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Natural Killer (NK) Cells Play a Critical Role in the Early Innate Immune Response to Chlamydophila abortus Infection in Mice
J. Comp. Path. 2004, Vol. 130, 48–57
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Natural Killer (NK) Cells Play a Critical Role in the Early Innate Immune Response to Chlamydophila abortus Infection in Mice A. J. Buendı´a, C. M. Martı´nez, N. Ortega*, L. Del Rı´o†, M. R. Caro*, M. C. Gallego*, J. Sa´nchez, J. A. Navarro, F. Cuello* and J. Salinas* Departamento de Histologı´a y Anatomı´a Patolo´gica and *Sanidad Animal, Facultad de Veterinaria, Universidad de Murcia, Campus de Espinardo, Murcia, Spain, and †Department of Microbiology and Immunology, Cornell University College of Veterinary Medicine, Ithaca, NY 14853, USA
Summary Chlamydophila abortus, the aetiological agent of ovine enzootic abortion, induces a strong inflammatory reaction that leads to the T helper cell (Th1) specific immune response necessary for the clearance of infection. Because the role of natural killer (NK) cells during the first stages of this response has received little attention, this study focused on determining the function of these cells in a mouse model of infection. The location of NK cells in the liver and spleen of infected mice was examined immunohistochemically with an anti-Ly49G monoclonal antibody. The number of NK cells increased during the infection both in spleen and liver. In subsequent experiments, an anti-asialo GM1 polyclonal antibody was injected to deplete the NK cells. NK-depleted mice showed a substantial increase in their susceptibility to C. abortus infection, with high mortality rates and an increased burden of bacteria in the liver. Histopathological studies showed that inflammatory foci, composed mainly of neutrophils, were greater in size and number in depleted mice, while numerous chlamydial inclusions were associated with the foci. Serum concentrations of IFN-gamma, a key cytokine in the control of C. abortus infection, were substantially reduced in the NK-depleted mice. To establish the relationship between NK cells and other components of the innate immune response, neutrophils were depleted with the RB6-8C5 antibody. These cells were shown to be crucial in the recruitment of NK cells to the inflammatory foci. q 2003 Elsevier Ltd. All rights reserved. Keywords: abortion; bacterial infection; Chlamydophila abortus; immunity to C. abortus; NK (natural killer) cells; sheep
Introduction Chlamydophila abortus (formerly Chlamydia psittaci serotype 1), an obligate intracellular bacterium, is responsible for enzootic abortion in ewes and is the most commonly diagnosed cause of abortion in several European countries (Rodolakis et al., 1998; Buxton and Henderson, 1999). It is also capable of causing serious human infections (Buxton, 1986). Mouse models have been widely used to study the pathogenesis of C. abortus infections and the Correspondence to: J. Salinas. 0021–9975/$ - see front matter doi: 10.1016/S0021-9975(03)00069-0
immune response (Anderson, 1986; Buzoni-Gatel et al., 1990; Buendı´a et al., 1998; Del Rı´o et al., 2000). In experimental murine infections, the systemic spread of C. abortus is followed by the establishment of an effective immune response capable of eliminating the infection from every organ except the placenta, where the bacteria multiply, inducing abortion (Buendı´a et al., 1998). C. abortus infection is ultimately controlled by a specific helper T cell 1 (Th1) immune response which is, at least partly, interleukin (IL)-12-independent (Del Rı´o et al., 2001) and characterized by q 2003 Elsevier Ltd. All rights reserved.
NK Cells in the Immune Response to C. abortus
the early production of high concentrations of interferon (IFN)-g (McCafferty et al., 1994; Buendı´a et al., 1999b) and the presence of Tcells, particularly CD8þ T cells (Buzoni-Gatel et al., 1992; Del Rı´o et al., 2000). However, exacerbated production of cytokines in response to C. abortus infection can induce pathological changes (Del Rı´o et al., 2001), and abortion has been associated with the detrimental effect of inflammatory cytokines (IFN-g, tumour necrosis factor [TNF]-a) induced by the infection in the placenta (Entrican, 2002). In addition, Buendı´a et al. (1999a) demonstrated the important role of innate (non-specific) immunity, especially that associated with neutrophils, in the early stages of a primary infection. In recent years, it has been established that innate immunity not only acts as a first line of defence, but is also capable of leading to specific immunity through the secretion of different cytokines (Romani et al., 1997; Su et al., 2001). Buendı´a et al. (1999a) reported that neutrophil depletion resulted in increased mortality and early abortion in pregnant mice. Neutrophils also influence the recruitment of other leucocyte populations, especially CD8þ Tcells (Montes de Oca et al., 2000a); however, the importance of neutrophils during a secondary infection is limited (Montes de Oca et al., 2000b). While research into neutrophils has progressed, the role of another important component of the innate immunity, the natural killer (NK) cell, has been poorly studied in C. abortus infection. NK cells constitute an important source of IFN-g during bacterial and viral infections and have a cytotoxic function against infected cells (Biron et al., 1999). Both functions are necessary for the clearance of C. abortus infection and have been found to play a role in the early control of infection by the related species Chlamydia trachomatis (Tseng and Rank, 1998). Hence, to increase our knowledge of the immune mechanisms that contribute to the early control of C. abortus, the potential role of NK cells requires investigation. In this study, emphasis is placed not only on that role but also on the relationship between NK cells and other components of innate immunity, such as neutrophils.
Materials and Methods Mice Eight-week-old female C57BL/6J (H-2b) mice were purchased from Harlan UK Limited (Blackthorn, UK). They were free of common viral and bacterial pathogens according to the results of routine screening procedures performed by the suppliers.
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Experimental Design To evaluate the role of NK cells in the C. abortus infection in mice, three main experiments were carried out, each being repeated with the same numbers of animals to confirm reproducibility of the results. Experiment 1. The purpose was to investigate the distribution of NK cells in the liver and spleen in two groups of 10 mice, one group uninfected and the other group infected with C. abortus. Subgroups of five animals were killed for examination 2 and 4 days after inoculation. Experiment 2. The purpose was to investigate the effect of NK cell depletion on the course of C. abortus infection. In this experiment, the following features were examined: weight loss, survival time after infection, isolation of C. abortus from the liver, presence of IFN-g in serum, histopathology and immunohistochemistry of the liver, and concentrations of alanine transaminase in serum. Two groups of 18 infected mice were used, one group having been subjected to NK cell depletion. Sub-groups of five animals were killed for examination 2 and 4 days after infection. The remaining mice, which were used to assess survival time and weight loss, were killed at day 15 after infection unless they had already died. Experiment 3. The purpose was to investigate the effect of neutrophil depletion on C. abortus infection and on NK cells recruitment to inflammatory foci. In this experiment additional features examined were the same as those in experiment 2. Three groups of 16 infected mice were used, one group having been subjected to NK cell depletion, and a second group having been subjected to neutrophil depletion. Sub-groups of five mice were killed for examination 2 and 4 days after infection. The remaining mice, if still alive, were killed at day 15 after infection. Micro-organisms and Infection Mice were infected with the abortion-causing C. abortus strain AB7, isolated from ovine abortion (Salinas et al., 1995). The bacteria were propagated in the yolk sacs of developing chick embryos, and titrated by enumerating inclusion-forming units (IFUs) on McCoy cells, as described by Buendı´a et al. (1999a). Standardized aliquots were frozen at 280 8C until used. Mice were infected by intraperitoneal (i.p.) inoculation with 106 IFUs of C. abortus in 0.2 ml of phosphate-buffered saline (PBS), pH 7.2, 0.1 M.
