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Infection and Immunity, March 2006, p. 1837-1845, Vol. 74, No. 3
0019-9567/06/$08.00+0     doi:10.1128/IAI.74.3.1837-1845.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

T Cells Regulate the Early Inflammatory Response to Bordetella pertussis Infection in the Murine Respiratory Tract

O. Zachariadis,^1,2 J. P. Cassidy,^1* J. Brady,¹ and B. P. Mahon²

Department of Veterinary Pathology, Faculty of Veterinary Medicine, University College Dublin, Belfield, Dublin 4, Ireland,¹ Mucosal Immunology Laboratory, Institute of Immunology, NUI Maynooth, County Kildare, Ireland²

Received 30 August 2005/ Returned for modification 21 October 2005/ Accepted 2 December 2005

Top Abstract Introduction Materials and Methods Results Discussion References

 
The role of T cells in the regulation of pulmonary inflammation following Bordetella pertussis infection was investigated. Using a well-characterized murine aerosol challenge model, inflammatory events in mice with targeted disruption of the T-cell receptor -chain gene ( TCR^–/– mice) were compared with those in wild-type animals. Early following challenge with B. pertussis, TCR^–/– mice exhibited greater pulmonary inflammation, as measured by intra-alveolar albumin leakage and lesion histomorphometry, yet had lower contemporaneous bacterial lung loads. The larger numbers of neutrophils and macrophages and the greater concentration of the neutrophil marker myeloperoxidase in bronchoalveolar lavage fluid from TCR^–/– mice at this time suggested that differences in lung injury were mediated through increased leukocyte trafficking into infected alveoli. Furthermore, flow cytometric analysis found the pattern of recruitment of natural killer (NK) and NK receptor⁺ T cells into airspaces differed between the two mouse types over the same time period. Taken together, these findings suggest a regulatory influence for T cells over the early pulmonary inflammatory response to bacterial infection. The absence of T cells also influenced the subsequent adaptive immune response to specific bacterial components, as evidenced by a shift from a Th1 to a Th2 type response against the B. pertussis virulence factor filamentous hemagglutinin in TCR^–/– mice. The findings are relevant to the study of conditions such as neonatal B. pertussis infection and acute respiratory distress syndrome where T cell dysfunction has been implicated in the inflammatory process.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Early interactions between host defense cells and bacterial pathogens are critical in determining the nature and extent of inflammation and the subsequent resolution of infection. Previous studies have suggested that T cells have an important role in regulating the initial inflammatory response to microbial invasion and, in so doing, prevent excessive tissue injury (15, 24, 12, 43, 44, 47). Experimental evidence suggests T cells execute their regulatory function through influencing the movement and function of key inflammatory effector cells such as neutrophils (15, 11), macrophages (12, 47), and NK cells (27, 49). T cells expressing the receptor develop early in ontogeny and are disproportionately abundant within epithelial surfaces, including those in the lung (18). Their intraepithelial distribution and capacity to recognize nonprotein antigens, sometimes in a non-major histocompatibility complex-restricted fashion, has lead to their consideration as part of the first line of the host defense. Furthermore, the common overlap of pathogen-encoded cell antigens with molecules expressed by stressed host cells has led to the view that T-cell responses are driven more by nonspecific markers of cell injury than by the overt presence of microbial or other foreign antigens (18).

The early activation of T lymphocytes can lead to gamma interferon (IFN-) production that is instrumental in the upregulation of both macrophage and natural killer (NK) cell functions central to early antibacterial protection prior to the ß T-cell response (18). IFN- also influences the downstream acquisition of a Th1 phenotype by ß T cells (25, 45). T cells thus bridge the innate and acquired immune response by providing initial protection of epithelia and tissues from invasion and injury in instances where ß T cells are not yet operational and then down-regulating the antigen-specific adaptive immune response after the danger has passed to minimize potential immune-mediated injury (43). Experimental data suggest that this T-cell population exerts its influence through chemokine and cytokine production that in turn regulates the movement and activation of neutrophils, macrophages, NK cells, and NK receptor-positive (NKR⁺) T cells (15, 24, 12, 43, 47). NK cells are part of the innate immune response, effecting cytolysis and releasing inflammatory cytokines (48, 35). NKR⁺ T cells, typically defined as NK1.1⁺ ßTCR⁺ cells in mice, are a novel lymphoid lineage distinct from NK cells (41). On stimulation, this T lymphocyte subtype can rapidly secrete large amounts of both Th1 (IFN- and tumor necrosis factor beta) (8) and Th2 (interleukin-4 [IL-4] and IL-5) (52) cytokines in a nonspecific manner prior to the development of the acquired immune response and are a proposed link between the innate and adaptive immune responses, in a manner analogous to T cells (2, 16). Roles for NK cells and NKR⁺ T cells in airway inflammation have recently been described (21, 23, 50). However, the distinction between and NKR⁺ T cells is often indistinct, and their relative functions during immune responses are unclear. Consistent with a regulatory role for T cells, in vivo and in vitro production of the proinflammatory cytokines IL-6, IL-12, and IFN- are increased in TCR^–/– mice during the innate, early-phase host response to Listeria monocytogenes infection (43), while other studies suggest T cells influence macrophage and neutrophil trafficking and activation through production of CCL3/MIP-1 (47) and IL-10 (19), respectively.

