Essential role for humoral immunity during Ehrlichia infection in immunocompetent mice

Although cellular immunity is essential for host defense during intracellular bacterial infections, humoral immunity can also play a significant role in host defense during infection by some intracellular bacteria, including the ehrlichiae. Antibodies can protect susceptible SCID mice from fatal Ehrlichia chaffeensis infection, an observation that has been hypothesized to involve the opsonization of bacteria released from host cells. To determine whether humoral immunity plays an essential role during ehrlichia infection in immunocompetent mice, we utilized a murine model of fatal monocytotropic ehrlichiosis caused by Ixodes ovatus ehrlichia. Mice lacking either B cells or FcgammaRI were unable to resolve a low-dose (sublethal) I. ovatus ehrlichia infection, which suggested that humoral immunity is essential for resistance. Polyclonal sera generated in I. ovatus ehrlichia-infected mice recognized a conserved ehrlichia outer membrane protein and, when administered to infected mice, caused a significant decrease in bacterial infection. Mice experimentally depleted of complement, or deficient for complement receptors 1 and 2, were also susceptible to sublethal I. ovatus ehrlichia infection, as were mice that lacked the phox91 subunit of NADPH oxidase. The data are consistent with a mechanism whereby bacteria released from infected cells are lysed directly by complement or undergo antibody-mediated FcgammaR-dependent phagocytosis and subsequent exposure to reactive oxygen intermediates. The findings suggest mechanisms whereby antibodies contribute to immunity against intracellular bacteria in immunocompetent mice.     

Antibody-mediated elimination of the obligate intracellular bacterial pathogen Ehrlichia chaffeensis during active infection

It is generally accepted that cellular, but not humoral immunity, plays an important role in host defense against intracellular bacteria. However, studies of some of these pathogens have provided evidence that antibodies can provide immunity if present during the initiation of infection. Here, we examined immunity against infection by Ehrlichia chaffeensis, an obligate intracellular bacterium that causes human monocytic ehrlichiosis. Studies with mice have demonstrated that immunocompetent strains are resistant to persistent infection but that SCID mice become persistently and fatally infected. Transfer of immune serum or antibodies obtained from immunocompetent C57BL/6 mice to C57BL/6 scid mice provided significant although transient protection from infection. Bacterial clearance was observed when administration occurred at the time of inoculation or well after infection was established. The effect was dose dependent, occurred within 2 days, and persisted for as long as 2 weeks. Weekly serum administration prolonged the survival of susceptible mice. Although cellular immunity is required for complete bacterial clearance, the data show that antibodies can play a significant role in the elimination of this obligate intracellular bacterium during active infection and thus challenge the paradigm that humoral responses are unimportant for immunity to such organisms.     

Cytopathic effect, plaque formation, and lysis of Ehrlichia chaffeensis grown on continuous cell lines.

Ehrlichiae are strict intracellular bacterial pathogens that parasitize leukocytes or other blood cells. Only six agents of the tribe Ehrlichieae, namely, Cowdria ruminantium, Neorickettsia helminthoeca, Ehrlichia risticii, Ehrlichia sennetsu, Ehrlichia canis, and Ehrlichia chaffeensis, have been adapted to growth in continuous cell lines. E. chaffeensis, the agent of human ehrlichiosis, has been cultured only in a cell line of canine origin. We adapted purified cell-free E. chaffeensis for growth in human embryonic lung (HEL) fibroblasts (HEL 299), green monkey kidney cells (Vero), and a human cervical epithelioid carcinoma (HeLa) cell line. We observed a cytopathic effect with both Vero cells and HEL cells and plaque formation with cellular lysis when infected Vero cells were cultured in agar. Human fibroblasts are already commonly used for the isolation of viruses, coexiellae, and rickettsiae. Furthermore, the capability of these cells to support the growth of ehrlichiae suggests that they may be useful for primary isolation of ehrlichiae as well. The cytopathic effect produced in Vero or HEL cells offers a very helpful indicator of the infection. Plaque formation in Vero cells is a new phenomenon not yet reported for ehrlichiae and will allow the titration of inocula and clonal purification of this bacterium.     