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Distribution of NK Cells in the Liver and Spleen Uninfected or infected mice at days 2 and 4 after infection were killed by CO2 euthanasia. Liver and spleen samples from non-infected and infected mice were snap-frozen with 2-methylbutane cooled with liquid nitrogen for studying the distribution of NK cells in the tissue. Cryosections (5 mm) were immunolabelled with an avidin – biotin – peroxidase complex technique (Del Rı´o et al., 2000). Anti-Ly49G (clone 4D11) monoclonal antibody (mAb), recently described as the only antibody of value for the immunohistochemical detection of NK cells in frozen sections (Dokun et al., 2001), was purchased from BD Pharmingen (San Diego, CA, USA). A goat biotinylated anti-rat mouse-adsorbed polyclonal antibody (Caltag, Burlingame, CA, USA) was used as secondary antibody. The number of NK cells was determined in each of 20 fields (17 000 mm2/field) in sections of liver and spleen from each mouse. In-vivo Depletion of NK Cells NK cells were depleted at days 21, 1 and 3 (in relation to i.p. infection) by the intravenous injection of 2 mg of the rabbit anti-asialo GM1 polyclonal antibody in 100 ml (Wako Chemicals GmbH, Neuss, Germany). Non-depleted (control) mice were treated instead with normal rabbit serum (Sigma, Madrid, Spain). The efficacy of depletion was confirmed by flow cytometry of spleen cells, as described by Montes de Oca et al. (2000a), with an anti-NK1.1 mAb (clone PK-136; Caltag). It was found that the spleens of anti-asialo GM1-treated mice had less than 10% of the NK cells of the control mice. Isolation of C. abortus The course of infection was also evaluated by counting IFUs from the liver after isolation of C. abortus on McCoy cell monolayers, following the method previously described by Buendı´a et al. (1999a). One lobe of the liver was examined and the number of IFUs/g was calculated at days 2 and 4 post-infection (p.i.), the detection limit being 2.6 log IFUs per sample. Analysis of Serum for IFN-g Serum samples were taken by cardiac puncture at the time of euthanasia (day 2 or 4 p.i.). IFN-g concentrations were determined by the sandwich ELISA procedure. The capture antibody used was R4-6A2, and the biotinylated detection antibody
was XMG1.2, both purchased from BD PharMingen. Biotin-conjugated antibody was detected with a horseradish-peroxidase streptavidin (BD PharMingen) and a soluble substrate (ABTS; Sigma). The optical density was read at 405 nm. Histopathology and Immunohistochemistry The livers of infected mice were collected and fixed in formalin 10% in PBS, dehydrated and embedded in paraffin wax at 56 8C. Sections (5 mm) were cut, stained with haematoxylin and eosin (HE) and examined for histopathological changes. To demonstrate chlamydial antigen in paraffin wax sections, immunohistochemical labelling was carried out as previously described (Buendı´a et al., 1999b). Serum Transaminase Assay To measure the liver-associated enzyme alanine transaminase (ALT) in serum, a procedure based on a commercial kit (Sigma) was employed. Briefly, in a 96-well plate, 4 ml of serum were added to 20 ml of DL -alanine (0.2 mol/litre) and a-ketoglutaric acid (1.8 mmol/litre); the plate was shaken and the mixture incubated for 30 min at 37 8C. Then, 20 ml of 2,4-dinitrophenylhydrazine (DNP) were added and the mixture was incubated for a further 20 min at room temperature. Finally, the reaction was halted by the addition of 200 ml of 0.4N NaOH, and sample absorbances were measured at 492 nm after 5 min. In-vivo Depletion of Neutrophils The hybridoma producing the RB6-8C5 mAb (Tepper et al., 1992) was provided by Dr R.L. Coffman (DNAX Research Institute, Palo Alto, CA, USA). The rat IgG2b RB6-8C5 mAb was obtained from the ascitic fluid of pristane-primed homozygous nude mice (Harlan) inoculated with 107 hybridoma cells. The mAb was precipitated by saturation with ammonium sulphate, followed by dialysis against PBS pH 7.2 and filtration (0.22 mm). The mAb was purified by chromatography on a protein G column (Sigma) according to the manufacturer’s instructions. Protein concentration was estimated by a modification of the Lowry method (BCA-1 procedure; Sigma). Mice to be depleted of neutrophils received 1 mg of RB6-8C5 mAb intravenously 6 h before infection and on days 1 and 3 p.i. Infected but non-depleted (control) mice received rat IgG (Sigma) at the same time, by the same route, and at the same dosage. Depletion
NK Cells in the Immune Response to C. abortus
was assessed by flow cytometry analysis, as described by Montes de Oca et al. (2000a). Statistical Analysis The significance of differences between groups of mice for each time point investigated was determined by the Student’s t test. A probability value of P , 0:05 was considered significant.
Results
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anti-Ly49G mAb. In infected mice, numerous labelled NK cells were observed in blood vessels (Fig. 1a), and some such cells appeared in the inflammatory foci in the liver parenchyma (Fig. 1b) at day 4 p.i. In the spleen, some positive NK cells were observed in the red pulp of the uninfected mice, mainly in the marginal areas surrounding the lymphoid follicles (Fig. 1c). However, at day 4 p.i., they were outnumbered by NK cells in the marginal areas of the infected mice, in which NK cells were also observed within lymphoid follicles (Fig. 1d).
NK Cell Distribution in the Liver and Spleen of Infected and Non-infected Mice (Experiment 1)
Course of C. abortus Infection in NK Cell-depleted Mice (Experiment 2)
No NK cells were observed in the liver of the uninfected mice after immunolabelling with the
To determine the role of NK cells in the clearance of C. abortus infection, mice were treated with
Fig. 1a– d. Cryostat sections showing the distribution of NK cells. NK cells were detected in the liver of infected mice in (a) blood vessels, and (b) inflammatory foci. NK cells were also detected in (c) the splenic red pulp (RP) of uninfected mice, and (d) RP and lymphoid follicles (LF) of infected mice at day 4 p.i. Cryofixed sections were immunolabelled with the antiLy-49G monoclonal antibody (clone 4D11) and an avidin –biotin complex (ABC) technique. £ 300.
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anti-asialo GM1 antibody and subsequently infected with 106 IFUs. NK cell depletion induced striking mortality. Thus, 50% (8/16) of the depleted mice had succumbed to C. abortus infection by day 5 p.i (Fig. 2a). In contrast, no mortality was observed in the non-depleted control mice during the experiment, although signs of illness, such as ruffled fur, lethargy and huddling were observed from day 3 to day 8 p.i. Unlike the controls, NK-depleted mice showed little or no weight loss during the first 3 days p.i., but this difference had disappeared by day 4 p.i. (Fig. 2b). Isolation of C. abortus from the liver at days 2 and 4 p.i. revealed a significantly higher level of infection in the NK-depleted mice than in their non-depleted counterparts (Fig. 2c). A
characteristic feature of early C. abortus infection in C57BL/6 mice is the high concentration of IFN-g in serum, and in this study both NKdepleted and control mice showed similarly high concentrations at day 2 p.i. At day 4 p.i., however, NK-depleted mice, unlike the controls, showed only residual levels of serum IFN-g (Fig. 2d). Histopathology of C. abortus infection in the liver. At day 2 p.i., the liver of NK-depleted mice showed inflammatory foci composed mainly of neutrophils; both the number and size of the foci were greater than in the non-depleted controls. At day 4 p.i., the size of the foci had increased in both groups, but to a greater degree in the NK-depleted group (Fig. 3a and b). In the control mice these foci consisted of neutrophils and a substantial
Fig. 2a– d. Progression of the C. abortus infection in infected control mice (black symbols and columns) and NK-depleted mice (white symbols and columns). (a) Survival of infected control and NK-depleted mice after infection. Groups of eight mice were infected with 106 IFUs of C. abortus. Mice were monitored daily and mortality rates were calculated. (b) Effect of C. abortus infection on weight loss of infected control and NK-depleted mice. Groups of eight mice were infected with 106 IFUs of C. abortus and body weight changes were monitored daily. (c) IFU counts on homogenized liver tissue collected at 2 and 4 days p.i. The number of inclusions was assessed by indirect immunofluorescence. The results are the means and standard deviation for five mice. (d) Presence of IFN-g in the serum of infected control and NK-depleted mice at 2 and 4 days p.i. The results are the means and standard deviation for five mice. All the experiments were repeated with similar results. * Significant differences ðP , 0:05Þ between control and NK-depleted mice at the same day p.i.
