In vitro and in vivo characterization of smooth small colony variants of Brucella abortus S19.

Brucella abortus is known to produce chronic infections in both humans and a variety of animal species. However, the mechanisms underlying the persistence of the bacteria in the presence of an ongoing immune response are still unknown. In this respect we made use of the observation that in vitro grown B. abortus S19 exhibits heterogenicity in colony size when plated onto TS agar, while experimental infection of mice uniformly results in the in vivo selection of the small colony variant. We demonstrate that the spontaneous smooth small colony variant is characterized not only by a slower growth rate in vitro but also by an increased tolerance to hyperosmotic medium and, most importantly, a less effective clearance from spleens and livers of experimentally infected mice. On a molecular level, a gene with homology to a formerly described galactoside transport ATP binding protein (mglA) was differentially expressed in small versus large colonies of B. abortus S19.

Delayed type hypersensitivity-associated disruption of splenic periarteriolar lymphatic sheaths coincides with temporary loss of IFN-gamma production and impaired eradication of bacteria in Brucella abortus-infected mice.

A major problem of infections with facultative intracellular bacteria is their chronic course. We comprehensively evaluated the host response in murine brucellosis to study mechanisms contributing to bacterial persistence in the presence of an established immune response. Evidence is presented that the decrease in eradication kinetics, reproducibly occurring 18 d after infection of mice with Brucella abortus S19, is related to a state of downregulation of defense mechanisms. This is not due to a Th1 to Th2 switch or prostaglandin-mediated suppression by macrophages but is most probably caused by a severe disruption of spleen morphology at the height of Brucella-induced delayed type hypersensitivity. This results in a profound depletion of both CD4(+) and CD8(+) T cells in periarteriolar lymphatic sheaths, a consecutive deleterious shift in the relation of permissive macrophages and protective lymphocytes and an impaired capacity of splenocytes to produce IFN-gamma in response to soluble Brucella antigen.

Bartonella henselae-specific cell-mediated immune responses display a predominantly Th1 phenotype in experimentally infected C57BL/6 mice.

Immune responses of the immunocompetent host to Bartonella henselae infection were investigated in the murine infection model using C57BL/6 mice. Following intraperitoneal infection with human-derived B. henselae strain Berlin-1, viable bacteria could be recovered from livers and spleens during the first week postinfection, while Bartonella DNA remained detectable by PCR in the liver for up to 12 weeks after infection. Granulomatous lesions developed in livers of infected mice, reached maximal density at 12 weeks after infection, and persisted for up to 20 weeks, indicating that B. henselae induced a chronic granulomatous hepatitis in the immunocompetent murine host. T-cell-mediated immune responses were analyzed in vitro by means of spleen cell proliferation and cytokine release assays as well as analysis of immunoglobulin G (IgG) isotypes. Spleen cells from infected mice proliferated specifically upon stimulation with heat-killed Bartonella antigen. Proliferative responses were mainly mediated by CD4+ T cells, increased during the course of infection, peaked at 8 weeks postinfection, and decreased thereafter. Gamma interferon, but not interleukin-4, was produced in vitro by spleen cells from infected animals upon stimulation with Bartonella antigens. Bartonella-specific IgG was detectable in serum of infected mice by 2 weeks, and the antibody concentration peaked at 12 weeks postinfection. IgG2b was the prominent isotype among the Bartonella-specific serum IgG antibodies. These data indicate that B. henselae induces cell-mediated immune responses with a Th1 phenotype in immunocompetent C57BL/6 mice.

First Report of Beech Bark Disease in Michigan.