CD4 T-cell epitopes associated with protective immunity induced following vaccination of mice with an ehrlichial variable outer membrane protein

The ehrlichiae express variable outer membrane proteins (OMPs) that play important roles in both pathogenesis and host defense. Previous studies revealed that OMPs are immunodominant B-cell antigens and that passive transfer of anti-OMP antibodies can protect SCID mice from fatal ehrlichial infection. In this study, we used a model of fatal monocytotropic ehrlichiosis caused by Ehrlichia bacteria from Ixodes ovatus (IOE) to determine whether OMP immunization could generate protective immunity in immunocompetent mice. Immunization of C57BL/6 mice with a purified recombinant OMP expressed by IOE omp19 generated protection from fatal IOE infection and elicited robust humoral and CD4 T-cell responses. To identify CD4 T-cell epitopes within OMPs, we performed enzyme-linked immunospot analyses for gamma interferon (IFN-gamma) production using a panel of overlapping 16-mer peptides from IOE OMP-19. Five immunoreactive peptides comprising residues 30 to 45, 77 to 92, 107 to 122, 197 to 212, and 247 to 264 were identified; the strongest response was generated against OMP-19(107-122). Most of the peptides are conserved between E. muris and E. chaffeensis OMP-19, and they elicited IFN-gamma production in CD4 T cells from E. muris-infected mice, indicating that T-cell epitope cross-reactivity likely contributes to heterologous immunity. Accordingly, CD4 T-cell responses to both OMP-19 and OMP-19(107-122) were of greater magnitude following high-dose IOE challenge of mice that had been immunized by prior infection with E. muris. Our studies cumulatively identify B- and T-cell epitopes that are associated with protective homologous and heterologous immunity during ehrlichial infection.     

Cultivation of Ehrlichia chaffeensis in mouse embryo, Vero, BGM, and L929 cells and study of Ehrlichia-induced cytopathic effect and plaque formation.

We successfully propagated Ehrlichia chaffeensis in mouse embryo, Vero, BGM, and L929 cells inoculated with host cell-free ehrlichiae, indicating that E. chaffeensis is capable of entry, survival, and growth in a relatively wide range of cell types derived from different species. We demonstrated rapid adaptation of E. chaffeensis in these cell lines, so that typical morulae could be detected as early as 5 days after inoculation. E. chaffeensis-induced cytopathic effect with different morphological characteristics in mouse embryo, Vero, and L929 cells. The earliest cytopathic effect appeared in untreated and irradiated mouse embryo cells at 4 days postinoculation. As the infected foci gradually expanded, the center of the foci showed necrotic cells with pyknotic nuclei and degraded morulae. E. chaffeensis caused cell lysis in untreated and irradiated L929 cells, with formation of distinct, round macroscopic plaques at 18 days postinoculation. In untreated and irradiated Vero cells, E. chaffeensis produced infected foci composed of loosely interwoven necrotic cells, spaces of detached cells, cells filled with morulae, and uninfected cells, resulting in characteristic reticular foci. Irradiated cells generally contained many large morulae and presented larger cytopathic foci. DH82 and BGM cells did not develop obvious cytopathic foci under the conditions employed. The findings reported herein offer the opportunity to study the pathogenic mechanism of cell injury by E. chaffeensis, the basis for quantification of infectious E. chaffeensis, improved approaches for recovery of ehrlichiae from human patients and tick hosts, and additional methods for cultivation of E. chaffeensis for molecular analysis.     

Enhanced clearance of lactic dehydrogenase-5 in severe combined immunodeficiency (SCID) mice: effect of lactic dehydrogenase virus on enzyme clearance.