NK Cells in the Immune Response to C. abortus
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Fig. 3a– d. Livers of control (non-depleted) and NK-depleted mice at day 4 p.i. Note the differences in the number and size of the inflammatory foci between (a) control mice and (b) NK-depleted mice. (c) Detail of a typical inflammatory focus in the liver of a control mouse, showing a mixed inflammatory infiltrate of mononuclear cells with some neutrophils. (d) Detail of a typical inflammatory focus in the liver of a NK-depleted mouse, formed mainly of neutrophils with a few mononuclear cells, and showing a large basophilic chlamydial inclusion (arrowhead). HE. £ 300 (a and b); £ 1200 (c and d).
number of mononuclear cells, but in the NKdepleted mice they consisted almost exclusively of neutrophils (Fig. 3c and d). Furthermore, in the NK-depleted mice but not in the controls, large C. abortus inclusions appeared next to the inflammatory foci (Fig. 3d). To assess whether the observed inclusions were a genuine product of C. abortus infection, sections of liver were immunolabelled with an anti-C. abortus lipopolysaccharide (LPS) mAb. The control mice showed labelled C. abortus antigen in sparse inflammatory foci (Fig. 4a), while in the NK-depleted mice the immunoreaction was observed both in the numerous large inflammatory foci and in the numerous inclusions within hepatocytes (Fig. 4b). The increased histopathological changes observed in the liver of NKdepleted mice was associated with the significantly ðP , 0:05Þ higher serum concentrations of the enzyme ALT (242 ^ 22 IU/ml) in NK-depleted
mice than in control mice (56 ^ 8 IU/ml) at day 4 p.i. The baseline concentrations of ALT in noninfected mice was 18 ^ 4 IU/ml. Effect of Neutrophil Depletion on NK Cell Recruitment to the Inflammatory Foci (Experiment 3) Neutrophils were depleted with the RB6-8C5 mAb to study their influence on the NK cell response to C. abortus infection. Neutrophil-depleted mice infected with C. abortus showed 91% mortality (11/12) at day 6 p.i., as compared with 58% (7/12) in NK-depleted mice and no mortality in infected but non-depleted mice (Fig. 5a). The degree of infection, as revealed by hepatic IFU counts, was similar in neutrophil-depleted and NKdepleted mice, but significantly lower in nondepleted mice (Fig. 5b). The main histopathological feature of the livers of neutrophil-depleted mice
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Fig. 4. Immunoreaction against C. abortus antigen in the liver of infected non-depleted (control) and NK-depleted mice at day 4 p.i. (a) Sparse antigen in an inflammatory focus of the liver of a control mouse. (b) Abundant antigen in the larger inflammatory foci, and positive immunoreaction in the chlamydial inclusions within hepatocytes (arrowheads) in the liver of a NK-depleted mouse. ABC. £ 600.
was the lack of inflammatory foci; however, immunohistochemical analysis with anti-C. abortus LPS mAb revealed numerous labelled inclusions in the liver parenchyma (data not shown). Immunolabelling showed that in neutrophil-depleted mice, recruitment of NK cells to the liver parenchyma was less than in infected but non-depleted mice (Fig. 5c). No NK cells were observed in the liver of NK-depleted mice.
Discussion The normal distribution of NK cells in the spleen and the liver, established previously by Dokun et al.
(2001), was confirmed immunohistochemically at the outset of this study, with mAb 4D11 (an antibody that detects the Ly49G antigen expressed by about 50% of murine NK cells). NK cells were located in the red pulp of the spleen (adjacent to splenic lymphoid follicles), but not in the liver parenchyma. C. abortus infection altered this distribution: thus, NK cells increased in number in the red pulp, and were also observed infiltrating the lymphoid follicles. In the liver, a substantial degree of NK cell recruitment was observed in the inflammatory foci associated with C. abortus infection. These changes in NK cell distribution strongly suggest that NK cells play a role in the early
Fig. 5a– c. Progression of the C. abortus infection in the non-depleted (control) mice (black symbols and columns), NK-depleted mice (white symbols and columns) and neutrophil-depleted mice (grey symbols and columns). (a) Survival after infection. Groups of six mice were infected with 106 IFUs of C.abortus. Mice were monitored daily, and mortality rates were calculated. (b) IFU counts on homogenized liver tissues collected at 2 and 4 days p.i. The number of inclusions was assessed by indirect immunofluorescence. The results are the means and standard deviation for five mice. (c) Number of NK cells in the liver parenchyma of control and neutrophil-depleted mice; for each mouse, the number of cells labelled with anti-Ly49G mAb was counted in 20 areas of 17 000 mm2. The graph shows that the depletion of neutrophils by treatment with the anti-granulocyte RB6-8C5 mAb induced a significant decrease in the number of NK cells in the liver during C. abortus infection. The results are the means and standard deviation for five mice. All the experiments were repeated with similar results. * Significant differences ðP , 0:05Þ between control and depleted mice at the same day p.i.