T cells can modulate either the severity of disease or the protective immune response in murine models of tuberculosis, malaria, and herpes simplex virus type 1 encephalitis (45). In humans, large expansion of the T-cell population during infection with L. monocytogenes, Mycobacterium tuberculosis, Brucella melitensis, and Ehrlichia chaffeensis suggest their importance in the host response (45). Of particular interest in the context of pulmonary injury is a recent study implicating temporary functional suppression of T cells in the uncontrolled inflammation associated with acute respiratory distress syndrome (ARDS) triggered by sepsis (19).

Bordetella pertussis is a gram-negative bacterium that causes whooping cough, a respiratory disease that results in infant morbidity and mortality. The murine B. pertussis respiratory challenge model is an established, well-characterized experimental system particularly useful in the study of bacterium-mediated pulmonary inflammation (22, 32, 33). Although little is known about the role of T cells during B. pertussis infection, a role for T cells in the response of infected children has been suggested by the migration of circulating T cells to the airways (3), and murine studies have reported airway T cells following exposure to B. pertussis (30).

We investigated the role of T cells in B. pertussis infection by comparing the degree of pulmonary inflammation following infection of mice with targeted disruption of the T-cell receptor -chain gene ( TCR^–/–) and conventional C57BL/6 (wild type [WT]) mice. Measures of inflammation included bronchoalveolar lavage fluid (BALF) albumin and myeloperoxidase (MPO) levels, leukocyte cytology, and flow cytometric analysis in addition to lung lesion morphometry. TCR^–/– mice exhibited greater degrees of pulmonary inflammation mediated through increased neutrophil exudation. This increased response accounted for the apparent paradox of finding smaller bacterial loads early postinfection in the lungs of mice lacking T cells. TCR^–/– mice also had a significantly greater Th2 response to the B. pertussis virulence factor filamentous hemagglutinin (FHA), not evident in controls, suggesting that these cells influence the quality of adaptive immunity. Taken together, these findings suggest T cells play a nonredundant role in the initial pulmonary inflammatory response to bacterial infection and also influence the induction of cell-mediated immunity to specific bacterial antigens.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Mice. Male and female C57BL/6 T-cell receptor knockout ( TCR^–/–) (Jackson Laboratory, Bar Harbor, Maine) and C57BL/6 WT (Harlan Co., United Kingdom) mice were at least 8 weeks old at the initiation of experiments and were maintained in a specific-pathogen-free biosafety facility with free access to water and standard mouse rations. Animals were maintained under the guidelines of the Irish Department of Health and the research ethics committee of the National University of Ireland, Maynooth.

Bacterial inoculum and aerosol infection. Bordetella pertussis (strain W28) was grown under agitation conditions at 36°C in Stainer-Scholte liquid medium. Bacteria from a 48-h culture were resuspended at a concentration of approximately 2 x 10¹⁰ CFU per ml in physiological saline containing 1% casein. The challenge inoculum was administered as an aerosol over a period of 12 min with a nebulizer directed into an aerosol chamber containing groups of 20 to 24 mice. All experiments were performed at least twice.

Enumeration of pulmonary bacterial loads. Infected mice were euthanized at fixed time points after experimental challenge (four mice per time point), and the lungs were aseptically removed and homogenized in 1 ml of sterile physiological saline with 1% casein on ice. One hundred microliters of undiluted homogenate or of serially diluted homogenate from individual lungs was spotted in triplicate onto each of three Bordet-Gengou agar plates, and the number of CFU was estimated after 5 days of incubation. Results are reported as the mean number of B. pertussis CFU for individual lungs from the four mice sacrificed at each time point. Results are plotted on a logarithmic scale, with a limit of detection of approximately 0.3 log₁₀ CFU per lung.