Beech bark disease of American beech (Fagus grandifolia) is caused by the interaction of an introduced scale insect (Cryptococcus fagisuga) and the native fungus Nectria galligena, or N. coccinea var. faginata, which is thought to be introduced. Infestations of the insect precede development of the disease, and N. galligena is often found before N. coccinea var. faginata. Previously published records indicate that the beech scale extends as far west as eastern Ohio (2) and southern Ontario (Sajan, personal communication). The scale is now well established in several locations in both the Upper Peninsula (UP) and Lower Peninsula (LP) of Michigan. The scale insect has been found in beech stands throughout three counties (Oceana, Mason, and Muskegon) along the Lake Michigan shore in the LP, extending for at least 100 km from north to south, and occurring up to about 80 km inland. In the eastern UP, beech scale has been found in four counties (Alger, Chippewa, Luce, and Schoolcraft). The heaviest beech scale infestations are distributed around Ludington State Park in the west central LP and the Bass Lake Forest Campground in the eastern UP. The extent of the insect infestation suggests that it has been present in Michigan for many years, with anecdotal accounts placing the first observations of beech scale at Ludington State Park around 1990. These infection centers are distant from previously known beech scale infestations and are located in heavily used recreation areas, suggesting that the insect may have been transported by human activity. Perithecia of N. coccinea var. faginata were found in Ludington State Park in the LP at N 44° 1.951', W 86° 29.956' and perithecia of N. galligena were found at the Bass Lake site in the UP, at N 46° 27.748', W 85° 42.478'. The identity of the fungi collected from each location was confirmed by measurements of perithecium and ascospore morphology (1). Perithecia at both sites were scarce and difficult to find. Surveys planned for the summer and fall of 2001 will further delimit the occurrence and distribution of the pathogens involved in the disease in Michigan. To our knowledge, this is the first report of the beech scale and beech bark disease in Michigan, with N. galligena and N. coccinea var. faginata identified as the pathogens. References: (1) H. V. T. Cotter and R. O. Blanchard. Plant Dis. 65:332-334, 1981. (2) M. E. Mielke et al. Plant Dis. 69:905, 1985.

Evidence for a disease-promoting effect of Staphylococcus aureus-derived exotoxins in atopic dermatitis.

BACKGROUND: The skin of patients with atopic dermatitis (AD) exhibits a striking susceptibility to colonization with Staphylococcus aureus. Some strains of S aureus secrete exotoxins with T-cell superantigen activity (toxigenic strains), and abnormal T-cell functions are known to play a critical role in AD. OBJECTIVE: Our purpose was to examine the impact of superantigen production by skin-colonizing S aureus on disease severity. METHODS: In a cross-sectional study of 74 children with AD, the presence and density of toxigenic and nontoxigenic strains of S aureus was correlated with disease severity. In a subgroup of patients the T-cell receptor Vbeta repertoire of peripheral blood and lesional T cells was investigated and correlated with individual superantigen activity of skin-colonizing S aureus. RESULTS: Fifty-three percent of children with AD were colonized with toxigenic strains of S aureus producing staphylococcal enterotoxin C, staphylococcal enterotoxin A, toxic shock syndrome toxin-1, staphylococcal enterotoxin B, and staphylococcal enterotoxin D in decreasing frequency. Children colonized with toxigenic S aureus strains had higher disease severity compared with the nontoxigenic and S aureus-negative groups. Patients colonized with toxigenic S aureus exhibited shifts in the intradermal T-cell receptor Vbeta repertoire that correspond to the respective superantigen-responsive T-cell subsets. CONCLUSION: The data demonstrate that S aureus-released exotoxins can modulate disease severity and dermal T-cell infiltration.

Murine model of Bartonella henselae infection in the immunocompetent host.

Bartonella henselae is an emerging pathogen causing cat scratch disease, bacillary angiomatosis, and peliosis hepatis. Progress in understanding the pathogenesis of and the immune response to these infections has been limited by the lack of an animal model. Following intraperitoneal infection of C57BL/6 mice with B. henselae, organs were cleared of cultivatable bacteria within 6 days. In contrast, B. henselae DNA could be detected in liver tissue for at least 3 months. Liver tissue showed granulomatous inflammation reaching its highest degree of intensity during the fourth week of infection and resolving within 12 weeks postinfection. This mouse model is applicable to the study of the pathogenesis of B. henselae and the immune response to this pathogen in the immunocompetent host.

Granulocyte colony-stimulating factor worsens the outcome of experimental Klebsiella pneumoniae pneumonia through direct interaction with the bacteria.

