INFECTION AND IMMUNITY, Oct. 2001, p. 6427–6433 0019-9567/01/$04.00⫹0 DOI: 10.1128/IAI.69.10.6427–6433.2001 Copyright © 2001, American Society for Microbiology. All Rights Reserved.
Vol. 69, No. 10
Bartonella henselae-Specific Cell-Mediated Immune Responses Display a Predominantly Th1 Phenotype in Experimentally Infected C57BL/6 Mice MARDJAN ARVAND,* RALF IGNATIUS, THOMAS REGNATH,† HELMUT HAHN, AND MARTIN E. A. MIELKE‡ Department of Medical Microbiology, Institute of Infectious Diseases, Free University of Berlin, Berlin, Germany Received 16 March 2001/Returned for modification 4 May 2001/Accepted 15 June 2001
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 CD4ⴙ 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. B. henselae and its recognition as the main causative agent of CSD, the induction of a delayed-type hypersensitivity reaction, a hallmark of CMI, was used to diagnose CSD clinically (14). Cats are considered the natural host of B. henselae and source of infection for humans. There are few reports on experimental infection of cats with B. henselae; however, the course of disease in cats differs from that in humans, and the results for clinical manifestations and histopathological findings are conflicting (7, 9, 11, 21, 23), possibly as a result of using different B. henselae strains and/or different mechanisms of inoculation. In addition, characterization of the induced immune responses has been difficult because of limitations of immunological tools in feline models. In contrast, murine infection models have been shown to be often advantageous for immunological studies (16). In the present study, we used a murine model of B. henselae infection established in our laboratory (22) to investigate the immune responses induced in the immunocompetent host. Following intraperitoneal (i.p.) infection of C57BL/6 mice with B. henselae, the cellular and humoral immune responses were analyzed at multiple time points until 20 weeks postinfection (p.i.). Proliferative responses were studied in vitro by means of a splenocyte proliferation assay. The roles of different T-cell subsets were investigated by administration of monoclonal antibodies (MAbs) to CD4 and CD8 T cells in proliferation assays. In cytokine release studies, Th cells involved in CMI against B. henselae were further characterized. Humoral immune responses were studied by enzyme-linked immunoassay (ELISA) for Bartonellaspecific immunoglobulin G (IgG) antibodies, and the IgG isotypes were determined. In addition, development and alter-
Clinical presentation of infections caused by Bartonella henselae ranges from a relatively mild lymphadenopathy with few additional symptoms, seen in cat scratch disease (CSD) in immunocompetent patients, to life-threatening systemic disease in immunocompromised individuals, such as bacillary angiomatosis and peliosis (BAP) associated with bacteremia and fever (2). Little is known about the pathogenesis of B. henselae infections and the induced immune responses, mainly because of the lack of a suitable animal model. Several observations point to a crucial role of cell-mediated immunity (CMI) in the pathogenesis and control of B. henselae infections. In human immunodeficiency virus (HIV)-infected patients with AIDS, the clinical manifestations of disease are more severe than the mild and self-limiting course of infection in immunocompetent patients. Recently, Mohle-Boetani et al. (19) reported that HIV-infected individuals with CD4⫹ T-cell counts of ⬍50 l⫺1 are at highest risk for developing BAP and suggested than BAP be considered an AIDS-defining opportunistic infection. Also, B. henselae has been shown to induce granuloma formation in experimentally infected animals (9, 22) and in lymph nodes, spleens, and livers of human patients suffering from CSD (8, 12, 13, 27), indicating the induction of CMI in immunocompetent hosts. In addition, in the era prior to detection of * Corresponding author. Present address: Hygiene-Institut, University of Heidelberg, Im Neuenheimer Feld 324, 69120 Heidelberg, Germany. Phone: 49 6221 567807. Fax: 49 6221 565627. E-mail:
[email protected]. † Present address: Labor Enders and Partners, 70193 Stuttgart, Germany. ‡ Present address: Robert-Koch-Institut, 13353 Berlin, Germany. 6427
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ations of granulomatous lesions in livers of infected mice were monitored histologically. The present study demonstrates that a virulent B. henselae strain is capable of inducing a long-lived inflammation of the liver and strong Th1 type immune responses in immunocompetent C57BL/6 mice.
INFECT. IMMUN. TABLE 1. Detection of B. henselae DNA by amplification of the htrA gene in liver tissue of B. henselae-infected mice Expt. no.