NK Cells in the Immune Response to C. abortus
defensive response to C. abortus. A similar conclusion was reached by Tseng and Rank (1998) in respect of the related species, C. trachomatis; these authors, however, did not describe the location of the NK cells, reported in our investigation. To study their functional role, NK cells were depleted with the anti-asialo GM1 polyclonal antibody. This resulted in increased mortality, a higher C. abortus burden in the liver, and changes in morbidity. Histopathological examination of the liver of depleted mice revealed large inflammatory foci composed mainly of neutrophils, with some mononuclear cells; in addition, hepatocytes with chlamydial inclusions were observed, some of which surrounded the inflammatory foci. The fact that the large numbers of neutrophils present in the liver were unable to prevent the active multiplication of C. abortus in hepatocytes is especially significant, since neutrophils have previously been defined as a crucial first line of defence against C. abortus infection (Buendı´a et al., 1999a); moreover, Del Rı´o et al. (2000) reported that a vigorous neutrophilic response was associated with early clearance of infection in two mouse strains showing different degrees of susceptibility. The increased number of neutrophils may have been related to the increased liver damage indicated by the ALT levels in the NK-depleted mice. Our findings suggest that, in the absence of NK cells, neutrophils were unable to control the early multiplication of C. abortus in the liver, and that the continuous and non-effective recruitment of neutrophils, together with this bacterial multiplication, was the cause of pathological changes that may have been responsible for the increased mortality observed in NK-depleted mice. NK cells are recognized as an important component of innate resistance against a wide range of intracellular pathogens (Mohan et al., 1997; Vester et al., 1999; Su et al., 2001). They may act against C. abortus through two different mechanisms, namely (1) the direct lysis of infected cells in a perforindependent process, and (2) the production of proinflammatory cytokines, especially IFN-g. This cytokine is known to play a key role in the response to C. abortus infection (McCafferty et al., 1994; Del Rı´o et al., 2001), exerting a potent early chlamydiostatic effect and probably playing an immunoregulatory role in the subsequent specific immune response (Tseng and Rank, 1998). In the present study, the concentration of IFN-g in the serum was strikingly reduced in NK-depleted mice at day 4 p.i. Previous studies in our laboratory demonstrated that IFN-g production in response to C. abortus is a complex process that is mainly IL-12-dependent; in
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IL-12 deficient mice, however, IFN-g production, albeit much reduced, is still sufficient to afford some degree of protection (Del Rı´o et al., 2001). Furthermore, it seems that several leucocyte subpopulations play a role in its production; thus, Buendı´a et al. (2002b) reported that a previously established Th-2 response, induced by a nematode infection, reduced antigen-specific IFN-g production but not the early presence of IFN-g in serum. Moreover, the production of IFN-g, together with other pro-inflammatory cytokines, is partly regulated by B cells (Buendı´a et al., 2002a). The present study confirmed that NK cells were associated with the presence of IFN-g at day 4 p.i. and that the lack of this cytokine was associated with uncontrolled multiplication of C. abortus. This relationship between NK cells and early IFN-g production and the control of infection has been reported for other intracellular pathogens, such as Plasmodium and Listeria (Dunn and North, 1991; De Souza et al., 1997); on the other hand, in infection with Brucella (also an intracellular bacterium) NK cells showed no more than a limited role in the early control of infection (Fernandes et al., 1995). In our model, however, the production of IFN-g at 2 days p.i. was independent of the presence of NK cells. Macrophages may also represent a possible source of IFN-g in C. abortus infection, since they have recently been reported as early IFN-g producers in response to the related species Chlamydophila pneumoniae (Gigliotti-Rothfuchs et al., 2001). To test the role of neutrophils, mice were depleted of this subpopulation by injecting a mAb (RB6-8C5). This resulted in a mortality rate even higher than that of NK-depleted mice. Histopathological analysis of the neutrophil-depleted mice revealed the complete absence of inflammatory foci, although numerous chlamydial inclusions were observed in hepatocytes. Low numbers of NK cells were observed in the livers of these mice. The findings suggest that neutrophils are essential for the early recruitment of NK cells into the inflammatory foci, and the recruited cells are then able to control the multiplication of C. abortus. The recruitment of another important leucocyte cell subpopulation, CD8þ T cells, has previously been described in C. abortus infection (Montes de Oca et al., 2000a). In recent years, several studies (Cassatella, 1995; Gasperini et al., 1999) have demonstrated that neutrophils produce chemokines such as macrophage inflammatory protein 1a (MIP-1a) and MIP-1b and IFN-g-inducible protein10 (IP-10), all of which have been reported to be chemotactic and activators of NK cells (Maghazachi et al., 1994; Loetscher et al, 1996).
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In conclusion, this study demonstrated the importance of NK cells in the early control of C. abortus infection and the close and complex relationship between NK cells and another component of the innate immune response, the neutrophil. A deeper knowledge of the innate defence mechanisms against C. abortus is required. This might also assist in the development of C. abortus vaccines, since the cellular components of the innate response play a role in the selective recruitment of effector cells, and hence may favour an effective immune response.
Acknowledgments We thank R.L. Coffman for the generous gift of RB6-8C5 hybridoma cells. This work was supported in part by a grant from MCyT (AGL2001-0627). C.M. Martı´nez and N. Ortega were the recipients of predoctoral grants from Ministerio Ciencia y Tecnologı´a and Universidad de Murcia, respectively.
References Anderson, I. E. (1986). Comparison of the virulence in mice of some ovine isolates of Chlamydia psittaci. Veterinary Microbiology, 12, 213–220. Biron, C. A., Nguyen, K. B., Pien, G. C., Cousens, L. P. and Salazar-Mather, T. P. (1999). Natural killer cells in antiviral defence: function and regulation by innate cytokines. Annual Reviews in Immunology, 17, 189 –220. Buendı´a, A. J., Del Rio, L., Ortega, N., Sa´nchez, J., Gallego, M. C., Caro, M. R., Navarro, J. A., Cuello, F. and Salinas, J. (2002a). B-cell-deficient mice show an exacerbated inflammatory response in a model of Chlamydophila abortus infection. Infection and Immunity, 70, 6911 – 6918. Buendı´a, A., Fallon, P. G., Del Rio, L., Ortega, N., Caro, M. R., Gallego, M. C. and Salinas, J. (2002b). Previous infection with the nematode Nippostrongylus brasiliensis alters the immune specific response against Chlamydophila abortus infection. Microbial Pathogenesis, 33, 7 –15. Buendı´a, A. J., Montes de Oca, R., Navarro, J. A., Sa´nchez, J., Cuello, F. and Salinas, J. (1999a). Role of polymorphonuclear neutrophils in a murine model of Chlamydia psittaci-induced abortion. Infection and Immunity, 67, 2110 –2116. Buendı´a, A. J., Sa´nchez, J., Del Rio, L., Garce´s, B., Gallego, M. C., Caro, M. R., Bernabe´, A. and Salinas, J. (1999b). Differences in the immune response against ruminant chlamydial strains in a murine model. Veterinary Research, 30, 495– 507. Buendı´a, A. J., Sa´nchez, J., Martı´nez, M. C., Ca´mara, P., Navarro, J. A., Rodolakis, A. and Salinas, J. (1998). Kinetics of infection and effects on placental cell
populations in a murine model of Chlamydia psittaciinduced abortion. Infection and Immunity, 66, 2128 –2134. Buxton, D. (1986). Potential danger to pregnant women of Chlamydia psittaci from sheep. Veterinary Record, 118, 510–511. Buxton, D. and Henderson, D. (1999). Infectious abortion in sheep. In Practice, 21, 360 –368. Buzoni-Gatel, D., Bernard, F., Andersen, A. A. and Rodolakis, A. (1990). Protective effects of polyclonal and monoclonal antibodies against abortion in mice infected by Chlamydia psittaci. Vaccine, 8, 342–346. Buzoni-Gatel, D., Guilloteau, L., Bernard, F., Bernard, S., Chardes, T. and Rocca, A. (1992). Protection against Chlamydia psittaci in mice conferred by Lyt-2þ T cells. Immunology, 77, 284–288. Cassatella, M. A. (1995). The production of cytokines by polymorphonuclear neutrophils. Immunology Today, 16, 21 –26. Del Rı´o, L., Buendı´a, A. J., Sa´nchez, J., Gallego, M. C., Caro, M. R., Ortega, N., Seva, J., Pallares, F. J., Cuello, F. and Salinas, J. (2001). Endogenous IL-12 is not required for resolution of Chlamydophila abortus (Chlamydia psittaci serotype 1) infection in mice. Infection and Immunity, 69, 4808 –4815. Del Rı´o, L., Buendı´a, A. J., Sa´nchez, J., Garce´s, B., Caro, M. R., Gallego, M. C., Bernabe´, A. and Salinas, J. (2000). Chlamydophila abortus (Chlamydia psittaci serotype 1) clearance is associated with the early recruitment of neutrophils and CD8 þ T cells in a mouse model. Journal of Comparative Pathology, 123, 171–181. De Souza, J. B., Williamson, K. E., Otani, T. and Playfair, J. H. L. (1997). Early interferon gamma responses in lethal and non-lethal murine blood-stage malaria. Infection and Immunity, 65, 1593 –1598. Dokun, A. O., Chu, D. T., Yang, L., Bendelac, A. S. and Yokoyama, W. M. (2001). Analysis of in situ NK cell responses during viral infections. Journal of Immunology, 167, 5286 –5293. Dunn, P. L. and North, R. J. (1991). Early gamma interferon production by natural killers is important in defense against murine listeriosis. Infection and Immunity, 59, 2892 – 2900. Entrican, G. (2002). Immune regulation during pregnancy and host—pathogen interactions in infectious abortion. Journal of Comparative Pathology, 126, 79–94. Fernandes, D. M., Benson, R. and Baldwin, C. L. (1995). Lack of a role for natural killer cells in early control of Brucella abortus 2308 infection in mice. Infection and Immunity, 63, 4029 – 4033. Gasperini, S., Marchi, M., Calzetti, F., Laudanna, C., Vicentini, L., Olsen, H., Murphy, M., Liao, F., Farber, J. and Cassatella, M. A. (1999). Gene expression and production of the monokine induced by IFN-g (MIG), IFN-inducible T cell a chemoattractant (ITAC), and IFN-g-inducible protein-10 (IP-10) chemokines by human neutrophils. Journal of Immunology, 162, 4928 –4937.