Besides its well-established effects on granulocytopoiesis, granulocyte colony-stimulating factor (G-CSF) has been shown to have direct effects on the recruitment and bactericidal ability of neutrophils, resulting in improved survival of experimentally infected animals. We studied the effect of G-CSF on the course of experimental pneumonia induced by Klebsiella pneumoniae, an important gram-negative bacillary pulmonary pathogen. Using a highly reproducible murine model, we here show the paradoxical finding that mortality from infection was significantly increased when animals received G-CSF before induction of pneumonia. Administration of G-CSF promoted replication of bacteria in the liver and spleen, thus indicating an impairment rather than an enhancement of antibacterial mechanisms. By contrast, a monoclonal antibody against Klebsiella K2 capsule significantly reduced bacterial multiplication in the lung, liver, and spleen, and abrogated the increased mortality caused by G-CSF. In vitro studies showed a direct effect of G-CSF on K pneumoniae resulting in increased capsular polysaccharide (CPS) production. When bacteria were coincubated with therapeutically achievable concentrations of G-CSF, phagocytic uptake and killing by neutrophils was impaired. Western blot analysis showed three binding sites of G-CSF to K pneumoniae. Binding of 125I-G-CSF to K pneumoniae was displaced by an excess of unlabeled G-CSF, whereas an unrelated cytokine, interleukin-1alpha, did not compete with G-CSF binding to the bacteria. Thus, in this model, the direct effect of G-CSF on a bacterial virulence factor, CPS production, outweighed any beneficial effect of G-CSF on recruitment and stimulation of leukocytes.

Cytokines in the induction and expression of T-cell-mediated granuloma formation and protection in the murine model of listeriosis.

Lymphocyte-mediated inflammation is a hallmark of autoimmune diseases, such as multiple sclerosis. Crohn's disease, rheumatoid arthritis and sarcoidosis. However, this type of inflammation probably developed under evolutionary pressure from pathogenic microorganisms, such as mycobacteria and other intracellular infective agents. One such pathogen, the gram-positive bacterium Listeria monocytogenes (L. monocytogenes), induces a cascade of tissue alterations that ultimately results in the eradication of the bacteria associated with a granulomatous response. Consequently, murine listeriosis has been established as a model to analyze not only T-cell-dependent antibacterial protection but also T-cell-mediated mononuclear inflammation in parenchymal organs. Extensive studies of the molecular basis of the latter phenomenon led to the conclusion that the most decisive step from non-specific microabscess formation to granulomatous inflammation is the activation of non-specifically invading CD4+ T cells, which results in high local concentrations of TNF-alpha and IFN-gamma in the presence of IL-2. This in turn induces CD11b-independent mechanisms of intraparenchymal monocyte accumulation. Because any attempt to neutralize the effects of TNF-alpha and IFN-gamma to modulate T-cell-mediated inflammation will also dramatically decrease host resistance, other anti-inflammatory strategies based on the modulation of TNF-alpha and IFN-gamma-induced mechanisms of monocyte accumulation must be developed. Recalling the classical work by Dienes & Schoenheit on the induction of bacterial allergies (1), the cytokine phenotype of granuloma formation also has implications as regards the most potent adjuvant environment for the development of a T-cell response. The murine listeriosis model is the basis for all conclusions in this article on the role of cytokines in the induction and expression of T-cell-mediated inflammation and, as we will show, promises to yield still more insights into the rational design of vaccines.

Mechanisms of granuloma formation in murine Mycobacterium avium infection: the contribution of CD4+ T cells.

Infection with the virulent Mycobacterium avium strain TMC 724 caused progressive infection in C57BL/6 and BALB/c mice, while infection with a less virulent strain (M. avium SE 01) resulted in chronically persistent bacterial loads. Livers of mice infected with TMC 724 were characterized by progressively expanding tumor-like infiltrations of epithelioid macrophages, while SE 01 induced well-developed, compact epithelioid granulomas that remained constant in size and number for at least 4 months. When C57BL/6 mice were depleted of CD4+ T cells by i.p. administration of specific mAb at the time of infection, their capacity to initiate granuloma formation was completely abrogated during the first 4 weeks of infection. Semi-quantitative competitive RT-PCR of liver homogenates obtained 3 weeks after infection revealed that depletion of CD4+ T cells was accompanied by a 25-fold reduced expression of IFN-gamma mRNA and a 5-fold reduced expression of tumor necrosis factor (TNF)-alpha mRNA when compared to control infected mice. Granuloma morphology in response to either TMC 724 or SE 01 was similar in immunodeficient SCID mice to that observed in syngeneic BALB/c mice. However, SCID mice developed granulomas in a delayed fashion and were less efficient in surrounding infected Kupffer cells with an inflammatory infiltration. The delayed kinetics of granuloma initiation in infected SCID mice was paralleled by a lower mRNA expression for IFN-gamma and TNF-alpha compared to that observed in infected BALB/c mice. mAb-mediated neutralization of IFN-gamma in BALB/c mice significantly reduced inflammatory infiltrations and granuloma formation. These data support the conclusion that CD4+ T cells accelerate granuloma formation by enhancing the production of TNF-alpha and IFN-gamma at the site of infection.