1 2 3
MATERIALS AND METHODS Bacteria. B. henselae strain Berlin-1, which was originally isolated from the cutaneous bacillary angiomatosis lesions of an HIV-infected patient (4), was used throughout this study. The primary isolate was inoculated in brucella broth supplemented with 250 mg of hemin/liter and 8% Fildes (24), grown to a logphase culture, and stored in aliquots at ⫺70°C. To prepare the inocula for the infection, aliquots were thawed and grown again to log-phase cultures in supplemented brucella broth, washed twice with phosphate-buffered saline (PBS), and resuspended in PBS to obtain a final concentration of (2 ⫾ 1) ⫻ 108 CFU/ml. One aliquot was plated in 10-fold serial dilutions on Columbia agar with 5% human blood to determine the colony count, and the remaining bacteria were used for inoculation of animals within 1 h after preparation. Mice. Female C57BL/6 mice raised in our breeding facilities were used at the age of 10 to 13 weeks. Animals were kept under specific-pathogen-free conditions (positive-pressure cabinet). Infection of animals. Animals were injected i.p. with 1 ⫻ 108 to 2 ⫻ 108 CFU of B. henselae in a volume of 1 ml of PBS or with 1 ml of PBS as a control. At 1 to 7 days and at 2, 4, 8, 12, and 20 weeks p.i., at least three mice per group were euthanized with CO2 asphyxiation. Livers were collected for histology, culture, and PCR, and spleens were removed aseptically for proliferation and cytokine release assays and culture. Serum samples were collected by standard procedures for serology and were stored at ⫺70°C until used. Detection of B. henselae by culture and PCR. Bacterial loads in livers and spleens were determined by plating 10-fold serial dilutions of organ homogenates on Columbia agar supplemented with 5% human blood. For the detection of Bartonella DNA, samples of liver tissue (0.1 to 0.2 mg) were snap-frozen in liquid nitrogen and stored at ⫺70°C until used. DNA was isolated by using the QIAamp tissue extraction kit (Qiagen, Hilden, Germany) according to the manufacturer’s protocol. A seminested PCR assay was performed to amplify part of the htrA gene (1) by using oligonucleotides CAT1 and CAT2 as the outer primers and CAT2 and RH1 as the inner primers (1). PCR was performed with Taq polymerase (In ViTek, Berlin, Germany) in a volume of 50 l in two sequential tubes. Amplification was accomplished for outer primers by predenaturing for 4 min at 94°C followed by 35 cycles of 94°C for 30 s, 60°C for 60 s, and 72°C for 45 s and a final extension step of 72°C for 10 min. The second amplification step was carried out under the same conditions, except that 45 cycles were performed. Positive and negative controls were included in each run. After electrophoresis through a 2% agarose gel and staining with ethidium bromide, the products were photographed. The presence of a 390-bp band was considered positive. The detection limit of this procedure was 10 CFU per organ. Histology. One liver lobe per mouse was fixed in 4% (vol/vol) formalin-PBS, embedded in paraffin, sectioned (3 m thick), and stained with hematoxylin and eosin. Numbers of granulomatous lesions were determined by counting focal mononuclear infiltrations per 0.25 cm2 at ⫻100 magnification. Spleen cell proliferation assay. T-cell proliferative responses to Bartonella antigens were studied by [3H]thymidine ([3H]TdR) incorporation. Single-cell suspensions of spleen cells were prepared as follows. Spleens were disrupted in RPMI 1640 (all cell culture products were purchased by Biochrom, Berlin, Germany) supplemented with penicillin (100 U/ml) and streptomycin (100 g/ ml). Erythrocytes were lysed by 60 s of incubation in H2O. Cells were washed twice and resuspended at a density of 107 cells/ml in RPMI 1640 supplemented with 0.2 mM L-glutamine; 100 U of penicillin, 100 g of streptomycin, and 20 mg of polymyxin B (Sigma, Deisenhofen, Germany)/ml; and 10% heat-inactivated fetal calf serum. The viability of cells was ⬎98% as determined by trypan blue exclusion. Samples of 0.1 ml (106 cells) were added to 0.1 ml of antigen suspension in 96-well flat-bottom tissue culture plates. As the antigen, we used heatkilled (45 min at 60°C) B. henselae (HKBH) at final concentrations of 104 to 107 CFU/ml, concanavalin A (5 g/ml), pokeweed mitogen (5 g/ml), or medium only. Experimental conditions were set up in triplicate. Cultures were incubated at 37°C in 5% CO2 for 3 days. This incubation period had been found in preliminary experiments to be appropriate for detection of splenocyte proliferative responses to HKBH. [3H]TdR (1 Ci/well; Amersham, Buckinghamshire, United Kingdom) was added to the wells for the final 18 h of incubation. Cells were then harvested onto glass fiber filters by a semiautomated harvester. In-
a
No. of PCR-positive mice/no. of tested mice at wk p.i.: 2
4
8
12
20
3/3 3/3 NDa
3/3 3/3 ND
3/3 2/3 2/3
2/3 3/3 2/3
0/3 0/3 0/3
ND, not done.