Bronchoalveolar lavage fluid collection and analysis. To assess the nature and severity of the pulmonary inflammatory response to aerosol B. pertussis challenge, BALF samples were obtained from euthanized mice by repeated administration and aspiration of 0.5-ml volumes of phosphate-buffered saline (PBS) via cannulation of the trachea. A total of 5 ml of sample was obtained from each mouse group at each time point through pooling of samples from six animals. Following aseptic erythrolysis by osmotic disruption, cells from the lavage fluid were recovered by centrifugation at 300 x g for 7 min and resuspended in PBS. Total leukocytes were counted, and cytospin preparations were stained with Giemsa to determine the differential cell count at each time point. The means and standard errors (SE) of the means of the absolute numbers of macrophages, neutrophils, and lymphocytes were determined following counting of 600 cells. The cell-free supernatant was analyzed for albumin and MPO content. Albumin levels were measured using a murine albumin enzyme-linked immunosorbent assay (ELISA) kit (mouse albumin ELISA kit; Universal Biologicals Ltd., Cambridge, United Kingdom). The detection limits were between 7.8 and 500 ng/ml. To measure MPO activity, supernatant samples were incubated in a 50 mM potassium phosphate buffer containing H₂O₂ (1.5 mol/liter) in the presence of o-dianisidine dihydrochloride (167 µg/ml; Sigma Aldrich). The enzymatic activity was determined spectrophotometrically by measuring the change of absorbance at 460 nm over 3 min using a 96-well plate reader (46). For flow cytometry, BALF samples (10⁵ cells) were incubated in 2% normal rabbit serum at 0°C for 20 min and normal mouse serum at 0°C for 15 min. Samples were then incubated in the dark on ice for 20 min with 100 µl of appropriately diluted Armenian hamster anti-mouse TCR antibody conjugated to fluorescein isothiocyanate, Armenian hamster anti-mouse NK1.1 antibody conjugated to phycoerythrin, or Armenian hamster anti-mouse CD3 antibody conjugated with peridinin chlorophyll protein (all sourced from PharMingen, San Diego, CA). Appropriate isotype control antibodies were employed in parallel samples. After labeling, cells were washed twice with PBS, and the relative fluorescence intensities were determined on a 4-decade log scale by flow cytometric analysis using FACSCalibur instrumentation (Becton Dickinson, Mountain View, CA). Data were analyzed using CellQuest software (Becton Dickinson).

Histopathology and lesion morphometry. To assess pulmonary inflammation, entire mouse lungs were fixed by immersion in 10% formalin, and following paraffin embedding, 3-µm longitudinal sections of each of the five lobes were cut, stained with hematoxylin and eosin, and examined (8 mice per group per time point). The areas of inflammatory lesions in each lung section were measured using computer-aided histomorphometry (Palmrobo software, version 1.2.3; Palm microlaser technologies AG Ltd., Bernried, Germany), and a mean lesion area was calculated for each mouse type at each time point. Assessments of histopathology and morphometry were performed without prior knowledge of sample identity.

Measurement of B. pertussis-specific immune responses. The concentration of B. pertussis-specific immunoglobulin G1 (IgG1), IgG2a, IgG2b, and IgG3 in collected sera from WT and TCR^–/– mice 43 days postchallenge were measured by ELISA as previously described (28). Briefly, plates were coated with heat-inactivated B. pertussis or with the B. pertussis virulence factor FHA (1 µg/ml). After blocking and the addition of serum samples, alkaline phosphatase-labeled rat anti-mouse IgG1, IgG2a, IgI2b, and IgG3 (PharMingen, San Diego, CA) were used to detect B. pertussis-specific antibody subclasses. Antibody levels were expressed as the mean endpoint titers calculated by regression of the straight part of the curve of optical density versus serum dilution to a cutoff of two standard deviations above background control values.

Spleen cells from infected WT and TCR^–/– mice 43 days after challenge (n = 4 per group per experiment, repeated at least twice) were resuspended at 2 x 10⁶ per ml in RPMI 1640 medium and tested for in vitro proliferation to B. pertussis antigens as previously described (28). Briefly, cells were cultured for 4 days with heat-inactivated B. pertussis (10⁴ CFU per ml equivalent), inactivated pertussis toxin (5 µg/ml), FHA (5 µg/ml), pertactin (5 µg/ml), concanavalin A (5 µg/ml, positive control), or medium alone (negative control). For the final 4 h of culture, [³H]thymidine was added and the results expressed as the mean cpm of thymidine incorporation (±SE). Spleen cells from infected WT and TCR^–/– mice 43 days after challenge were also cultured with B. pertussis antigens as described for the proliferation assay. At the 72-h time point, culture supernatants were assessed for the presence of IL-4, IL-5 and IFN- by ELISA (PharMingen) and calculated by comparison with known cytokine standards as previously described (26). Although the kinetics of cytokine production varies, the 72-h time point has previously been shown acceptable for detection of these cytokines (1).