Kinetics and dose dependence of macrophage colony-stimulating factor-induced proliferation and activation of murine mononuclear phagocytes in situ: differences between lungs, liver, and spleen.

Alveolar macrophages (AM) play an important role in antimicrobial defense mechanisms of the lung. It therefore seems reasonable to use macrophage colony-stimulating factor (M-CSF) to enhance local resistance mechanisms. However, little is known about the in vivo activity of M-CSF on macrophages in various organs. We determined the effect of a single subcutaneous dose of M-CSF (10, 50, 100, and 500 ng, respectively) on the number and functional status of AM as well as of macrophages in liver and spleen of mice. Organs were investigated immunohistochemically on days 1 and 3 after injection using monoclonal antibodies specific for F4/80, Ia antigen, and MAC-1. We found a significant increase in the number of F4/80+ AM, Kupffer cells, and splenic macrophages reaching its maximum 24 h after injection of low doses (10 and 50 ng per mouse, respectively) of M-CSF and decreasing to a level seen in untreated mice at 72 h after M-CSF in liver and spleen, whereas at a dose of 50 ng per mouse the number of AM remained high. In contrast, the numbers of AM, Kupffer cells, and splenic macrophages did not increase significantly when high doses were used (500 ng). The expression of Ia antigen and MAC-1 was increased on macrophages in the spleen but not on AM or Kupffer cells. TNF-alpha was elevated in bronchoalveolar (BAL) fluid after 3 h and IL-6 at 6, 12, and 24 h after M-CSF injection in dose-dependent manner. Nitric oxide production was not increased after injection of M-CSF. Our results point to regional differences in the response of macrophages to M-CSF. These may caused by differences in the M-CSF-induced production of TNF-alpha and IL-6. These findings may be important for the therapeutic use of M-CSF in microbial infections.

CD4+ T cell associated cytokine gene expression during experimental infection with Listeria monocytogenes: the mRNA phenotype of granuloma formation.

In murine listeriosis, elimination of bacteria and immunity to re-infection critically depend on Thy-1+CD4- cells, while cell-mediated inflammatory phenomena like delayed-type hypersensitivity and granuloma formation are mediated by CD4+ T cells. In an attempt to correlate T cell phenotype and function with a particular set of cytokines produced in vivo, we examined the cytokine gene expression profile associated with the presence or absence of CD4+ and/or CD8+ cells in the livers of mice during experimental infection with Listeria monocytogenes. T cell subset depletion was achieved by i.p. administration of saturating amounts of the appropriate mAbs, and mRNA detection was carried out using a qualitative and semi-quantitative polymerase chain reaction-based mRNA amplification protocol. In both primary and secondary infection, the presence of CD4+ cells was a prerequisite for granuloma formation, and was found to be closely associated with mRNA expression for IL-2, IL-3 and IL-4, a 5-fold increase in expression of tumor necrosis factor (TNF)-alpha and granulocyte macrophage colony stimulating factor, and a 25-fold increase in expression of IFN-gamma and TNF-beta mRNAs, suggesting a role for these cytokines in granuloma formation. In striking contrast, depletion of CD8+ cells did not result in reduced mRNA expression for any one of the cytokines studied, implying that CD8+ T cell mediated cure and prevention of listeriosis may operate via qualitatively distinct mechanisms.

The mRNA-phenotype of granuloma formation: CD4+ T cell-associated cytokine gene expression during primary murine listeriosis.

In murine listeriosis, elimination of bacteria and immunity to reinfection critically depend on Thy1+ CD4- cells, while cell-mediated inflammatory phenomena such as DTH and granuloma formation are mostly mediated by CD4+ T cells. In an attempt to correlate T cell phenotype and function with a particular set of cytokines produced, we examined the cytokine gene expression profile associated with the presence or absence of Thy1+, CD4+ and/or CD8+ cells in the livers of mice during a primary infection with L. monocytogenes. The presence of CD4+ cells was found to be closely associated with mRNA expression for IL-2, IL-3 and IL-4, a 5-fold increase in expression of TNF-alpha and GM-CSF and a 25-fold increase in expression of IFN-gamma and TNF-beta mRNAs, and temporally coincided with the development of granulomatous lesions. In vivo neutralization of TNF-alpha and, to a lesser extent, IFN-gamma resulted in abrogation of granuloma formation. A similar correlation between the presence of CD8+ cells and mRNA expression for any one of the cytokines studied did not exist, pointing to a qualitatively different mechanism of CD8+ T cell mediated cure of listeriosis.