corporation of [3H]TdR was determined in a liquid scintillation counter. Results were expressed as mean counts per minute for each animal. In some experiments, blocking MAbs to CD4 or CD8 or an isotype control antibody (Pharmingen, San Diego, Calif.) was added to the wells at a final concentration of 20 g/ml. Stimulation was performed with 106 CFU of HKBH/ml or with medium only. For the kinetics studies, stimulation indices (SIs) were calculated to facilitate comparison of the proliferation rates at different time points p.i. by dividing mean counts per minute of HKBH-containing wells (triplicates) by mean counts per minute for medium-only wells (triplicates) for each animal. Stimulation ratios (SR) were then calculated to facilitate comparison among groups by dividing the SI for each infected mouse by the mean SI for three uninfected control mice tested at the same time point (10). Measurement of cytokine production by spleen cells. The concentrations of gamma interferon (IFN-␥) and interleukin-4 (IL-4) in supernatants of spleen cell cultures were determined using commercially available ELISA kits (Pharmingen) according to the manufacturer’s recommendation. Supernatants were collected after 48 h of stimulation of spleen cells with antigens and stored at ⫺40°C until used. Cytokine levels were estimated by comparison with standard curves for recombinant murine IFN-␥ and IL-4. The detection limits of the assays were 100 pg/ml for IFN-␥ and 150 pg/ml for IL-4. Determination of Bartonella-specific IgG antibodies. Levels of Bartonella-specific total IgG and IgG subclasses IgG1 and IgG2b were determined in serum by means of ELISA. Microtiter plates were coated overnight at 4°C with outer membrane proteins (OMP) of B. henselae strain Berlin-1 (8a, 25) at a concentration of 10 g/ml in bicarbonate buffer (0.1 ml/well). Plates were washed and blocked with PBS containing 0.5% Tween 20 (Merck, Darmstadt, Germany) and 0.3% bovine serum albumin (Sigma) for 30 min at 37°C (0.2 ml/well). After three washes, 0.1-ml diluted serum samples were added to the wells and incubated for 30 min at 37°C. For the determination of total IgG, sera were diluted 1:10,000 in PBS-Tween. To analyze the IgG subclasses, fourfold serial dilutions of the sera were performed (1:200 to 1:3,200,000). After three washes, 0.1 ml of rabbit anti-mouse IgG-, IgG1-, or IgG2b-horseradish peroxidase conjugate (Dianova, Hamburg, Germany) was added and incubated for 30 min at 37°C. After six washes, 0.1 ml of tetramethylbenzidine substrate (Sigma) was added for 30 min at 37°C. Reactions were stopped by addition of 25 l of 2 M H2SO4, and the optical density was determined at 450 nm. Statistics. The unpaired two-tailed Student t test was performed with STATVIEW software. P values of ⬍0.05 were considered significant.
RESULTS Course of infection in C57BL/6 mice. To determine the course of infection with B. henselae strain Berlin-1 in mice, C57BL/6 mice were injected i.p. with 1 ⫻ 108 to 2 ⫻ 108 CFU of B. henselae/ml or PBS. At various time points p.i., three animals per group were euthanized and organs were analyzed for recovery of B. henselae by culture and PCR. Bacteria could be cultivated during the first 3 days p.i. from livers and up to 6 days p.i. from spleens (data not shown). Thus, the kinetics of clearance of viable bacteria from organs was similar to the one described previously by us (22) for B. henselae strain Houston-1. Liver tissue was also subjected to PCR amplification of the htrA gene (1). Here, Bartonella DNA could be detected in livers of infected animals up to 12 weeks p.i. (Table 1), while all tissue samples from PBS-injected control animals revealed
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FIG. 1. Granulomatous lesion in the liver tissue of B. henselae-infected mice. C57BL/6 mice were infected i.p. with 2 ⫻ 108 CFU of B. henselae for 2 (A) or 12 weeks (B). Sections were stained with hematoxylin and eosin. Original magnification, ⫻400.