Statistical analysis. Statistical analysis was performed with GraphPad PRISM 3 software. Analysis of variance (one and two way) with Bonferroni's multiple-comparison test was used assess the significance of differences between the two groups. P values of <0.05 were considered significant. Student's t test was used to determine significance between groups in antibody subclass and cytokine assays. P values of <0.05 were considered significant.

Top Abstract Introduction Materials and Methods Results Discussion References

 
T cells reduce the extent of early-stage Bordetella pertussis-mediated pulmonary inflammation through effects on neutrophil trafficking. To determine the potential regulatory role of T cells in the inflammatory response to aerosol Bordetella pertussis challenge, the degree of pulmonary inflammation was compared between TCR^–/– and WT mice at early time points postinfection. Quantitation of inflammation was achieved through measurement of albumin leakage into BALF and through computer-aided lesion histomorphometry. BALF albumin levels were higher (P < 0.01) and the mean area of foci of lung inflammation larger (P < 0.05) in TCR^–/– than in WT mice at 3 and 6 days postinfection, respectively (Fig. 1 and 2). In both mouse phenotypes at 3, 6, and 10 days postchallenge, pulmonary inflammation was characterized by multifocal aggregates of admixed intact and degenerate neutrophils and macrophages, frequently adjacent to small bronchioles. At day 6, the larger areas of inflammation in TCR^–/– mice contained greater numbers of degenerate neutrophils (Fig. 3). From 10 days, increased numbers of macrophages and lymphocytes and reduced numbers of neutrophils were features of the inflammatory response with occasional marginally located fibroblasts noted in both strains of mice.

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FIG. 1. Lung vascular permeability in TCR–/– (open bar) compared to WT (solid bar) mice following aerosol B. pertussis challenge. Mouse albumin levels were measured in BALF as an index of vascular leakage. Results are means ± SE from 6 mice per group. *, P < 0.01 (compared to the corresponding value for WT mice).

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FIG. 2. Pulmonary inflammation was more extensive in TCR–/– (open bar) compared to WT (solid bar) mice at day 6 following aerosol B. pertussis challenge. Computer-aided histomorphometry was used to quantify the mean surface area of inflammatory lesions in both mouse types. Results are means ± SE from 8 mice per group. *, P < 0.05 (compared to the corresponding value for WT mice).

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FIG. 3. Photomicrographs of lung sections illustrating quantitative differences in the inflammation in TCR–/– and WT mice at day 6 following B. pertussis infection. Mice were aerosol challenged, giving an initial pulmonary load of approximately 4.5 x 1010 CFU per lung. Focal infiltrates of admixed intact and degenerate neutrophils, macrophages, and lymphocytes efface alveolar architecture in WT (A and C) and TCR–/– (B and D) mice. Staining was done with hematoxylin and eosin. Magnification, x200 (A and B); x400 (C and D).

Further investigation of the inflammatory effector mechanisms influenced by T cells was carried out through quantitation of MPO levels and of leukocyte numbers in BALF. Increased BALF MPO at 6 days in the TCR^–/– compared to the WT mice (P < 0.05) (Fig. 4) and the detection of greater numbers of neutrophils in BALF at both 3 and 6 days postinfection (P < 0.01, respectively) (Table 1) corresponded to greater neutrophil exudation into the alveolar spaces in the absence of T cells. Measurements of macrophage numbers in BALF also indicated increased extravasation of these leukocytes in TCR^–/– compared to WT mice at 6 days postchallenge (P < 0.01). Taken together, these findings demonstrate that, in the absence of T cells, neutrophil and macrophage exudation into airspaces is enhanced, resulting in more extensive pulmonary inflammation.

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FIG. 4. Neutrophil accumulation in airspaces of TCR–/– (open bar) compared to WT (solid bar) mice following aerosol B. pertussis challenge. MPO levels were measured in BALF as an index of neutrophil influx into airspaces. Results are means ± SE from 6 mice per group. *, P < 0.05 (compared to the corresponding value for WT mice); OD, optical density.

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TABLE 1. Cytological examination of leukocytes present in BALF from B. pertussis-infected TCR–/– mice or controlsa

TCR^–/– mice have reduced pulmonary Bordetella pertussis load in the early phase of infection. One explanation for the increased inflammation in the TCR^–/– mice could have been increased bacterial carriage in these animals at early time points postchallenge. The relationship between the inflammatory response and the pulmonary bacterial load was assessed by comparing patterns of clearance of B. pertussis from the lungs of the TCR^–/– and WT mice. Mice lacking T cells with enhanced early inflammatory injury had significantly reduced lung bacterial counts at 3 days (P < 0.01) and 6 days (P < 0.01) postinfection compared to WT controls (Fig. 5). Thus, the increased inflammatory damage seen in this model cannot be accounted for by increased bacterial carriage. The fact that bacterial clearance rates were similar in both mouse types from day 10 suggested that T cells have an influence on the inflammatory response involved in bacterial killing in only the early phase following infection.