The lactic dehydrogenase (LDH) level in plasma and the clearance of LDH in C.B-17 scid (severe combined immunodeficiency; SCID) mice were compared with those in C.B-17 or BALB/cCrSlc mice with or without lactic dehydrogenase virus (LDV) infection. The resting enzyme level in SCID mice showed little difference from that in C.B-17 or BALB/cCrSlc mice. The degree of increased plasma LDH level in SCID mice was lower than that in C.B-17 and BALB/cCrSlc mice after LDV infection. To assess the mechanisms of decrease in LDH elevation in SCID mice infected with LDV, virus replication was compared in SCID and BALB/cCrSlc mice. The infectivity titre of plasma in SCID mice was higher (more than 10 times) than that in BALB/cCrSlc mice. Moreover, the percentage of virus antigen positive Kupffer cells was higher in SCID mice than that in BALB/cCrSlc mice. The level of endogenous LDH release as a result of carbon tetrachloride treatment was similar in the SCID and BALB/cCrSlc mice. The clearance rate of endogenous LDH was greater in SCID mice than in BALB/cCrSlc mice with or without LDV infection. The rate of clearance of intravenously injected porcine LDH-5, but not porcine LDH-1, was enhanced in SCID mice as compared with that in BALB/cCrSlc mice. Furthermore, carbon clearance was higher in SCID mice than that in BALB/cCrSlc mice. These results suggest that the smaller increase of plasma LDH after infection might be due, at least in part, to the enhanced LDH-5 clearance function by macrophages in SCID mice.     

Inoculation candidiasis in a murine model of severe combined immunodeficiency syndrome.

To further elucidate the importance of T- and B-lymphocyte-mediated responses in host defense against systemic infection with Candida albicans, we studied this infection in a murine model of severe combined immunodeficiency (SCID). The course of inoculation candidiasis in these mice, which lack functional T and B lymphocytes, was compared with that in immunologically normal BALB/c mice. Mice were inoculated intravenously with 10(5) yeast cells. Quantitative cultures of liver, spleen, and kidneys were performed with necropsy specimens obtained 1, 3, 7, 10, 14, and 21 days after this intravenous inoculation. The differences in the time courses of recovery of organisms from liver and spleen specimens were not significantly different in the SCID mice compared with the BALB/c mice. The recovery of C. albicans from the kidneys was significantly lower in the SCID mice, indicating less persistence of the organism in the kidneys of the SCID mice than in those of the BALB/c mice. These data indicate that defense mechanisms other than T- and B-lymphocyte-mediated mechanisms are primarily responsible for host defense against inoculation candidiasis.     

Involvement of CD4(+) Th1 cells in systemic immunity protective against primary and secondary challenges with Trypanosoma cruzi

In general, gamma interferon (IFN-gamma)-producing CD4(+) Th1 cells are important for the immunological control of intracellular pathogens. We previously demonstrated an association between parasite-specific induction of IFN-gamma responses and resistance to the intracellular protozoan Trypanosoma cruzi. To investigate a potential causal relationship between Th1 responses and T. cruzi resistance, we studied the ability of Th1 cells to protect susceptible BALB/c mice against virulent parasite challenges. We developed immunization protocols capable of inducing polarized Th1 and Th2 responses in vivo. Induction of parasite-specific Th1 responses, but not Th2 responses, protected BALB/c mice against virulent T. cruzi challenges. We generated T. cruzi-specific CD4(+) Th1 and Th2 cell lines from BALB/c mice that were activated by infected macrophages to produce their corresponding cytokine response profiles. Th1 cells, but not Th2 cells, induced nitric oxide production and inhibited intracellular parasite replication in T. cruzi-infected macrophages. Despite the ability to inhibit parasite replication in vitro, Th1 cells alone could not adoptively transfer protection against T. cruzi to SCID mice. In addition, despite the fact that the adoptive transfer of CD4(+) T lymphocytes was shown to be necessary for the development of immunity protective against primary T. cruzi infection in our SCID mouse model, protective secondary effector functions could be transferred to SCID mice from memory-immune BALB/c mice in the absence of CD4(+) T lymphocytes. These results indicate that, although CD4(+) Th1 cells can directly inhibit intracellular parasite replication, a more important role for these cells in T. cruzi systemic immunity may be to provide helper activity for the development of other effector functions protective in vivo.     