negative results (not shown). These findings suggest that, despite our failing to cultivate B. henselae from livers of infected mice beyond the first week p.i., bacterial antigens might persist in the liver tissue for several weeks p.i. Histopathological lesions in livers of infected mice. B. henselae strains have previously been shown to be able to induce inflammatory lesions during experimental infections of mice and cats (9, 21, 22). To further characterize the infection of mice with B. henselae strain Berlin-1, liver tissue from infected and uninfected mice was analyzed for histopathological alterations at various time points p.i. Only in liver tissues of infected mice could mononuclear cell infiltrations already be
detected at 2 weeks p.i. (Fig. 1A). The lesions expanded in size and number continuously and reached maximal density at 12 weeks p.i. (Fig. 2). They consisted mainly of lymphocytes and monocytes and were located in both perivascular (Fig. 1A) and intraparenchymal (Fig. 1B) regions. Although the size and number of the inflammatory foci decreased thereafter, some lesions were still detectable at the latest time point of observation, i.e., at 20 weeks p.i. (Fig. 2). Hence, the Berlin-1 strain induces a long-lived inflammatory reaction in the liver tissue of immunocompetent C57BL/6 mice. T-cell responses. (i) Proliferative responses of spleen cells. To determine the cell-mediated immune responses induced by
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FIG. 2. Kinetics of cellular inflammatory reactions in the livers of B. henselae-infected mice. C57BL/6 mice were injected i.p. with 2 ⫻ 108 CFU of B. henselae or PBS. At indicated time points, five animals per group were euthanized, liver tissue was stained for histology with hematoxylin and eosin, and lesions were counted. Results are presented as means ⫾ standard errors.
B. henselae, we investigated the proliferative responses of spleen cells from infected and uninfected mice upon in vitro stimulation with HKBH at 8 weeks p.i. Spleen cells of infected mice proliferated in a dose-dependent manner after stimulation with HKBH, while splenocytes of uninfected mice revealed very low proliferation rates (Fig. 3). The proliferative responses of splenocytes from infected mice were significantly higher (P ⬍ 0.05) than those from uninfected mice upon stimulation with HKBH at concentrations of 105 to 107 CFU/ml. Thus, B. henselae infection induces significant T-cell responses in vivo, which are detectable in vitro by means of spleen cell proliferation assays. (ii) Role of CD4ⴙ T cells in proliferative responses to B. henselae. To further determine the T-cell subset(s) which substantially contributes to the proliferative responses of
FIG. 3. Proliferative responses of spleen cells from B. henselaeinfected mice. C57BL/6 mice were injected i.p. with 2 ⫻ 108 CFU of B. henselae or PBS. At 8 weeks p.i., four animals per group were euthanized and spleen cells were isolated. Splenocytes were stimulated in vitro with 104 to 107 CFU of HKBH/ml or medium for 3 days, and proliferation was determined by [3H]TdR uptake. Results are means ⫾ standard errors. Single asterisk, P ⬍ 0.05; double asterisks, P ⬍ 0.001.
FIG. 4. Proliferative responses of splenocytes from infected mice in the presence of anti-CD4 or anti-CD8 antibodies. C57BL/6 mice were infected i.p. with 2 ⫻ 108 CFU of B. henselae for 8 weeks. Spleen cells were stimulated with 106 CFU of HKBH/ml for 3 days in the presence of 20 g of MAbs against CD4, CD8, or an isotype-matched control/ml or in the absence of antibodies. Results represent mean counts per minute ⫾ standard errors (three animals). The mean spontaneous proliferation rate without stimulation was 11,170 ⫾ 2,708 cpm.
