lymphocytes profoundly affects both their effector functions and their potential to recruit the appropriate leukocyte subsets to the sites of inflammation.     

Chagas' disease is attenuated in mice lacking gamma delta T cells.

The role of gamma delta T cells in the immunopathology of Chagas' disease is evaluated by monitoring the course of Trypanosoma cruzi infection in mice lacking gamma delta T cells after disruption of the T-cell receptor C delta locus. Levels of parasitemia, states of lymphocyte activation, and levels of lymphokine production as well as tissue pathology are compared in delta knockout mice and their littermates in acute and chronic phases of infection. Although the levels of circulating parasites do not significantly differ in the two groups, mortality scores and numbers of inflammatory lesions of skeletal and cardiac muscles are lower in gamma delta T cell-deficient m ice than in littermate controls. Furthermore, polyclonal lymphocyte activation, as measured by proliferative activities and numbers of B- and T-cell blasts in the spleen, are reduced in deficient mice in the acute and chronic phases of infection. Levels of gamma interferon mRNA obtained from total spleen cells, known to be a critical lymphokine in resistance to T. cruzi infection, are significantly higher in uninfected gamma delta T cell-deficient mice than in control animals and slightly above levels for littermates in the course of acute infection. Interestingly, however, in chronic phases, the levels of this lymphokine are not statistically different between the two groups of mice. These results indicate that gamma delta T cells do not play a crucial role in parasite clearance during the acute phase of the disease but contribute to the mechanisms leading to tissue damage and pathology.     

Evidence that gamma delta T cells play a limited role in resistance to murine listeriosis.

A role for alpha beta and gamma delta T cells in protection against primary and secondary infection with Listeria monocytogenes was studied. The results show that mice depleted of either gamma delta T cells with 3A10 monoclonal antibody (mAb), or alpha beta T cells with anti-CD4 plus anti-CD8 mAb, or both types of T cells, remained capable of controlling Listeria multiplication during the first 4 days of primary sublethal infection. Moreover, mice depleted of either or both types of T cells also remained capable of resolving primary infection, although the absence of alpha beta T cells, but not gamma delta T cells, caused resolution to be slower. Likewise, Listeria-immune mice depleted of either alpha beta or gamma delta T cells remained capable of resolving secondary infection with a large inoculum of L. monocytogenes, although depletion of alpha beta T cells, and to a much lesser extent gamma delta T cells, resulted in early exacerbation of infection. However, immune mice depleted of both types of T cells lost their ability to resist a lethal Listeria challenge. Taken together, the results show that whereas neither type of T cell is needed for resistance to sublethal primary listeriosis, alpha beta T cells may act in concert with gamma delta T cells in protecting mice against lethal secondary infection. In addition, the results indicate that the role of gamma delta T cells in anti-Listeria resistance is much less important than the role of alpha beta T cells, and can be demonstrated mainly in the absence of alpha beta T cells.     

Cytokine stimulation of T lymphocytes regulates their capacity to induce monocyte production of tumor necrosis factor-α, but not interleukin-10: Possible relevance to pathophysiology of rheumatoid arthritis

Previous studies in the laboratory have shown that the pro-inflammatory cytokine tumor necrosis factor (TNF)-α plays a pivotal role in the pathogenesis of rheumatoid arthritis (RA). The mechanisms involved in regulating monocyte/macrophage cytokine production are not yet fully understood, but are thought to involve both soluble factors and cell/cell contact with other cell types. We and others have previously demonstrated that T cells activated through the T cell receptor/CD3 complex induce monocyte TNF-α production by contact-mediated signals. In this report, we investigated further whether T cells activated by cytokines in the absence of T cell receptor stimulation also regulate monocyte cytokine production. T cells were activated in an antigen-independent manner using the cytokines interleukin (IL)-15 or IL-2 alone, or in combination with IL-6 and TNF-α. Subsequently, T cells were fixed and incubated with monocytes. Fixed, cytokine-stimulated T cells induced monocytes to secrete TNF-α in a dose-dependent manner, but did not induce secretion of IL-10, a potent endogenous down-regulator of TNF-α and other pro-inflammatory cytokines. Stimulation of monocyte TNF-α was markedly inhibited when T cells were physically separated from monocytes within the tissue culture well, confirming that T cell contact is necessary. T cell acquisition of monocyte-activating capacity was shown to be dependent on the period of cytokine stimulation, with T cells activated for 8 days more effective than T cells activated for shorter periods. Addition of interferon-γ or granulocyte/macrophage colony-stimulating factor to the T cell/monocyte cultures enhanced T cell induction of monocyte TNF-α by threefold and ninefold, respectively. The results from this model of cognate interaction suggest that cytokine-stimulated T cells, interacting with macrophages in the rheumatoid synovial membrane, may contribute to the continuous excessive production of TNF-α observed in the RA joint, and to the imbalance of pro-inflammatory cytokines over anti-inflammatory cytokines.     