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Gigliotti-Rothfuchs, A., Gigliotti, D., Palmblad, K., Andersson, U., Wigzell, H. and Rottenberg, M. E. (2001). IFN-ab-dependent, IFN-g secretion by bone marrow-derived macrophages controls an intracellular bacterial infection. Journal of Immunology, 167, 6453 –6461. Loetscher, M., Gerber, B., Loetscher, P., Jones, S. A., Piali, L., Clark-Lewis, I., Baggiolini, M. and Mose, B. (1996). Activation of NK cells by CC chemokines. Chemotaxis, Ca2þ mobilization, and enzyme release. Journal of Immunology, 156, 322– 327. Maghazachi, A. A., Al-Aoukaty, A. and Schall, T. J. (1994). C –C chemokines induce the chemotaxis of NK and IL-2-activated NK cells. Journal of Immunology, 153, 4969 –4977. McCafferty, M. C., Maley, S. W., Entrican, G. and Buxton, D. (1994). The importance of interferon-g in an early infection of Chlamydia psittaci in mice. Immunology, 81, 631 –636. Mohan, K., Moulin, P. and Stevenson, M. M. (1997). Natural killer cytokine production, not cytotoxicity, contributes to resistance against blood-stage Plasmodium chabaudi AS infection. Journal of Immunology, 159, 4990 –54998. Montes de Oca, R., Buendı´a, A. J., Del Rı´o, L., Sa´nchez, J., Salinas, J. and Navarro, J. A. (2000a). Polymorphonuclear neutrophils are necessary for the recruitment of CD8 þ T cells in the liver in a pregnant mouse model of Chlamydophila abortus (Chlamydia psittaci serotype 1) infection. Infection and Immunity, 68, 1746 –1751. Montes de Oca, R., Buendı´a, A. J., Sa´nchez, J., Del Rı´o, L., Seva, J., Navarro, J. A. and Salinas, J. (2000b). Limited role of polymorphonuclear neutrophils in a pregnant mouse model of secondary infection by
Chlamydophila abortus (Chlamydia psittaci serotype 1). Microbial Pathogenesis, 29, 319–327. Rodolakis, A., Salinas, J. and Papp, J. (1998). Recent advances in ovine chlamydial abortion. Veterinary Research, 29, 275 –288. Romani, L., Mencacci, A., Cenci, E., Del Sero, G., Bistoni, F. and Puccetti, P. (1997). An immunoregulatory role of neutrophils in CD4 þ T helper subset selection in mice with candidiasis. Journal of Immunology, 158, 2356 –2362. Salinas, J., Souriau, A., Cuello, F. and Rodolakis, A. (1995). Antigenic diversity of ruminant Chlamydia psittaci strains demonstrated by the indirect microimmunofluorescent test with monoclonal antibodies. Veterinary Microbiology, 43, 219 –226. Su, H. C., Nguyen, K. B., Salazar-Mather, T. P., Ruzek, M. C., Dalod, M. Y. and Biron, C. A. (2001). NK cell functions restrain T cell responses during viral infections. European Journal of Immunology, 31, 3048 –3055. Tepper, R. I., Coffman, R. L. and Leder, P. (1992). An eosinophil-dependent mechanism for the antitumor effect of interleukin-4. Science, 257, 548– 551. Tseng, C. K. and Rank, R. G. (1998). Role of NK cells in early host response to chlamydial genital infection. Infection and Immunity, 66, 5867 –5875. Vester, B., Mu¨ller, K., Solbach, W. and Laskay, T. (1999). Early gene expression of NK cell-activating chemokines in mice resistant to Leishmania major. Infection and Immunity, 67, 3155 –3159.
Received; January 6th; 2003 Accepted; July 1st; 2003
www.elsevier.com/locate/jcpa
Natural Killer (NK) Cells Play a Critical Role in the Early Innate Immune Response to Chlamydophila abortus Infection in Mice A. J. Buendı´a, C. M. Martı´nez, N. Ortega*, L. Del Rı´o†, M. R. Caro*, M. C. Gallego*, J. Sa´nchez, J. A. Navarro, F. Cuello* and J. Salinas* Departamento de Histologı´a y Anatomı´a Patolo´gica and *Sanidad Animal, Facultad de Veterinaria, Universidad de Murcia, Campus de Espinardo, Murcia, Spain, and †Department of Microbiology and Immunology, Cornell University College of Veterinary Medicine, Ithaca, NY 14853, USA
Summary Chlamydophila abortus, the aetiological agent of ovine enzootic abortion, induces a strong inflammatory reaction that leads to the T helper cell (Th1) specific immune response necessary for the clearance of infection. Because the role of natural killer (NK) cells during the first stages of this response has received little attention, this study focused on determining the function of these cells in a mouse model of infection. The location of NK cells in the liver and spleen of infected mice was examined immunohistochemically with an anti-Ly49G monoclonal antibody. The number of NK cells increased during the infection both in spleen and liver. In subsequent experiments, an anti-asialo GM1 polyclonal antibody was injected to deplete the NK cells. NK-depleted mice showed a substantial increase in their susceptibility to C. abortus infection, with high mortality rates and an increased burden of bacteria in the liver. Histopathological studies showed that inflammatory foci, composed mainly of neutrophils, were greater in size and number in depleted mice, while numerous chlamydial inclusions were associated with the foci. Serum concentrations of IFN-gamma, a key cytokine in the control of C. abortus infection, were substantially reduced in the NK-depleted mice. To establish the relationship between NK cells and other components of the innate immune response, neutrophils were depleted with the RB6-8C5 antibody. These cells were shown to be crucial in the recruitment of NK cells to the inflammatory foci. q 2003 Elsevier Ltd. All rights reserved. Keywords: abortion; bacterial infection; Chlamydophila abortus; immunity to C. abortus; NK (natural killer) cells; sheep
Introduction Chlamydophila abortus (formerly Chlamydia psittaci serotype 1), an obligate intracellular bacterium, is responsible for enzootic abortion in ewes and is the most commonly diagnosed cause of abortion in several European countries (Rodolakis et al., 1998; Buxton and Henderson, 1999). It is also capable of causing serious human infections (Buxton, 1986). Mouse models have been widely used to study the pathogenesis of C. abortus infections and the Correspondence to: J. Salinas. 0021–9975/$ - see front matter doi: 10.1016/S0021-9975(03)00069-0
immune response (Anderson, 1986; Buzoni-Gatel et al., 1990; Buendı´a et al., 1998; Del Rı´o et al., 2000). In experimental murine infections, the systemic spread of C. abortus is followed by the establishment of an effective immune response capable of eliminating the infection from every organ except the placenta, where the bacteria multiply, inducing abortion (Buendı´a et al., 1998). C. abortus infection is ultimately controlled by a specific helper T cell 1 (Th1) immune response which is, at least partly, interleukin (IL)-12-independent (Del Rı´o et al., 2001) and characterized by q 2003 Elsevier Ltd. All rights reserved.