splenocytes to Bartonella antigens, anti-CD4 and anti-CD8 antibodies were added to the spleen cell cultures prior to stimulation with HKBH. Administration of anti-CD4, but not of anti-CD8 or an isotype-matched control MAb, resulted in a more-than-60% reduction of proliferation of splenocytes from infected mice (Fig. 4), indicating that CD4⫹ T cells play a key role in CMI induced by B. henselae. (iii) Kinetics of the Bartonella-specific proliferative responses. To evaluate the development of cell-mediated immune responses, we decided to monitor the proliferative responses at various time points after infection. Splenocytes were isolated at 2 to 20 weeks p.i. and stimulated with HKBH, mitogens, or medium only. After 2 weeks of infection, splenocytes from infected mice revealed very high proliferation rates upon stimulation with HKBH, compared to those from uninfected animals (Table 2). However, at this time, the spontaneous proliferation in the absence of the antigen was also significantly higher for infected mice than for uninfected animals (Table 2). Thus, the early time point (2 weeks p.i.) does not seem to be appropriate for the analysis of Bartonella-specific CMI by means of the spleen cell proliferation assay because of high background proliferation. In contrast, at all later time points of observation, i.e., between 4 to 20 weeks p.i., the proliferative responses of splenocytes from infected and uninfected mice to stimulation with mitogens or medium were comparable (not shown). At those time points, splenocytes from infected animals revealed significantly higher proliferative responses upon stimulation with HKBH than those from uninfected mice. To facilitate comparison of the proliferative
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TABLE 2. Proliferative responses of splenocytes from B. henselae-infected and uninfected mice at 2 weeks p.i. Group (n)
Infected (5) Uninfected (5)
[3H]TdR incorporationa (cpm) after stimulation with: HKBH (106 CFU/ml)
ConAb (5 g/ml)
PWMc (5 g/ml)
Medium only
40,472 ⫾ 6,871 4,535 ⫾ 4,536 (0.0009)
161,927 ⫾ 12,750 116,538 ⫾ 27,302 (0.0210)
51,669 ⫾ 6,717 26,409 ⫾ 5,710 (0.1704)
4,580 ⫾ 1,122 632 ⫾ 149 (0.0082)
a
Values are means ⫾ standard errors. P values are in parentheses. Statistical significance was calculated by Student’s t test. ConA, concanavalin A. c PWM, pokeweed mitogen. b
responses at different time points p.i., SRs were determined. As shown in Fig. 5, the Bartonella-specific SRs increased continuously from the 2nd week p.i., reaching a maximum at 8 weeks p.i. and decreasing thereafter. Therefore, the most appropriate time for the analysis of Bartonella-specific CMI by means of a lymphoproliferation assay is at about 8 weeks p.i. (iv) IFN-␥ production by spleen cells of infected mice. The Th phenotypes of CD4⫹ T cells involved in CMI induced by an infectious agent can be differentiated by their cytokine secretion patterns. CD4⫹ T cells of the Th1 type predominantly produce IFN-␥, while Th2 cells secrete large amounts of IL-4 and IL-5. We therefore investigated the secretion of IFN-␥ and IL-4 by spleen cells of B. henselae-infected mice after in vitro stimulation with HKBH. Only spleen cells from infected mice responded in a dose-dependent manner to stimulation with HKBH by secretion of IFN-␥ (Fig. 6). Although we noticed considerable variation among animals within the infected group, the difference in IFN-␥ production was statistically significant (P ⬍ 0.05) at high antigen concentrations, i.e., at HKBH concentrations of 106 and 107 CFU/ml. In contrast,
FIG. 5. Kinetics of Bartonella-specific proliferation of spleen cells from infected mice. Spleen cell proliferation assays were performed as described for Fig. 3. SIs were calculated by dividing the mean counts per minute of wells (triplicates) containing antigen (HKBH at the indicated concentrations) by the mean counts per minute for wells without antigen. SRs were calculated by dividing the SI of each infected mouse by the mean SI of three uninfected control mice tested simultaneously. Results represent mean counts per minute ⫾ standard errors.
IL-4 was not detectable in the supernatants of spleen cells from infected and uninfected mice upon stimulation with HKBH (detection limit, 150 pg/ml). These data suggest that B. henselae predominantly induces Th1 type responses in C57BL/6 mice. Antibody responses. (i) Production of Bartonella-specific IgG antibodies. Antibody responses to infection with B. henselae were monitored by OMP ELISA for detection of serum IgG antibodies. As shown in Fig. 7, Bartonella-specific IgG was detected in serum samples of infected mice from 2 weeks p.i. through the whole period of observation. The levels of B. henselae-specific IgG peaked at 12 weeks p.i. (ii) Characterization of IgG isotypes among Bartonella-specific antibodies. In mice, IgG1 and IgE have been shown to be associated with a Th2 type response, whereas IgG2a and IgG2b are associated with Th1 type cell-mediated immune responses (20). We were therefore interested in the Th pattern of IgG isotypes in B. henselae-infected mice. Serum samples from mice infected for 4 weeks with B. henselae were serially diluted and subjected to the OMP ELISA to determine the Bartonellaspecific total IgG, IgG1, and IgG2b antibodies. The levels of Bartonella-specific IgG2b were found to be markedly higher than those of IgG1 (Fig. 8), further supporting the induction of Th1 type responses by B. henselae in C57BL/6 mice.