Cyclic dimeric GMP signaling regulates intracellular aggregation, sessility, and growth of Ehrlichia chaffeensis

Cyclic dimeric GMP (c-di-GMP), a bacterial second messenger, is known to regulate bacterial biofilm and sessility. Replication of an obligatory intracellular pathogen, Ehrlichia chaffeensis, is characterized by formation of bacterial aggregates called morulae inside membrane-bound inclusions. When E. chaffeensis matures into an infectious form, morulae become loose to allow bacteria to exit from host cells to infect adjacent cells. E. chaffeensis expresses a sensor kinase, PleC, and a cognate response regulator, PleD, which can produce c-di-GMP. A hydrophobic c-di-GMP antagonist, 2'-O-di(tert-butyldimethysilyl)-c-di-GMP (CDGA) inhibits E. chaffeensis internalization into host cells by facilitating degradation of some bacterial surface proteins via endogenous serine proteases. In the present study, we found that PleC and PleD were upregulated synchronously during exponential growth of bacteria, concomitant with increased morula size. While CDGA did not affect host cells, when infected cells were treated with CDGA, bacterial proliferation was inhibited, morulae became less compact, and the intracellular movement of bacteria was enhanced. Concurrently, CDGA treatment facilitated the extracellular release of bacteria with lower infectivity than those spontaneously released from sham-treated cells. Addition of CDGA to isolated inclusions induced dispersion of the morulae, degradation of an inclusion matrix protein TRP120, and bacterial intrainclusion movement, all of which were blocked by a serine protease inhibitor. These results suggest that c-di-GMP signaling regulates aggregation and sessility of E. chaffeensis within the inclusion through stabilization of matrix proteins by preventing the serine protease activity, which is associated with bacterial intracellular proliferation and maturation.     

The relative susceptibility of mouse strains to pulmonary Cryptococcus neoformans infection is associated with pleiotropic differences in the immune response

CBA/J mice were highly susceptible to intratracheal (i.t.) Cryptococcus neoformans infection relative to BALB/c mice, while both strains were equally susceptible to intravenous (i.v.) infection. Increased susceptibility in i.t. infection was associated with higher brain CFU, lower serum immunoglobulin M (IgM) and IgG responses to glucuronoxylomannan (GXM), lack of IgE regulation during infection, and alveolar macrophage permissiveness to intracellular replication in vitro. In contrast, for BALB/c mice, relative resistance was associated with increased interleukin-12 (IL-12) and decreased IL-10 pulmonary levels. In CBA/J mice, relative susceptibility was associated with a decreased proportion of CD4+ and CD8+ T cells and an increase in macrophage percentage in pulmonary infiltrates. In contrast, no significant differences in these cytokines or cell recruitment were observed in the i.v. model, consistent with no differences in the survival rate. Passive antibody (Ab) protection experiments revealed a prozone effect in the BALB/c mice with i.v. infection, such that Ab efficacy decreased at higher doses. In the i.t. model using CBA/J mice, low Ab doses were disease enhancing and protection was observed only at high doses. Our results show (i) that differences in mouse strain susceptibility are a function of the infection model, (ii) that susceptibility to pulmonary infection was associated with macrophage permissiveness for intracellular replication, and (iii) that the efficacy of passive Ab in pulmonary infection is a function of dose and mouse strain. The results highlight significant differences in the pathogenesis of cryptococcal infection among inbred mice and associate their relative susceptibility with differences in numerous components of the innate and adaptive immune responses.     

Immune responses to Cryptosporidium muris and Cryptosporidium parvum in adult immunocompetent or immunocompromised (nude and SCID) mice.

Adult murine models of Cryptosporidium infection involving Cryptosporidium muris and C. parvum were used to study immunity to cryptosporidiosis in the mammalian host. Immunocompetent BALB/c or C57BL/6 mice developed a highly patent infection with the RN 66 strain of C. muris but overcame the infection and were immune to reinfection. In contrast, severe combined immunodeficiency (SCID) mice or nude mice had a chronic infection lasting at least 109 days. The development of the C. muris infection appeared to be confined to the gastric epithelium in immunocompetent and immunocompromised mice. SCID mice injected intraperitoneally with histocompatible spleen or mesenteric lymph node cells from uninfected BALB/c mice were able to recover from the C. muris infection. The protective effect of donor spleen cells was not reduced by depletion of the B cell population but was significantly reduced by depletion of Thy.1 cells. Treatment of C57BL/6 or BALB/c mice during infection with a gamma interferon-neutralizing monoclonal antibody, but not a tumor necrosis factor-neutralizing monoclonal antibody, resulted in a significant increase in oocyst production. In the C. parvum model, a severe and eventually fatal chronic infection with a cervine isolate was established in SCID mice, with parasitization occurring in the ileum, cecum, and colon. SCID mice injected with unprimed BALB/c spleen cells prior to inoculation of C. parvum oocysts were resistant to infection. These results suggested that the two animal models should be valuable in the study of immunity to cryptosporidial infection.     