NK Cells in the Immune Response to C. abortus
the early production of high concentrations of interferon (IFN)-g (McCafferty et al., 1994; Buendı´a et al., 1999b) and the presence of Tcells, particularly CD8þ T cells (Buzoni-Gatel et al., 1992; Del Rı´o et al., 2000). However, exacerbated production of cytokines in response to C. abortus infection can induce pathological changes (Del Rı´o et al., 2001), and abortion has been associated with the detrimental effect of inflammatory cytokines (IFN-g, tumour necrosis factor [TNF]-a) induced by the infection in the placenta (Entrican, 2002). In addition, Buendı´a et al. (1999a) demonstrated the important role of innate (non-specific) immunity, especially that associated with neutrophils, in the early stages of a primary infection. In recent years, it has been established that innate immunity not only acts as a first line of defence, but is also capable of leading to specific immunity through the secretion of different cytokines (Romani et al., 1997; Su et al., 2001). Buendı´a et al. (1999a) reported that neutrophil depletion resulted in increased mortality and early abortion in pregnant mice. Neutrophils also influence the recruitment of other leucocyte populations, especially CD8þ Tcells (Montes de Oca et al., 2000a); however, the importance of neutrophils during a secondary infection is limited (Montes de Oca et al., 2000b). While research into neutrophils has progressed, the role of another important component of the innate immunity, the natural killer (NK) cell, has been poorly studied in C. abortus infection. NK cells constitute an important source of IFN-g during bacterial and viral infections and have a cytotoxic function against infected cells (Biron et al., 1999). Both functions are necessary for the clearance of C. abortus infection and have been found to play a role in the early control of infection by the related species Chlamydia trachomatis (Tseng and Rank, 1998). Hence, to increase our knowledge of the immune mechanisms that contribute to the early control of C. abortus, the potential role of NK cells requires investigation. In this study, emphasis is placed not only on that role but also on the relationship between NK cells and other components of innate immunity, such as neutrophils.
Materials and Methods Mice Eight-week-old female C57BL/6J (H-2b) mice were purchased from Harlan UK Limited (Blackthorn, UK). They were free of common viral and bacterial pathogens according to the results of routine screening procedures performed by the suppliers.
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Experimental Design To evaluate the role of NK cells in the C. abortus infection in mice, three main experiments were carried out, each being repeated with the same numbers of animals to confirm reproducibility of the results. Experiment 1. The purpose was to investigate the distribution of NK cells in the liver and spleen in two groups of 10 mice, one group uninfected and the other group infected with C. abortus. Subgroups of five animals were killed for examination 2 and 4 days after inoculation. Experiment 2. The purpose was to investigate the effect of NK cell depletion on the course of C. abortus infection. In this experiment, the following features were examined: weight loss, survival time after infection, isolation of C. abortus from the liver, presence of IFN-g in serum, histopathology and immunohistochemistry of the liver, and concentrations of alanine transaminase in serum. Two groups of 18 infected mice were used, one group having been subjected to NK cell depletion. Sub-groups of five animals were killed for examination 2 and 4 days after infection. The remaining mice, which were used to assess survival time and weight loss, were killed at day 15 after infection unless they had already died. Experiment 3. The purpose was to investigate the effect of neutrophil depletion on C. abortus infection and on NK cells recruitment to inflammatory foci. In this experiment additional features examined were the same as those in experiment 2. Three groups of 16 infected mice were used, one group having been subjected to NK cell depletion, and a second group having been subjected to neutrophil depletion. Sub-groups of five mice were killed for examination 2 and 4 days after infection. The remaining mice, if still alive, were killed at day 15 after infection. Micro-organisms and Infection Mice were infected with the abortion-causing C. abortus strain AB7, isolated from ovine abortion (Salinas et al., 1995). The bacteria were propagated in the yolk sacs of developing chick embryos, and titrated by enumerating inclusion-forming units (IFUs) on McCoy cells, as described by Buendı´a et al. (1999a). Standardized aliquots were frozen at 280 8C until used. Mice were infected by intraperitoneal (i.p.) inoculation with 106 IFUs of C. abortus in 0.2 ml of phosphate-buffered saline (PBS), pH 7.2, 0.1 M.
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Distribution of NK Cells in the Liver and Spleen Uninfected or infected mice at days 2 and 4 after infection were killed by CO2 euthanasia. Liver and spleen samples from non-infected and infected mice were snap-frozen with 2-methylbutane cooled with liquid nitrogen for studying the distribution of NK cells in the tissue. Cryosections (5 mm) were immunolabelled with an avidin – biotin – peroxidase complex technique (Del Rı´o et al., 2000). Anti-Ly49G (clone 4D11) monoclonal antibody (mAb), recently described as the only antibody of value for the immunohistochemical detection of NK cells in frozen sections (Dokun et al., 2001), was purchased from BD Pharmingen (San Diego, CA, USA). A goat biotinylated anti-rat mouse-adsorbed polyclonal antibody (Caltag, Burlingame, CA, USA) was used as secondary antibody. The number of NK cells was determined in each of 20 fields (17 000 mm2/field) in sections of liver and spleen from each mouse. In-vivo Depletion of NK Cells NK cells were depleted at days 21, 1 and 3 (in relation to i.p. infection) by the intravenous injection of 2 mg of the rabbit anti-asialo GM1 polyclonal antibody in 100 ml (Wako Chemicals GmbH, Neuss, Germany). Non-depleted (control) mice were treated instead with normal rabbit serum (Sigma, Madrid, Spain). The efficacy of depletion was confirmed by flow cytometry of spleen cells, as described by Montes de Oca et al. (2000a), with an anti-NK1.1 mAb (clone PK-136; Caltag). It was found that the spleens of anti-asialo GM1-treated mice had less than 10% of the NK cells of the control mice. Isolation of C. abortus The course of infection was also evaluated by counting IFUs from the liver after isolation of C. abortus on McCoy cell monolayers, following the method previously described by Buendı´a et al. (1999a). One lobe of the liver was examined and the number of IFUs/g was calculated at days 2 and 4 post-infection (p.i.), the detection limit being 2.6 log IFUs per sample. Analysis of Serum for IFN-g Serum samples were taken by cardiac puncture at the time of euthanasia (day 2 or 4 p.i.). IFN-g concentrations were determined by the sandwich ELISA procedure. The capture antibody used was R4-6A2, and the biotinylated detection antibody
was XMG1.2, both purchased from BD PharMingen. Biotin-conjugated antibody was detected with a horseradish-peroxidase streptavidin (BD PharMingen) and a soluble substrate (ABTS; Sigma). The optical density was read at 405 nm. Histopathology and Immunohistochemistry The livers of infected mice were collected and fixed in formalin 10% in PBS, dehydrated and embedded in paraffin wax at 56 8C. Sections (5 mm) were cut, stained with haematoxylin and eosin (HE) and examined for histopathological changes. To demonstrate chlamydial antigen in paraffin wax sections, immunohistochemical labelling was carried out as previously described (Buendı´a et al., 1999b). Serum Transaminase Assay To measure the liver-associated enzyme alanine transaminase (ALT) in serum, a procedure based on a commercial kit (Sigma) was employed. Briefly, in a 96-well plate, 4 ml of serum were added to 20 ml of DL -alanine (0.2 mol/litre) and a-ketoglutaric acid (1.8 mmol/litre); the plate was shaken and the mixture incubated for 30 min at 37 8C. Then, 20 ml of 2,4-dinitrophenylhydrazine (DNP) were added and the mixture was incubated for a further 20 min at room temperature. Finally, the reaction was halted by the addition of 200 ml of 0.4N NaOH, and sample absorbances were measured at 492 nm after 5 min. In-vivo Depletion of Neutrophils The hybridoma producing the RB6-8C5 mAb (Tepper et al., 1992) was provided by Dr R.L. Coffman (DNAX Research Institute, Palo Alto, CA, USA). The rat IgG2b RB6-8C5 mAb was obtained from the ascitic fluid of pristane-primed homozygous nude mice (Harlan) inoculated with 107 hybridoma cells. The mAb was precipitated by saturation with ammonium sulphate, followed by dialysis against PBS pH 7.2 and filtration (0.22 mm). The mAb was purified by chromatography on a protein G column (Sigma) according to the manufacturer’s instructions. Protein concentration was estimated by a modification of the Lowry method (BCA-1 procedure; Sigma). Mice to be depleted of neutrophils received 1 mg of RB6-8C5 mAb intravenously 6 h before infection and on days 1 and 3 p.i. Infected but non-depleted (control) mice received rat IgG (Sigma) at the same time, by the same route, and at the same dosage. Depletion
NK Cells in the Immune Response to C. abortus
was assessed by flow cytometry analysis, as described by Montes de Oca et al. (2000a). Statistical Analysis The significance of differences between groups of mice for each time point investigated was determined by the Student’s t test. A probability value of P , 0:05 was considered significant.