BCG-induced immunomodulation of DTH to heterologous erythrocytes leads to Mac-1-independent myelomonocytic cell recruitment.

BCG(mycobacterium bovis)-modulated delayed-type hypersensitivity (DTH) to sheep red blood cells (SRBC) differs from that in nonmodulated mice with respect to kinetics of expression, cellular composition of inflammatory foci, and susceptibility to specific suppressor mechanisms. We investigated whether the differences between these two types of SRBC-specific DTH reactions are based on different T cell subpopulations involved or on differences in the mechanisms of myelomonocytic cell recruitment induced by the same T cell subset. We demonstrate that both types of DTH are exclusively mediated by CD4+ T cells, but significantly differ in the mechanisms of inflammatory cell extravasation. While the expression of nonmodulated DTH to SRBC is markedly inhibited by anti-Mac-1 mAb 24 hr after challenge, the BCG-modulated DTH is totally resistant to such treatment. Thus, BCG modulation of the DTH response to SRBC most probably results in the generation of qualitatively different, antigen-specific CD4+ T cells, which induce the activation of adhesion molecules able to circumvent the Mac-1 dependency of the nonmodulated skin response.

The role of cytokines in experimental listeriosis.

Listeria monocytogenes is a Gram-positive, intracytoplasmatically replicating pathogen that elicits host reactions which are very similar in man and rodents. Using murine listeriosis as a highly reproducible and convenient experimental model for studying the immune response to infections with facultative intracellular bacteria, Mackaness developed the concept of T cell-mediated macrophage activation as the pivotal mechanism in host defense against this type of infectious agents. Continued research in listeriosis itself, however, provided paradoxical findings that challenged the original dogma. In particular, the finding that T cell-mediated inflammatory events, like DTH and granuloma formation, can be dissociated from protective effector mechanisms has provided a new impetus and experimental access to characterizing the molecular mediators responsible for these diverging phenomena. This review first summarizes the cellular basis for the dichotomy of immunological phenomena outlined above and will then relate recent findings on cytokine expression in infected tissues to these dual categories of the host response to infection. The authors will focus on data obtained from in vivo experiments and draw on evidence from in vitro systems only when appropriate in vivo verification is still lacking. The data presented will cover the developments made in the field of cytokine research since our previous review in 1981 (Rev. Infect. Dis. 3: 1221-1250). Detectable numbers of listeria-specific T cells become apparent on day 4 to 5 of a primary infection. Whereas the localized and sustained release of TNF and IFN-gamma mediated by CD4+ cells seems to be the focusing event triggering mononuclear cell accumulation, the coincidental eradication of bacteria critically depends on CD8+ and/or CD4-CD8-Thy1+ cells. Their effector functions, however, remain obscure, since cytokines cannot be identified that will substitute for their presence. None of the cytokines studied thus far has been demonstrated to effectively cure an established infection. In addition, the increased production of cytokines characteristic of an anamnestic response (IL-2, IL-3, IL-4, IFN-gamma and TNF) can be dramatically reduced by depleting CD4+ T cells without any effect on the animal's ability to eradicate high lethal doses of bacteria and Listeria-specific CD8+ T cells can mediate protection even in the presence of neutralizing antibodies to IFN-gamma. In conclusion, the murine model of Listeria infection provides an interesting experimental approach for the development of immunotherapeutic strategies aimed at reducing T cell-mediated immunopathology without interfering with innate resistance and T cell-mediated cure and prevention of disease.

Kinetic analysis of cytokine gene expression in the livers of naive and immune mice infected with Listeria monocytogenes. The immediate early phase in innate resistance and acquired immunity.