Intranasal infection of beige mice with Mycobacterium avium complex: role of neutrophils and natural killer cells.

Beige mice show increased susceptibility to intranasal infection with organisms of the Mycobacterium avium complex (MAC) compared with their immunocompetent congenics, C57BL/6 mice. This increased susceptibility was clear 2 weeks postinfection, before the activation of the specific immune response. T lymphocytes from 4-week infected beige mice, cultured in vitro, produced amounts of gamma interferon similar to those found in cells from C57BL/6 mice. Macrophage activation, as judged by NO production and lysis of the macrophage target P815, occurred in the lungs of beige mice. Despite the inability of bone marrow-derived NK cells from beige mice to lyse NK-susceptible YAC-1 cells, their gamma interferon production was normal. Monoclonal antibody to NK1.1 was used to deplete C57BL/10 mice of lytic activity against YAC-1 cells without exacerbating infection between 2 and 6 weeks of observation, making it unlikely that any deficiency in NK cells was the cause of susceptibility in beige mice. There was a striking influx of neutrophils in the lungs of beige mice compared with C57BL/6. More than half of the MAC organisms appeared associated with the neutrophils of beige mice, while in C57BL/6 mice, most MAC organisms were associated with cells of macrophage/monocyte morphology. Injection of monoclonal antibody specific for neutrophils failed to eliminate those cells from the lungs of beige mice. However, in C57BL/6 mice, neutrophil numbers were reduced by 95% without exacerbating the infection. We conclude that, although neutrophils are not essential to the relative resistance of C57BL/6 mice, the known deficiencies in both neutrophils and macrophages account for the susceptibility of beige mice.     

Trophoblast-macrophage interactions: a regulatory network for the protection of pregnancy

PROBLEM: Macrophages are one of the first immune cells observed at the implantation site. Their presence has been explained as the result of an immune response toward paternal antigens. The mechanisms regulating monocyte migration and differentiation at the implantation site are largely unknown. In the present study, we demonstrate that trophoblast cells regulate monocyte migration and differentiation. We propose that trophoblast cells 'educate' monocytes/macrophages to create an adequate environment that promote trophoblast survival. METHOD OF STUDY: CD14(+) monocytes were isolated from peripheral blood using magnetic beads. Co-culture experiments were conducted using a two-chamber system. Monocytes were stimulated with lipopolysaccharide (LPS) and cytokine levels were determined using multiplex cytokine detecting assay. RESULTS: Trophoblast cells increase monocyte migration and induce a significant increase in the secretion and production of the pro-inflammatory cytokines [interleukin-6 (IL-6), IL-8, tumor necrosis factor-alpha] and chemokines (growth-related oncogen-alpha, monocyte chemoattractant protein-1, macrophage inflammatory protein-1beta, RANTES). Furthermore, the response of monocytes to LPS was different in monocytes pre-exposed to trophoblast cells. CONCLUSION: The results of this study suggest that trophoblast cells are able to recruit and successfully educate monocytes to produce and secrete a pro-inflammatory cytokine and chemokine profile supporting its growth and survival. Furthermore we demonstrate that trophoblast cells can modulate monocytes response to bacterial stimuli.     

Rapid lipopolysaccharide-induced differentiation of CD14(+) monocytes into CD83(+) dendritic cells is modulated under serum-free conditions by exogenously added IFN-gamma and endogenously produced IL-10.