Results
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anti-Ly49G mAb. In infected mice, numerous labelled NK cells were observed in blood vessels (Fig. 1a), and some such cells appeared in the inflammatory foci in the liver parenchyma (Fig. 1b) at day 4 p.i. In the spleen, some positive NK cells were observed in the red pulp of the uninfected mice, mainly in the marginal areas surrounding the lymphoid follicles (Fig. 1c). However, at day 4 p.i., they were outnumbered by NK cells in the marginal areas of the infected mice, in which NK cells were also observed within lymphoid follicles (Fig. 1d).
NK Cell Distribution in the Liver and Spleen of Infected and Non-infected Mice (Experiment 1)
Course of C. abortus Infection in NK Cell-depleted Mice (Experiment 2)
No NK cells were observed in the liver of the uninfected mice after immunolabelling with the
To determine the role of NK cells in the clearance of C. abortus infection, mice were treated with
Fig. 1a– d. Cryostat sections showing the distribution of NK cells. NK cells were detected in the liver of infected mice in (a) blood vessels, and (b) inflammatory foci. NK cells were also detected in (c) the splenic red pulp (RP) of uninfected mice, and (d) RP and lymphoid follicles (LF) of infected mice at day 4 p.i. Cryofixed sections were immunolabelled with the antiLy-49G monoclonal antibody (clone 4D11) and an avidin –biotin complex (ABC) technique. £ 300.
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anti-asialo GM1 antibody and subsequently infected with 106 IFUs. NK cell depletion induced striking mortality. Thus, 50% (8/16) of the depleted mice had succumbed to C. abortus infection by day 5 p.i (Fig. 2a). In contrast, no mortality was observed in the non-depleted control mice during the experiment, although signs of illness, such as ruffled fur, lethargy and huddling were observed from day 3 to day 8 p.i. Unlike the controls, NK-depleted mice showed little or no weight loss during the first 3 days p.i., but this difference had disappeared by day 4 p.i. (Fig. 2b). Isolation of C. abortus from the liver at days 2 and 4 p.i. revealed a significantly higher level of infection in the NK-depleted mice than in their non-depleted counterparts (Fig. 2c). A
characteristic feature of early C. abortus infection in C57BL/6 mice is the high concentration of IFN-g in serum, and in this study both NKdepleted and control mice showed similarly high concentrations at day 2 p.i. At day 4 p.i., however, NK-depleted mice, unlike the controls, showed only residual levels of serum IFN-g (Fig. 2d). Histopathology of C. abortus infection in the liver. At day 2 p.i., the liver of NK-depleted mice showed inflammatory foci composed mainly of neutrophils; both the number and size of the foci were greater than in the non-depleted controls. At day 4 p.i., the size of the foci had increased in both groups, but to a greater degree in the NK-depleted group (Fig. 3a and b). In the control mice these foci consisted of neutrophils and a substantial
Fig. 2a– d. Progression of the C. abortus infection in infected control mice (black symbols and columns) and NK-depleted mice (white symbols and columns). (a) Survival of infected control and NK-depleted mice after infection. Groups of eight mice were infected with 106 IFUs of C. abortus. Mice were monitored daily and mortality rates were calculated. (b) Effect of C. abortus infection on weight loss of infected control and NK-depleted mice. Groups of eight mice were infected with 106 IFUs of C. abortus and body weight changes were monitored daily. (c) IFU counts on homogenized liver tissue collected at 2 and 4 days p.i. The number of inclusions was assessed by indirect immunofluorescence. The results are the means and standard deviation for five mice. (d) Presence of IFN-g in the serum of infected control and NK-depleted mice at 2 and 4 days p.i. The results are the means and standard deviation for five mice. All the experiments were repeated with similar results. * Significant differences ðP , 0:05Þ between control and NK-depleted mice at the same day p.i.
NK Cells in the Immune Response to C. abortus
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Fig. 3a– d. Livers of control (non-depleted) and NK-depleted mice at day 4 p.i. Note the differences in the number and size of the inflammatory foci between (a) control mice and (b) NK-depleted mice. (c) Detail of a typical inflammatory focus in the liver of a control mouse, showing a mixed inflammatory infiltrate of mononuclear cells with some neutrophils. (d) Detail of a typical inflammatory focus in the liver of a NK-depleted mouse, formed mainly of neutrophils with a few mononuclear cells, and showing a large basophilic chlamydial inclusion (arrowhead). HE. £ 300 (a and b); £ 1200 (c and d).
number of mononuclear cells, but in the NKdepleted mice they consisted almost exclusively of neutrophils (Fig. 3c and d). Furthermore, in the NK-depleted mice but not in the controls, large C. abortus inclusions appeared next to the inflammatory foci (Fig. 3d). To assess whether the observed inclusions were a genuine product of C. abortus infection, sections of liver were immunolabelled with an anti-C. abortus lipopolysaccharide (LPS) mAb. The control mice showed labelled C. abortus antigen in sparse inflammatory foci (Fig. 4a), while in the NK-depleted mice the immunoreaction was observed both in the numerous large inflammatory foci and in the numerous inclusions within hepatocytes (Fig. 4b). The increased histopathological changes observed in the liver of NKdepleted mice was associated with the significantly ðP , 0:05Þ higher serum concentrations of the enzyme ALT (242 ^ 22 IU/ml) in NK-depleted
mice than in control mice (56 ^ 8 IU/ml) at day 4 p.i. The baseline concentrations of ALT in noninfected mice was 18 ^ 4 IU/ml. Effect of Neutrophil Depletion on NK Cell Recruitment to the Inflammatory Foci (Experiment 3) Neutrophils were depleted with the RB6-8C5 mAb to study their influence on the NK cell response to C. abortus infection. Neutrophil-depleted mice infected with C. abortus showed 91% mortality (11/12) at day 6 p.i., as compared with 58% (7/12) in NK-depleted mice and no mortality in infected but non-depleted mice (Fig. 5a). The degree of infection, as revealed by hepatic IFU counts, was similar in neutrophil-depleted and NKdepleted mice, but significantly lower in nondepleted mice (Fig. 5b). The main histopathological feature of the livers of neutrophil-depleted mice
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Fig. 4. Immunoreaction against C. abortus antigen in the liver of infected non-depleted (control) and NK-depleted mice at day 4 p.i. (a) Sparse antigen in an inflammatory focus of the liver of a control mouse. (b) Abundant antigen in the larger inflammatory foci, and positive immunoreaction in the chlamydial inclusions within hepatocytes (arrowheads) in the liver of a NK-depleted mouse. ABC. £ 600.
was the lack of inflammatory foci; however, immunohistochemical analysis with anti-C. abortus LPS mAb revealed numerous labelled inclusions in the liver parenchyma (data not shown). Immunolabelling showed that in neutrophil-depleted mice, recruitment of NK cells to the liver parenchyma was less than in infected but non-depleted mice (Fig. 5c). No NK cells were observed in the liver of NK-depleted mice.