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FIG. 5. The early pulmonary bacterial load is reduced in TCR–/– mice following aerosol challenge with B. pertussis. TCR–/– and WT mice were challenged by aerosol inoculation with B. pertussis. The pulmonary bacterial load was monitored at intervals after challenge by performing viable counts on lungs. Results are mean ± SE CFU counts performed on individual lungs in triplicate for 4 mice per group at each time point. *, P < 0.01 (compared to the corresponding values for WT mice).

T cells participate in the early host response to aerogenous B. pertussis challenge and influence the pulmonary recruitment of NK cells and of NKR⁺ T and CD3⁺ T cells. We examined the kinetics of T cell, NK cell, NKR⁺ CD3⁺ T cell, and NKR^– CD3⁺ T cell recruitment into the pulmonary airspaces in response to B. pertussis infection through flow cytometric analysis of BALF. As expected, T cells could not be detected in TCR^–/– mice, whereas infection of WT mice resulted in an influx of T cells that persisted over 3, 6, and 10 days postinfection (Table 2), indicating that this cell type responds rapidly to B. pertussis infection. Recruitment of NK cells, of NKR⁺ CD3⁺ T cells, and of NKR^– CD3⁺ T cells into airspaces differed between the two mouse groups over this time period. Increased numbers of NK cells were noted in BALF from TCR^–/– mice compared to WT mice at 3 (P < 0.05), 6 (P < 0.01), and 10 (P < 0.01) days postinfection. Increased numbers of NKR^– CD3⁺ T cells were also detected in the TCR^–/– mice at day 3 (P < 0.01). Numbers of NKR⁺ CD3⁺ T cells were reduced at day 6 (P < 0.01) and 10 (P < 0.01) in lavage samples from TCR^–/– mice. Taken together, these differences in responses in the two mouse types indicate that T cells have a critical role in the coordination of the early immune response in the airways.

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TABLE 2. Flow cytometric analysis of cells present in BALF from B. pertussis-infected TCR–/– mice or controlsa

T cells influence the quality of the adaptive immune response to B. pertussis. In addition to the coordination of the early host response to airway pathogens, it was likely that T cells would also influence the development of the subsequent adaptive immune response. The influence of T cells on both the humoral and cellular components of the adaptive response to B. pertussis challenge was therefore assessed by the measurement of B. pertussis- or FHA-specific antibody subclasses in mouse sera or B. pertussis induction of T-cell responses in spleens obtained 43 days postinfection, at which point bacteria had been cleared from the lungs. While the overall response in both TCR^–/– mice and WT controls was Th1 in character, dominated by IgG2a (Fig. 6A) and IFN- production (Fig. 7D), as previously reported (32), an important variation was observed. TCR^–/– mice had significantly increased levels of FHA-specific IgG1 and reduced IgG2a (Fig. 6B) compared to WT mice (P < 0.01) and had a significantly greater IL-4 response to FHA (P < 0.01) than WT animals (Fig. 7B). This finding suggests that T cells play a role in directing the quality of adaptive immunity and influence the induction of cell-mediated immunity to specific antigens.

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FIG. 6. Antibody responses in B. pertussis-infected WT or TCR–/– mice against inactivated whole bacteria (A) or the B. pertussis virulence factor FHA (B). Increased IgG1 and reduced IgG2a was noted in TCR–/– compared to WT mice. Subclasses of B. pertussis- or FHA-specific IgG were measured from serum sampled 43 days after aerosol challenge of TCR–/– or WT mice. IgG1 (open bar), IgG2a (solid bar), IgG2b (vertical stripes), and IgG3 (horizontal stripes) were measured by ELISA. Results are means ± SE of ELISA titers from 4 mice per group measured in quadruplicate. *, P < 0.01 (comparing the IgG1 and IgG2a responses).

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FIG. 7. Enhanced IL-4 production by splenocytes from TCR–/– mice in response to the B. pertussis virulence factor FHA, compared to WT mice. Proliferation (A), IL-4 (B), IL-5 (C), and IFN- (D) responses to either medium control (open bar), heat-inactivated B. pertussis (104 CFU per ml equivalent) (horizontal stripes), inactivated pertussis toxin (diagonal stripes), FHA (vertical stripes), pertactin (hatching), or concanavalin A positive control (solid bar) at 5 µg/ml are shown. Results are the means ± SE for 4 mice per group, assayed individually in triplicate. *, P < 0.01 (compared to the corresponding value for WT mice); Conc, concentration.