An Essential Role for Gamma Interferon in Innate Resistance to Shigella flexneri Infection

Shigella spp. are the major cause of bacillary dysentery worldwide. To identify immune effectors associated with protection of the naive host during infection, the susceptibility to pulmonary Shigella infection of each of various mouse strains that have a targeted deletion in a specific aspect of the immune system was evaluated. Our results demonstrate that mice deficient in gamma interferon are 5 orders of magnitude more susceptible to Shigella than are wild-type mice, whereas mice deficient in B and T lymphocytes or in T lymphocytes alone exhibit no difference in susceptibility. Significantly lower numbers of shigellae were recovered from immunocompetent compared with gamma-interferon-deficient mice after infection. While immunocompetent mice were able to clear a sublethal Shigella inoculum by day 5 postinfection, progressively increasing numbers of shigellae were cultured from the lungs of gamma interferon-deficient mice over the same period. Histopathology of the lungs from immunocompetent mice infected with a sublethal Shigella inoculum showed mild inflammatory changes, whereas the lungs from gamma interferon-deficient mice demonstrated progressively worsening acute bronchiolitis with ulceration. Further, the time to death in gamma interferon-deficient mice correlates inversely with the size of the Shigella inoculum. To identify the cellular source of gamma interferon, we infected SCID mice, T-cell-receptor-deficient mice, beige mice (a mouse strain deficient in natural killer [NK] cell activity), and mice depleted of NK cells using anti-asialo-GM₁. Each NK cell-deficient mouse strain exhibited a 10-fold-greater susceptibility to Shigella infection than immunocompetent mice. To test the protective effects of gamma interferon in vitro, survival of intracellular Shigella was examined in primary macrophages from wild-type mice, primary macrophages from gamma interferon-deficient mice, a macrophage cell line, and a fibroblast cell line. Following activation with gamma interferon, each cell type eradicated intracellular Shigella, while nonactivated macrophages fostered Shigella replication and nonactivated fibroblast cells fostered both Shigella replication and intercellular spread. Taken together, these data establish that NK cell-mediated gamma interferon is essential to resistance following primary Shigella infection.     

A Murine Model for Infection with Burkholderia cepacia with Sustained Persistence in the Spleen

Burkholderia cepacia is an opportunistic pathogen that causes severe systemic infections in patients with chronic granulomatous disease (CGD) or with cystic fibrosis (CF), but its mechanisms of virulence are poorly understood. We developed a murine model of systemic infection in wild-type (WT) and gamma interferon knockout (GKO) BALB/c mice to facilitate dissection of components of pathogenicity and host defense. Both WT and GKO mice were susceptible to chronic splenic infection with B. cepacia, but not with Pseudomonas aeruginosa. B. cepacia strains from patients with CGD persisted longer than those from CF patients. C57BL/6 mice were the most susceptible murine strain; bacteria persisted in the spleen for 2 months. DBA/2, BALB/c, and A/J strains of mice were relatively resistant to infection. Certain strains of B. cepacia complex can persist in the murine spleen after systemic infection; this may provide clues to its virulence in compromised hosts, such as those with CGD and CF.     