We showed previously that about half of purified CD14(+) peripheral blood monocytes cultured under serum-free conditions and treated with GM-CSF and bacterial LPS rapidly (2 - 4 day) differentiate into CD83(+) dendritic cells (DC). The remaining cells retain the CD14(+)/CD83(-) monocyte/macrophage phenotype. In order to identify factors that influence whether monocytes differentiate into DC or remain on the monocyte/macrophage developmental pathway, we evaluated the effects of exogenously added IFN-gamma and endogenously produced IL-10 on the proportion and function of CD14(+) monocytes that adopt DC characteristics in response to LPS. IFN-gamma priming dramatically increased the proportion of monocytes that adopted stable DC characteristics in response to LPS, improved their T cell allosensitizing capacity, and enhanced levels of secreted IL-12 heterodimer. IFN-gamma priming also suppressed the production of IL-10, a cytokine known to have inhibitory effects on DC differentiation. When monocytes were treated with LPS plus IL-10-neutralizing antibodies, dramatically enhanced DC differentiation, IL-12 secretion, and T cell allosensitizing capacity were observed, mimicking in many respects the effects of IFN-gamma priming. IFN-gamma primed cells still displayed appreciable sensitivity to exogenously added IL-10, suggesting that attenuated IL-10 secretion is partially responsible for the enhancing effects of IFN-gamma. These studies therefore identify IFN-gamma as a DC differentiation co-factor for CD14(+) monocytes, and IL-10 as an autocrine/paracrine inhibitor of DC differentiation, linking these agents for the first time as mutually opposed regulators that govern whether CD14(+) cells differentiate into DC upon contact with LPS or remain on the monocyte/macrophage developmental pathway.     

Mycobacterial 65-kD heat shock protein induces release of proinflammatory cytokines from human monocytic cells.

Monocytes having phagocytosed mycobacteria are known to present the bacterial 65-kD heat shock protein (hsp) on their cell surface to alpha beta and gamma delta T lymphocytes. Cytotoxic CD4+ cells may then lyse monocytes expressing mycobacterial 65-kD hsp. However, it is not known whether 65-kD hsp directly stimulates monocyte functions other than antigen presentation. This study has demonstrated that following extraction of bacterial lipopolysaccharide, purified recombinant mycobacterial 65-kD hsp may directly activate THP-1 cells, a human monocytic line, to accumulate mRNA for and secrete tumour necrosis factor (TNF), a cytokine important in granuloma formation, the characteristic host immune response to mycobacterial infection. TNF gene expression and secretion following stimulation by hsp was dose-dependent and abolished by heat-induced proteolysis. Subsequently, THP-1 cells secreted IL-6 and IL-8, cytokines involved in recruitment and differentiation of T lymphocytes. The data indicate that secretion of proinflammatory cytokines from monocytes activated by mycobacterial 65-kD hsp may be important in the host immune response and in the development of antigen-specific T cell-mediated immunity.     

The early monocytic response to cytomegalovirus infection is MyD88 dependent but occurs independently of common inflammatory cytokine signals

Cytomegalovirus latently infects myeloid cells; however, the acute effects of the virus on this cell subset are poorly characterised. We demonstrate that systemic cytomegalovirus infection induced rapid activation of monocytes in the bone marrow, characterised by upregulation of CD69, CD11c, Ly6C and M‐CSF receptor. Activated bone marrow monocytes were more sensitive to M‐CSF and less sensitive to granulocyte‐monocyte colony stimulating factor in vitro, resulting in the generation of more macrophages and fewer dendritic cells, respectively. Monocyte activation was also observed in the periphery and resulted in significant accumulation of monocytes in the spleen. MyD88 expression was required within the haematopoietic compartment to initiate monocyte activation and recruitment. However, monocytes lacking MyD88 were activated and recruited in the presence of MyD88‐sufficient cells in mixed bone marrow chimeras, indicating that once initiated, the process was MyD88 independent. Interestingly, we found that monocyte activation occurred in the absence of the common inflammatory cytokines, namely type I interferons (IFNs), IL‐6, TNF‐α and IL‐1 as well as the NLRP3 inflammasome adaptor protein, ASC. We also excluded a role for the chemokine‐like protein MCK‐2 (m131/129) expressed by murine CMV. Taken together, these results challenge the notion that a single inflammatory cytokine mediates activation and recruitment of monocytes in response to infection.     