Top Abstract Introduction Materials and Methods Results Discussion References

 
This study examined the role of T cells in the early host response to B. pertussis, an important respiratory pathogen. We show that in the first days of infection, prior to the development of adaptive immunity, T cells play a nonredundant role in influencing pulmonary inflammation. Specifically, in their absence, the influx of neutrophils, macrophages, NK cells, and NKR⁺ T cells into airways is altered and pulmonary inflammation, as measured by histomorphometry, BALF albumin and MPO levels, is increased. Intriguingly, these early events also have longer term influences on the quality of adaptive immunity in that the pattern of cytokine and antibody subclass production was altered to the bacterial virulence factor FHA, suggesting that the early encounter between T cells and pathogen shapes subsequent immunity.

The apparent paradox of finding decreased pulmonary bacterial loads in mice missing T cells, a key component of the host first line of defense is also accounted for by the increased early inflammatory response in TCR^–/– animals, but as our lung histomorphometry and BALF albumin data clearly illustrate, this reduction in bacterial load is at the expense of increased pulmonary injury. It is important to consider that the small BALF albumin increase in TCR^–/– mice at day 3, although statistically significant, may not have biological significance if interpreted in isolation. However, as part of a range of inflammatory parameters including histopathology, fluorescence-activated cell sorting, BALF cytology, and MPO measurements, the BALF albumin increase contributes to the evidence pointing to increased inflammation.

BALF cytology and MPO measurement show that enhanced neutrophil trafficking into and activation within airspaces accounts for the early, accentuated inflammatory response in TCR^–/– mice. The larger amounts of reactive oxygen metabolites and lysosomal enzymes that result from increased neutrophil migration and activation are likely to account for the increased parenchymal injury seen in the TCR^–/– animals. The absence of T cells also results in enhanced macrophage extravasation to the interstitium and alveoli. This event could have further contributed to pulmonary injury, given that T cells can prevent macrophage-mediated tissue damage by lysing activated macrophages (12). Alternatively, this increased macrophage influx could reflect a compensatory response to remove the increased numbers of neutrophils and debris in the airspaces of TCR^–/– mice. Taken together, the data support evidence from previous studies that T cells play a nonredundant, integrated role in early immune interactions. In their absence, potentially harmful inflammatory responses mediated by neutrophils (11, 15, 34, 43, 44) and macrophages (12, 47) are enhanced (4). It must also be considered that BALF cytology may underestimate the extent of macrophage extravasation in response to infection, as many macrophages remain in the interstitial space rather than migrating directly into alveoli in a manner similar to neutrophils (42).

In previous studies using -T-cell-depleted mice, the more extensive hepatic and pulmonary neutrophil infiltration observed following L. monocytogenes (15) and M. tuberculosis (11) infection, respectively, suggested T cells influence neutrophil migration. In line with current findings, intraperitoneal inoculation of -T-cell-depleted mice with L. monocytogenes by Fu et al. (15) resulted in more extensive, neutrophil-rich lesions than controls within 10 days postinfection. Furthermore, these -T-cell-depleted mice exhibited reduced bacterial loads. It is likely that T cells form part of an integrated signaling network that result in complex interactions with a multitude of other host immune and stromal cells. This may be affected by -T-cell production of a range of chemokines and cytokines, including CCL3/MIP-1, CCL4/MIP-1ß, CCL5/RANTES, lymphotactin, IFN-, IL-6, IL-10, or IL-12, that influence leukocyte trafficking and activation (14, 17, 19, 20, 29, 38, 43, 47). While this study has shown that TCR^–/– limit pulmonary injury, the precise mechanism operating here awaits further study. In particular, it is known that specific subpopulations of T cells such as V1 T cells perform highly specific and often opposing functions in different tissues and during different infections (7). The murine respiratory challenge model of B. pertussis infection would be an ideal system to study the function of these subpopulations in a clinically relevant model. It is also likely that the absence of T cells results in other changes in the integrated functions of the immune response. For example, T cells are important in homeostasis and the modulation of inflammation (6); then the lack of these functions throughout the development of these mice may lead to differential responses from various other cells that are involved in resolving inflammation.