Analysis of Host Cells Associated with the Spv-Mediated Increased Intracellular Growth Rate of Salmonella typhimurium in Mice

The 90-kb virulence plasmid of Salmonella typhimurium encodes five spv genes which increase the growth rate of the bacteria within host cells within the first week of systemic infection of mice (P. A. Gulig and T. J. Doyle, Infect. Immun. 61:504–511, 1993). The presently described study was aimed at identifying the host cells associated with Spv-mediated virulence by manipulating the mouse host and the salmonellae. To test the effects of T cells and B cells on the Spv phenotype, salmonellae were orally inoculated into nude and SCID BALB/c mice. Relative to normal BALB/c mice, nude and SCID BALB/c mice were unaffected for splenic infection with either the Spv⁺ or Spv⁻ S. typhimurium strains at 5 days postinoculation. When mice were pretreated with cyclophosphamide to induce granulocytopenia, there was a variable increase in total salmonella infection, but the relative splenic CFU of Spv⁺ versus Spv⁻ S. typhimurium was not changed after oral inoculation. In contrast, depletion of macrophages from mice by treatment with cyclophosphamide plus liposomes containing dichloromethylene diphosphate resulted in equivalent virulence of Spv⁺ and Spv⁻ salmonellae. To examine if the spv genes affected the growth of salmonellae in nonphagocytic cells, an invA::aphT mutation was transduced into Spv⁺ and Spv⁻ S. typhimurium strains. InvA⁻ Spv⁺ salmonellae were not significantly affected for splenic infection after subcutaneous inoculation compared with the wild-type strain, and InvA⁻ Spv⁻ salmonellae were only slightly attenuated relative to InvA⁺ Spv⁻ salmonellae. Invasion-defective salmonellae still exhibited the Spv phenotype. Therefore, infection of nonphagocytes is not involved with the Spv virulence function. Taken together, these data demonstrate that macrophages are essential for suppressing the infection by Spv⁻ S. typhimurium, by serving as the primary host cell for Spv-mediated intracellular replication and possibly by inhibiting the replication of salmonellae within other macrophages.     

Protective responses against skin-dwelling microfilariae of Onchocerca lienalis in severe combined immunodeficient mice.

Inoculation of severe combined immunodeficient (SCID) mice with microfilariae of Onchocerca lienalis results in a sustained infection of the skin, extending for months beyond the point at which the parasites are eliminated from immunocompetent BALB/c controls. Reconstitution of SCID mice with spleen cells, thymocytes, or CD4+-cell-enriched splenocytes from naive BALB/c donors confers the ability to mount a protective immune response, leading to the rapid elimination of microfilariae. High levels of interleukin-5 and low levels of gamma interferon in the sera of reconstituted SCID mice during the destruction of microfilariae suggest that this protective immune response is directed by Th2 lymphocytes, mirroring that observed in immunocompetent controls. Unexpectedly, abbreviation of primary infections of unreconstituted SCID mice with the drug ivermectin induces resistance to reinfection with microfilariae at a level equivalent to that induced in secondarily infected, immunocompetent controls. In contrast to protection mediated by adoptive reconstitution, resistance induced by ivermectin-abbreviated infection occurs in the absence of T cells and in association with negligible levels of serum interleukin-5 and gamma interferon. This points to the activation of some alternative host defense mechanism that operates after the clearance of therapeutic levels of drug. Such a response could have important implications for the treatment of human onchocerciasis and may go some way in explaining the long-term suppression of microfilariae observed in patients after treatment with ivermectin.     

A 'safe-site' for Salmonella typhimurium is within splenic cells during the early phase of infection in mice

Salmonella typhimurium infection in mice is focused on the spleen and liver, and prolonged infection can lead to sepsis and death. After intravenous infection with a moderate dose of S. typhimurium, the few bacteria that survive in the spleen and liver grow in a 'safe-site' where they are protected from immune destruction. In this study, we demonstrated that the lack of killing of resident salmonella in the spleen and liver was not because the salmonella were transformed within the host and became resistant to killing, or because the infected mice lost the ability to kill salmonella. We showed that the salmonella were within an intracellular 'safe-site' that protected them from killing. Brief treatment of salmonella-infected mice with gentamicin reduced the numbers of salmonella in the blood but had no effect on the numbers in the liver and spleen, suggesting an intracellular location of the salmonella. After dissociation of spleen cells from recently infected mice, 60% of the salmonella remained cell associated. These cell-associated salmonella, unlike cell-free salmonella, were resistant to killing by gentamicin. The cell-associated salmonella were rendered susceptible to gentamicin after sonication, providing confirmation of their previous intracellular location.     