Discussion The normal distribution of NK cells in the spleen and the liver, established previously by Dokun et al.
(2001), was confirmed immunohistochemically at the outset of this study, with mAb 4D11 (an antibody that detects the Ly49G antigen expressed by about 50% of murine NK cells). NK cells were located in the red pulp of the spleen (adjacent to splenic lymphoid follicles), but not in the liver parenchyma. C. abortus infection altered this distribution: thus, NK cells increased in number in the red pulp, and were also observed infiltrating the lymphoid follicles. In the liver, a substantial degree of NK cell recruitment was observed in the inflammatory foci associated with C. abortus infection. These changes in NK cell distribution strongly suggest that NK cells play a role in the early
Fig. 5a– c. Progression of the C. abortus infection in the non-depleted (control) mice (black symbols and columns), NK-depleted mice (white symbols and columns) and neutrophil-depleted mice (grey symbols and columns). (a) Survival after infection. Groups of six mice were infected with 106 IFUs of C.abortus. Mice were monitored daily, and mortality rates were calculated. (b) IFU counts on homogenized liver tissues collected at 2 and 4 days p.i. The number of inclusions was assessed by indirect immunofluorescence. The results are the means and standard deviation for five mice. (c) Number of NK cells in the liver parenchyma of control and neutrophil-depleted mice; for each mouse, the number of cells labelled with anti-Ly49G mAb was counted in 20 areas of 17 000 mm2. The graph shows that the depletion of neutrophils by treatment with the anti-granulocyte RB6-8C5 mAb induced a significant decrease in the number of NK cells in the liver during C. abortus infection. The results are the means and standard deviation for five mice. All the experiments were repeated with similar results. * Significant differences ðP , 0:05Þ between control and depleted mice at the same day p.i.
NK Cells in the Immune Response to C. abortus
defensive response to C. abortus. A similar conclusion was reached by Tseng and Rank (1998) in respect of the related species, C. trachomatis; these authors, however, did not describe the location of the NK cells, reported in our investigation. To study their functional role, NK cells were depleted with the anti-asialo GM1 polyclonal antibody. This resulted in increased mortality, a higher C. abortus burden in the liver, and changes in morbidity. Histopathological examination of the liver of depleted mice revealed large inflammatory foci composed mainly of neutrophils, with some mononuclear cells; in addition, hepatocytes with chlamydial inclusions were observed, some of which surrounded the inflammatory foci. The fact that the large numbers of neutrophils present in the liver were unable to prevent the active multiplication of C. abortus in hepatocytes is especially significant, since neutrophils have previously been defined as a crucial first line of defence against C. abortus infection (Buendı´a et al., 1999a); moreover, Del Rı´o et al. (2000) reported that a vigorous neutrophilic response was associated with early clearance of infection in two mouse strains showing different degrees of susceptibility. The increased number of neutrophils may have been related to the increased liver damage indicated by the ALT levels in the NK-depleted mice. Our findings suggest that, in the absence of NK cells, neutrophils were unable to control the early multiplication of C. abortus in the liver, and that the continuous and non-effective recruitment of neutrophils, together with this bacterial multiplication, was the cause of pathological changes that may have been responsible for the increased mortality observed in NK-depleted mice. NK cells are recognized as an important component of innate resistance against a wide range of intracellular pathogens (Mohan et al., 1997; Vester et al., 1999; Su et al., 2001). They may act against C. abortus through two different mechanisms, namely (1) the direct lysis of infected cells in a perforindependent process, and (2) the production of proinflammatory cytokines, especially IFN-g. This cytokine is known to play a key role in the response to C. abortus infection (McCafferty et al., 1994; Del Rı´o et al., 2001), exerting a potent early chlamydiostatic effect and probably playing an immunoregulatory role in the subsequent specific immune response (Tseng and Rank, 1998). In the present study, the concentration of IFN-g in the serum was strikingly reduced in NK-depleted mice at day 4 p.i. Previous studies in our laboratory demonstrated that IFN-g production in response to C. abortus is a complex process that is mainly IL-12-dependent; in
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IL-12 deficient mice, however, IFN-g production, albeit much reduced, is still sufficient to afford some degree of protection (Del Rı´o et al., 2001). Furthermore, it seems that several leucocyte subpopulations play a role in its production; thus, Buendı´a et al. (2002b) reported that a previously established Th-2 response, induced by a nematode infection, reduced antigen-specific IFN-g production but not the early presence of IFN-g in serum. Moreover, the production of IFN-g, together with other pro-inflammatory cytokines, is partly regulated by B cells (Buendı´a et al., 2002a). The present study confirmed that NK cells were associated with the presence of IFN-g at day 4 p.i. and that the lack of this cytokine was associated with uncontrolled multiplication of C. abortus. This relationship between NK cells and early IFN-g production and the control of infection has been reported for other intracellular pathogens, such as Plasmodium and Listeria (Dunn and North, 1991; De Souza et al., 1997); on the other hand, in infection with Brucella (also an intracellular bacterium) NK cells showed no more than a limited role in the early control of infection (Fernandes et al., 1995). In our model, however, the production of IFN-g at 2 days p.i. was independent of the presence of NK cells. Macrophages may also represent a possible source of IFN-g in C. abortus infection, since they have recently been reported as early IFN-g producers in response to the related species Chlamydophila pneumoniae (Gigliotti-Rothfuchs et al., 2001). To test the role of neutrophils, mice were depleted of this subpopulation by injecting a mAb (RB6-8C5). This resulted in a mortality rate even higher than that of NK-depleted mice. Histopathological analysis of the neutrophil-depleted mice revealed the complete absence of inflammatory foci, although numerous chlamydial inclusions were observed in hepatocytes. Low numbers of NK cells were observed in the livers of these mice. The findings suggest that neutrophils are essential for the early recruitment of NK cells into the inflammatory foci, and the recruited cells are then able to control the multiplication of C. abortus. The recruitment of another important leucocyte cell subpopulation, CD8þ T cells, has previously been described in C. abortus infection (Montes de Oca et al., 2000a). In recent years, several studies (Cassatella, 1995; Gasperini et al., 1999) have demonstrated that neutrophils produce chemokines such as macrophage inflammatory protein 1a (MIP-1a) and MIP-1b and IFN-g-inducible protein10 (IP-10), all of which have been reported to be chemotactic and activators of NK cells (Maghazachi et al., 1994; Loetscher et al, 1996).
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A. J. Buendı´a et al.
In conclusion, this study demonstrated the importance of NK cells in the early control of C. abortus infection and the close and complex relationship between NK cells and another component of the innate immune response, the neutrophil. A deeper knowledge of the innate defence mechanisms against C. abortus is required. This might also assist in the development of C. abortus vaccines, since the cellular components of the innate response play a role in the selective recruitment of effector cells, and hence may favour an effective immune response.
Acknowledgments We thank R.L. Coffman for the generous gift of RB6-8C5 hybridoma cells. This work was supported in part by a grant from MCyT (AGL2001-0627). C.M. Martı´nez and N. Ortega were the recipients of predoctoral grants from Ministerio Ciencia y Tecnologı´a and Universidad de Murcia, respectively.
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Received; January 6th; 2003 Accepted; July 1st; 2003