A clearer understanding of the particular influence of T cells on the trafficking of neutrophils may have broader significance in whooping cough and in the pathogenesis of conditions such as ARDS. In the nonimmunized neonate, B. pertussis infection has a very high mortality and is characterized by elevated neutrophil counts and by necrotizing pulmonary inflammation (40) but no reported bacterial dissemination. Interestingly, this is a period in which there is a switch from a predominant V1 to a V2 population in humans (10). However, recent work has shown that V1 T cells require dendritic or accessory cells to recognize antigen (9, 26), and it is well documented that dendritic cell populations in the neonate are quantitatively and qualitatively impaired (36, 37). Thus, the susceptibility of human neonates to B. pertussis and the associated high morbidity and mortality may be associated with a period when T cell responses are suboptimal. Our observation of increased neutrophil-mediated inflammation in TCR^–/– mice is consistent with that theory. Similarly, the implication that a temporary suppression of -T-cell activity in the lung results in the uncontrolled inflammation associated with ARDS is relevant to the findings of the current study. Previous work has suggested that pulmonary T cells were essential to control inflammation through the anti-inflammatory impact of IL-10 on neutrophils (19).

The current study also demonstrates that the absence of T cells alters pulmonary recruitment of particular lymphocyte subtypes. Flow cytometry of BALF indicated differences in the movement of NK, NKR⁺ T, and NKR^– T cells into airways in the absence of T cells. NKR⁺ T cells and T cells are often erroneously considered to perform identical functions, serving merely to amplify adaptive immunity or provide endogenous danger signals. However, this study and others indicate that both and NKR⁺ T cells perform nonredundant functions in immunity. This is seen in the striking contrast between the present study and a previous study (5). Using a similar approach, that study demonstrated that deletion of NKR⁺ cells resulted in a disseminating and lethal B. pertussis infection in mice, reminiscent of that seen in IFN- knockout mice (28). In contrast, we found that absence of T cells did not result in disseminated infection, atypical extrapulmonary pathology, or increased mortality (data not shown). Taken together, these data indicate that, in the murine model of B. pertussis infection, the principle function of NKR⁺ T cells is to provide IFN- early in the host response to infection to prevent bacterial dissemination, whereas T cells appear to have a greater influence on inflammatory cell trafficking.

The observation that the cytokine response to B. pertussis antigens is dominated by IFN- in both WT and TCR^–/– mice suggests that T cells are not essential for the development of a Th1 response. However, this study suggests that these cells do influence the quality of adaptive immunity. TCR^–/– mice had a significantly greater Th2 response to the B. pertussis virulence factor FHA, not evident in WT controls. This supports findings of a previous study that demonstrated a role for T cells in antigen presentation and IL-12 production, suggesting this cell population may contribute to the Th1 response evoked by infection (39). However, the observed IL-4-dominated response to FHA seen in this study may reflect a loss of a T cell-dendritic cell interaction involving FHA. This would support previous findings suggesting that such an interaction results in IL-10 production (13, 31, 51).

The findings of this study suggest that, although less than 3% of the pulmonary leukocyte population in mice (19), T cells have a critical, nonredundant role in the initial inflammatory response to a gram-negative bacterial infection. Current evidence suggests that their function includes the prevention of exaggerated, harmful inflammatory host responses through their influence over the movement of key inflammatory effector cells and the regulation of the host response to bacterial antigens. Such findings are relevant to the study of conditions such as ARDS where temporary suppression of -T-cell function is thought to contribute to increased pulmonary inflammation in response to sepsis (19).

 
We acknowledge the excellent technical assistance of Darren Ennis, Derek Doherty, Laura Madrigas y Estebas, Christy King, Brian Cloak, and Bernie Ruane.

The study was funded by The Wellcome Trust, and O.Z. was supported by The Greek State Scholarship Foundation.

 
* Corresponding author. Mailing address: Department of Veterinary Pathology, Faculty of Veterinary Medicine, University College Dublin, Belfield, Dublin 4, Ireland. Phone: 353 1 7166151. Fax: 353 1 7166157. E-mail: Joseph.Cassidy{at}ucd.ie.

Editor: D. L. Burns

Top Abstract Introduction Materials and Methods Results Discussion References

 