Regulation of chemokine/cytokine network during in vitro differentiation and HIV-1 infection of human monocytes: possible importance in the pathogenesis of AIDS

The monocyte/macrophage lineage represents heterogeneous cell populations characterized by major differences in the phenotype and functional activities. These cells are a major source of soluble factors, such as cytokines and chemokines, which can both affect HIV replication and AIDS pathogenesis. Although monocytes/macrophages are unanimously considered important targets of HIV-1 infection, the HIV-induced alterations in their physiological functions at different stages of differentiation are still matter of debate. In this article, we review our data on the regulation of chemokine/cytokine network with regard to macrophage differentiation and HIV-1 infection, in comparison with studies from other groups. The ensemble of the results emphasizes that: 1) macrophages markedly differ with respect to monocytes for a variety of responses potentially important in the pathogenesis of HIV infection; and 2) the experimental conditions can influence the HIVmonocyte/macrophage interactions, reflecting the possible in vivo existence of a spectrum of responses among macrophage populations.     

Proliferation of a subpopulation of human peripheral blood monocytes in the presence of colony stimulating factors may contribute to the inflammatory process in diseases such as rheumatoid arthritis

Apart from acting on hemopoietic progenitor cells, colony stimulating factors (CSFs) have been shown to be involved in the activation, survival, proliferation and differentiation of more mature cells of the monocyte/macrophage lineage. There is evidence that a proportion of human peripheral blood monocytes can proliferate in response to CSF-1, (also known as M-CSF) and granulocyte-macrophage-CSF (GM-CSF). CSFs have been shown to be at elevated levels in the synovial fluid of RA patients and thus local proliferation of monocyte/macrophage within an inflamed lesion may contribute to the local tissue hyperplasia evident in inflammatory conditions. Flow cytometric analysis of surface antigen expression and cytokine production in response to lipopolysaccharide stimulation has been used to characterise the proliferating subpopulation of monocytes. Further characterization and subsequent isolation of this subpopulation of monocytes may provide new and important information necessary in understanding inflammatory diseases such as rheumatoid arthritis, where local proliferation at the site of inflammation may be a key factor contributing to the chronicity of the disease.     

Signaling via the MyD88 adaptor protein in B cells suppresses protective immunity during Salmonella typhimurium infection

The myeloid differentiation primary response gene 88 (Myd88) is critical for protection against pathogens. However, we demonstrate here that MyD88 expression in B cells inhibits resistance of mice to Salmonella typhimurium infection. Selective deficiency of Myd88 in B cells improved control of bacterial replication and prolonged survival of the infected mice. The B cell-mediated suppressive pathway was even more striking after secondary challenge. Upon vaccination, mice lacking Myd88 in B cells became completely resistant against this otherwise lethal infection, whereas control mice were only partially protected. Analysis of immune defenses revealed that MyD88 signaling in B cells suppressed three crucial arms of protective immunity: neutrophils, natural killer cells, and inflammatory T?cells. We further show that interleukin-10 is an essential mediator of these inhibitory functions of B cells. Collectively, our data identify a role for MyD88 and B cells in regulation of cellular mechanisms of protective immunity during infection.     

Increase of heat-shock protein and induction of gamma/delta T cells in peritoneal exudate of mice after injection of live Fusobacterium nucleatum.

Fusobacterium nucleatum and Actinobacillus actinomycetemcomitans are Gram-negative rod periodontal pathogens. The peritoneal cavity of Institute of Cancer Research (ICR) mice was used as the local infection model. In vivo production of heat-shock proteins (hsp) was studied by injection of 1/10 minimum lethal dose (MLD) of each live bacteria into mice. Heat-shock proteins 70 and 60 were examined in the extract of peritoneal exudate cells (PEC) from mice injected intraperitoneally with either F. nucleatum or A. actinomycetemcomitans by using sodium dodecylsulphate-polyacrylamide gel electrophoresis and immunoblotting analysis. Although hsp are present in PEC without injection of the bacteria, both hsp increased and reached a peak on day 3 after F. nucleatum injection but not after A. actinomycetemcomitans. Kinetic study of gamma/delta cells in PEC after injection of bacteria showed that the increase of gamma/delta T cells was observed only in the PEC from mice injected with F. nucleatum but not A. actinomycetemcomitans. The gamma/delta T cells in PEC were either CD3+ and CD4+ or CD3+ and CD8+. The differential cell count of PEC suggested that gamma/delta T-cell induction is related to the expansion of the macrophage population. The phagocytic and chemiluminescence responses of macrophages against the same bacteria were compared after intensive immunization with live F. nucleatum and A. actinomycetemcomitans. Elevations of chemiluminescence response and phagocytic function by immunization were observed in the macrophages of mice immunized with F. nucleatum. These results suggest the sequential appearance of hsp, gamma/delta T cells and macrophage activation after fusobacterial infection.