FIG. 6. IFN-␥ secretion by spleen cells from B. henselae-infected mice. C57BL/6 mice were injected i.p. with 2 ⫻ 108 CFU of B. henselae or PBS. At 12 weeks p.i., five animals per group were euthanized and spleens were collected. Spleen cells were stimulated in vitro for 48 h with 104 to 107 CFU of HKBH/ml or medium only, and IFN-␥ release was determined by ELISA. Results are means ⫾ standard errors. Asterisk, P ⬍ 0.05.
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FIG. 7. Production of B. henselae-specific IgG antibodies in infected mice. C57BL/6 mice were injected i.p. with 2 ⫻ 108 CFU of B. henselae or PBS. At indicated time points, five animals per group were euthanized and serum samples were subjected to OMP ELISA. Results are means ⫾ standard errors. OD 450, optical density at 450 nm.
DISCUSSION The aim of the present study was to characterize the immune responses induced by B. henselae in the immunocompetent murine host in order to improve the understanding of the pathogenesis and immunological control of this infection. We used a murine infection model established in our laboratory (22) and chose B. henselae isolate Berlin-1, which was recently isolated by our group (4) and which therefore had not been subjected to multiple in vitro or in vivo passages, for this study. The course of infection was similar to the course that we previously observed for the Houston-1 strain (22). Viable bacteria were cleared within 1 week from organs of infected mice, while Bartonella DNA remained detectable for up to 12 weeks p.i., suggesting that bacterial antigens might persist for a long time in the infected host and stimulate its immune system. Differences regarding the kinetics of the granulomatous inflammation in the liver were observed. In the present study, the lesions peaked later, reached a lower absolute number, and persisted longer. This was most likely due to differences in the virulence of the B. henselae strains, since the mouse strain, the route of infection, and the infectious dose were identical. Little is known so far about differences in virulence among B. henselae strains; however, recent data from human and feline infections point to possible differences (3, 6, 21). Interestingly, the Berlin-1 strain and the Houston-1 strain have been shown to be indistinguishable by means of pulsed-field gel electrophoresis analysis and 16S rRNA typing (3, 4), suggesting that these isolates might be genetically identical. Given this, it appears to be likely that multiple passages of the Houston-1 strain might have led to a down-regulation of virulence factors in this strain. Accordingly, Batterman et al. (5) reported that multiple passages of B. henselae led to a loss of pilus expression by the bacteria, which was associated with reduced adhesion to and entry into epithelial cells. Hence, our data indicate that the primary isolate of a human-
INFECT. IMMUN.
derived B. henselae strain induces a chronic inflammation in the livers of immunocompetent mice and might therefore be useful for future studies on chronic Bartonella infection. The role of CMI in the pathogenesis and control of murine B. henselae infection is poorly understood, and the T-cell subsets involved in cell-mediated immune responses of mice against B. henselae have not been characterized. Therefore, we studied the cell-mediated immune responses of infected mice in splenocyte proliferation and cytokine release assays and analyzed the IgG isotypes of the Bartonella-specific antibody responses. Induction of Bartonella-specific T cells by B. henselae in C57BL/6 mice could be demonstrated by means of a spleen cell proliferation assay. The Bartonella-specific proliferative responses were shown to be mainly mediated by CD4⫹ T cells. We further studied the kinetics of proliferative responses in order to determine the time point of maximal cellular immune responses against B. henselae. At the earliest time point of investigation, i.e., after 2 weeks of infection, splenocytes from infected animals revealed high proliferative responses upon stimulation with HKBH. However, significant differences between infected and uninfected animals were also seen in the absence of B. henselae antigens. Therefore, the proliferative responses at this early phase of infection were not considered specific for HKBH. A possible explanation for this finding might include the in vivo stimulatory effect of the inoculated bacteria on the lymphocytes of infected mice, which might still be detectable in vitro at 2 weeks p.i. In contrast, we did not find any high background proliferation at the later time points p.i., i.e., at 4 weeks or thereafter. Comparison of proliferative responses at different time points revealed that Bartonella-specific proliferation increased continuously from the 2nd week p.i., peaked by 8 weeks, and decreased slowly thereafter. Therefore, about 8 weeks p.i. appears to be the most appropriate time point to study Bartonella-induced cell-mediated immune responses. In cytokine release assays, Bartonella-specific secretion of IFN-␥, but not IL-4, by spleen cells from infected animals was
FIG. 8. Analysis of IgG isotypes among Bartonella-specific serum antibodies in infected mice. C57BL/6 mice were infected i.p. with 2 ⫻ 8 10 CFU of B. henselae for 4 weeks. Serum samples were serially diluted and analyzed by OMP ELISA. Results are means ⫾ standard errors (five animals per group). OD 450, optical density at 450 nm.