4-1BB (CD137) is required for rapid clearance of Listeria monocytogenes infection

4-1BB (CD137), a member of the tumor necrosis factor receptor superfamily, is a T-cell-costimulatory receptor that is expressed on activated T cells, dendritic cells, and NK cells. Little has been reported about its role in early host defense against bacterial infection. In this study, we report that 4-1BB-deficient (4-1BB(-/-)) mice are much more susceptible to Listeria monocytogenes (intracellular bacteria) infections than wild-type mice. Upon L. monocytogenes infection, 4-1BB(-/-) mice showed a lower survival rate, a higher bacterial burden in organs, and larger hepatic microabscesses than 4-1BB(+/+) mice. 4-1BB(-/-) mice also had impairment in clearance of bacteria from the bloodstream. Neutrophils from 4-1BB(+/+) mice constitutively expressed 4-1BB, which could be activated to induce intracellular Ca(2+) influx by ligation with anti-4-1BB antibody. On the other hand, neutrophils from 4-1BB(-/-) mice were defective in reactive oxygen species generation, phagocytic activities, and intracellular Ca(2+) mobilization. In addition, mice pretreated with anti-4-1BB monoclonal antibody were much more resistant to L. monocytogenes infection than control antibody-treated mice. Our results support the notion that 4-1BB may play a major role in host defense against intracellular pathogens through neutrophil activation.     

Early Gene Expression of NK Cell-Activating Chemokines in Mice Resistant to Leishmania major

Susceptibility of mice to Leishmania major is associated with an insufficient NK cell-mediated innate immune response. We analyzed the expression of NK cell-activating chemokines in vivo during the first days of infection in resistant and susceptible mice. The mRNA expression of gamma interferon-inducible protein 10 (IP-10), monocyte chemoattractant protein 1 (MCP-1), and lymphotactin was upregulated 1 day after infection in the draining lymph nodes of resistant C57BL/6 mice but not in those of susceptible BALB/c mice. In vivo local treatment of BALB/c mice with recombinant IP-10 shortly after infection resulted in an enhanced NK cell activity in the draining lymph node. The data suggest that although the recruitment of NK cells is normal in susceptible mice, the lack of NK cell-activating chemokines is a factor resulting in a suboptimal NK cell-mediated defense.     

Cooperation between reactive oxygen and nitrogen intermediates in killing of Rhodococcus equi by activated macrophages

Rhodococcus equi is a facultative intracellular bacterium of macrophages which can infect immunocompromised humans and young horses. In the present study, we examine the mechanism of host defense against R. equi by using a murine model. We show that bacterial killing is dependent upon the presence of gamma interferon (IFN-gamma), which activates macrophages to produce reactive nitrogen and oxygen intermediates. These two radicals combine to form peroxynitrite (ONOO(-)), which kills R. equi. Mice deficient in the production of either the high-output nitric oxide pathway (iNOS(-/-)) or the oxidative burst (gp91(phox-/-)) are more susceptible to lethal R. equi infection and display higher bacterial burdens in their livers, spleens, and lungs than wild-type mice. These in vivo observations, which implicate both nitric oxide (NO) and superoxide (O(2)(-)) in bacterial killing, were reexamined in cell-free radical-generating assays. In these assays, R. equi remains fully viable following prolonged exposure to high concentrations of either nitric oxide or superoxide, indicating that neither compound is sufficient to mediate bacterial killing. In contrast, brief exposure of bacteria to ONOO(-) efficiently kills virulent R. equi. The intracellular killing of bacteria in vitro by activated macrophages correlated with the production of ONOO(-) in situ. Inhibition of nitric oxide production by activated macrophages by using N(G)-monomethyl-L-arginine blocks their production of ONOO(-) and weakens their ability to control rhodococcal replication. These studies indicate that peroxynitrite mediates the intracellular killing of R. equi by IFN-gamma-activated macrophages.