The anamnestic response to infection with Listeria monocytogenes is characterized by the rapid elimination of normally lethal doses of bacteria and accelerated granuloma formation. These phenomena are mediated by listeria-specific memory T cells within the first 24 h after reinfection. In order to elucidate the mechanisms operative during this decisive phase of infection, we conducted a comprehensive kinetic and quantitative analysis of cytokine gene expression in the livers of naive and immune mice. Organs were removed at 30 min, and 1, 2, 6, and 24 h after primary and secondary infections, and PCR3-assisted messenger RNA (mRNA) amplification was performed on matched samples using primers specific for IL-1 beta, IL-6, M-CSF, GM-CSF, TNF-alpha, IFN-gamma, IL-10, IL-4, IL-2, IL-3 and I1-2Rp55. The cytokine pattern characteristic of secondarily infected animals differed qualitatively by the expression of mRNA for IL-2, IL-2Rp55, IL-3, and IL-4, demonstrating the accumulation and activation of specific T cells in the livers as early as 1 to 2 h after reinfection. Combined in vivo depletion of both CD4+ and CD8+ T cells before reinfection almost completely abrogated the differentiated cytokine profile typical of the anamnestic response. Using competitive PCR for semiquantitative determination of mRNA levels, the amount of IL-1 beta and IL-6 mRNA was found to be very similar during primary and secondary infection, whereas TNF-alpha mRNA was found to be increased by approximately 10-fold 2 h and IFN-gamma mRNA by approximately 50 to 100-fold 6 h after reinfection when compared with a primary challenge. Combined in vivo depletion of both CD4+ and CD8+ T cells before reinfection resulted in a substantial (approximately 10-fold) decrease in IFN-gamma mRNA expression. To correlate these findings with cytokine secretion, spleen cells from naive and immune as well as normal and CD4+ and CD8+ cell depleted mice infected 6 h previously were cultured for 48 h, and supernatants were analyzed for the amount of the above mentioned cytokines. Semiquantitative PCR-assisted mRNA amplification is demonstrated to be a superior tool in dissociating the mediators of innate resistance from those operative in protective immunity and granuloma formation.

Monoclonal antibody against Klebsiella capsular polysaccharide reduces severity and hematogenic spread of experimental Klebsiella pneumoniae pneumonia.

Klebsiella pneumoniae is an important nosocomial pathogen causing severe pulmonary infections. The majority of clinical Klebsiella isolates produce a high-molecular-weight capsular polysaccharide (CPS) which is one of the dominant virulence factors. In the present study, we examined the potency of a murine immunoglobulin M monoclonal antibody (MAb) with specificity to Klebsiella type 2 CPS to protect rats against experimental Klebsiella pneumonia. The MAb did not prevent the invasion of virulent bacteria into the interalveolar space. However, the resolution of infection was accelerated in MAb-treated animals. This was demonstrated by (i) less severe weight loss and (ii) markedly reduced inflammatory reactions in the lung. The elimination of bacteria was significantly increased not only in the lungs but also in the livers of antibody-treated rats. This was reflected by reduced levels of circulating, soluble CPS and MAb-bound CPS. A mixture of human MAbs with specificity to CPS of clinically important Klebsiella serotypes may prove to be a useful tool for the prevention or supportive treatment of Klebsiella pneumonia.

Protective immunity and granuloma formation are mediated by two distinct tumor necrosis factor alpha- and gamma interferon-dependent T cell-phagocyte interactions in murine listeriosis: dissociation on the basis of phagocyte adhesion mechanisms.

Listeria-immune mice are able to express protective immunity in the absence of CD4+ T cells and an apparent granulomatous inflammation. Using a monoclonal antibody (5C6) able to inhibit the recruitment of myelomonocytic cells into inflammatory foci by binding to complement receptor type 3 (CR3/CD11b), we could show that protective immunity and granuloma formation indeed depend on two distinct types of T cell-phagocyte interactions. Listeria-specific CD8+ T lymphocytes, possibly in collaboration with CD4- CD8- T cells, rapidly interact with myelomonocytic cells infiltrating infected tissues in a CR3/CD11b-dependent manner. This interaction results in potent antilisterial protection but not in granuloma formation. On the contrary, CD4+ T cells are able to induce adhesion mechanisms that allow the accumulation of monocytes in granulomatous lesions even in the presence of monoclonal antibody 5C6. However, the protective capacity of these CR3/CD11b-independent T cell-mediated immune mechanisms is low in listeriosis. Tumor necrosis factor alpha and gamma interferon, known to be essential for the expression of both resistance and acquired immunity, are shown to be necessarily involved in granuloma formation, too. It therefore remains to be explained why CD8+ T cells, able to secrete both cytokines, do not induce granuloma formation. The data point to the presence of an as yet undefined CD4+ T cell-derived granuloma-inducing factor and favor the hypothesis that CD8+ T cells, in collaboration with circulating phagocytes, mediate immunity by rapidly liberating listeriae from permissive cells or protecting them from becoming infected.