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demonstrated, while none of these cytokines were produced by splenocytes from uninfected mice. Recently, Karem et al. (10) showed that spleen cells from BALB/c mice secreted large amounts of IFN-␥ upon in vitro stimulation with B. henselae antigen. However, this was observed regardless of prior exposure of the animals to B. henselae. The induction of nonspecific cellmediated immune responses in murine spleen cell cultures stimulated with Helicobacter felis (18) or Listeria monocytogenes (17) antigens has previously been found. In contrast, our results demonstrate a Bartonella-specific IFN-␥ secretion that was restricted to spleen cells from B. henselae-infected mice. Therefore, B. henselae induces CD4⫹, IFN-␥-secreting T cells in infected C57BL/6 mice, indicative of a predominantly Th1 type response. In an attempt to further confirm the Th1 type of Bartonellaspecific immune responses in C57BL/6 mice, we determined the IgG isotype pattern of the Bartonella-specific serum antibodies. We found that IgG2b rather than IgG1 was the predominant isotype among the Bartonella-specific serum IgG antibodies. Therefore, both cytokine secretion and IgG isotype patterns indicate that Bartonella-specific cell-mediated immune responses in C57BL/6 mice display a predominantly Th1 phenotype. Induction of Th1 type immune responses has been shown for other intracellular pathogens including Leishmania (26), Listeria (17), and, more recently, Ehrlichia spp. (15). Analysis of the kinetics of Bartonella-induced inflammatory lesions, Bartonella-specific proliferative responses, and specific antibody production revealed that the peak of proliferation preceded maximal inflammation in the liver as well as the highest titers of Bartonella-specific antibodies. This underlines a clonal expansion of Bartonella-specific CD4⫹ T cells as a prerequisite for both the attraction and activation of mononuclear phagocytes for granuloma formation, as well as T helper functions for B-cell stimulation. In conclusion, our study demonstrates that a human-pathogenic strain of B. henselae induces a long-lived inflammation in livers of immunocompetent C57BL/6 mice. Concomitantly, B. henselae elicits cell-mediated immune responses in infected animals that are mainly mediated by CD4⫹ Th1 cells. These data contribute to the understanding of the pathogenesis and control of Bartonella infection in the immunocompetent murine host and should be helpful for further animal studies, e.g., under conditions of immunosuppression. ACKNOWLEDGMENTS We thank Dagmar Piske and Antje Finke for excellent technical assistance. REFERENCES 1. Anderson, B., K. Sims, R. Regnery, L. Robinson, M. J. Schmidt, S. Goral, C. Hager, and K. Edwards. 1994. Detection of Rochalimaea henselae DNA in specimens from cat scratch disease patients by PCR. J. Clin. Microbiol. 32:942–948. 2. Anderson, B. E., and M. A. Neuman. 1997. Bartonella spp. as emerging human pathogens. Clin. Microbiol. Rev. 10:203–219. 3. Arvand, M., A. J. Klose, D. Schwartz-Porsche, H. Hahn, and C. Wendt. 2001. Genetic variability and prevalence of Bartonella henselae in cats in Berlin, Germany, and analysis of its genetic relatedness to a strain from Berlin that is pathogenic for humans. J. Clin. Microbiol. 39:743–746. 4. Arvand, M., C. Wendt, T. Regnath, R. Ullrich, and H. Hahn. 1998. Characterization of Bartonella henselae isolated from bacillary angiomatosis lesions in a human immunodeficiency virus-infected patient in Germany. Clin. Infect. Dis. 26:1296–1299.
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