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1. Barnard, A., B. P. Mahon, J. Watkins, K. Redhead, and K. H. G. Mills. 1996. Th1/Th2 cell dichotomy in acquired immunity to Bordetella pertussis: variables in the in vivo priming and in vitro cytokine detection techniques affect the classification of T cell subsets as Th1, Th2 or Th0. Immunology 87:372-380.[CrossRef][Medline]
2
2. Bendelac, A., M. N. Rivera, S.-H. Park, and J. H. Roark. 1997. Mouse CD1-specific NK1 T cells: development, specificity and function. Annu. Rev. Immunol. 15:535-562.[CrossRef][Medline]
3
3. Bertotto, A., F. M. De Benedictis, C. Vagliasindi, M. Radicioni, F. Spinozzi, G. M. Fabietti, G. Castellucci, L. Ferraro, R. Cozzali, A. Niccoli, and R. Vaccaro. 1997. Gamma delta T cells are decreased in the blood of children with Bordetella pertussis infection. Acta Paediatr. 86:114-115.[Medline]
4
4. Born, W. K., M. Lahn, K. Takeda, A. Kanehiro, R. L. O'Brien, and E. W. Gelfand. 2000. Role of gd T cells in protecting normal airway function. Respir. Res. 1:151-158.[CrossRef][Medline]
5
5. Byrne, P., P. McGuirk, S. Todryk, and K. H. Mills. 2004. Depletion of NK cells results in disseminating lethal infection with Bordetella pertussis associated with a reduction of antigen-specific Th1 and enhancement of Th2, but not Tr1 cells. Eur. J. Immunol. 34:2579-2588.[CrossRef][Medline]
6
6. Carding, S. R., and P. J. Egan. 2000. The importance of gamma delta T cells in the resolution of pathogen-induced inflammatory immune responses. Immunol. Rev. 173:98-108.[CrossRef][Medline]
7
7. Carding, S. R., and P. J. Egan. 2002. T cells: functional plasticity and heterogeneity. Nat. Rev. Immunol. 2:336-345.[CrossRef][Medline]
8
8. Chen, H., H. Huang, and W. E. Paul. 1997. NK1.1⁺ CD4⁺ T cells lose NK1.1 expression upon in vitro activation. J. Immunol. 158:5112-5119.[Abstract]
9
9. Das, H., M. Sugita, and M. B. Brenner. 2004. Mechanisms of Vdelta1 gammadelta T cell activation by microbial components. J. Immunol. 172:6578-6586.[Abstract/Free Full Text]
10
10. De Rosa, S. C., J. P. Andrus, S. P. Perfetto, J. J. Mantovani, L. A. Herzenberg, and M. Roederer. 2004. Ontogeny of gamma delta T cells in humans. J. Immunol. 172:1637-1645.[Abstract/Free Full Text]
11
11. D'Souza, C. D., A. M. Cooper, A. A. Frank, R. J. Mazzaccaro, B. R. Bloom, and I. M. Orme. 1997. An anti-inflammatory role for gamma delta T lymphocytes in acquired immunity to Mycobacterium tuberculosis. J. Immunol. 158:1217-1221.[Abstract]
12
12. Egan, P. J., and S. R. Carding. 2000. Downmodulation of the inflammatory response to bacterial infection by gammadelta T cells cytotoxic for activated macrophages. J. Exp. Med. 191:2145-2158.[Abstract/Free Full Text]
13
13. Ennis, D. P., J. P. Cassidy, and B. P. Mahon. 2005. Acellular pertussis vaccine protects against exacerbation of allergic asthma due to Bordetella pertussis in a murine model. Clin. Diagn. Lab. Immunol. 12:409-417.
14
14. Ferrero, E., P. Biswas, Y. K. Vettoretto, M. Ferrarini, M. Uguccioni, L. Piali, B. E. Leone, B. Moser, C. Rugarli, and R. Pardi. 2003. Macrophages exposed to Mycobacterium tuberculosis release chemokines able to recruit selected leukocyte subpopulations: focus on cells. Immunology. 108:365-374.[CrossRef][Medline]
15
15. Fu, Y.-X., C. E. Roark, K. Kelly, D. Drevets, P. Campbell, R. O'Brien, and W. Born. 1994. Immune protection and control of inflammatory tissue necrosis by T cells. J. Immunol. 153:3101-3115.[Abstract]
16
16. Godfrey, D. I., K. J. L. Hammond, L. D. Poulton, M. J. Smyth, and A. G. Baxter. 2000. NKT cells: facts, functions and fallacies. Immunol. Today 21:573-583.[CrossRef][Medline]
17
17. Gong, J. H., and I. Clark-Lewis. 1995. Antagonists of monocyte chemoattractant protein 1 identified by modification of functionally critical NH2 terminal residues. J. Exp. Med. 181:631-640.[Abstract/Free Full Text]
18
18. Hayday, A. C. 2000. Cells: a right time and a right place for a conserved third way of protection. Annu. Rev. Immunol. 18:975-1026.[CrossRef][Medline]
19
19. Hirsh, M., L. Dyugovskaya, V. Kaplan, and M. M. Krausz. 2004. Response of lung T cells to experimental sepsis in mice. Immunology 112:153-160.[CrossRef][Medline]
20
20. Kanehiro, A., M. Lahn, M. J. Mäkelä, A. Dakhama, M. Fujita, A. Joetham, R. J. Mason, W. Born, and E. W. Gelfand. 2001. Tumour necrosis factor- negatively regulates airway hyperresponsiveness through T cells. Am. J. Respir. Crit. Care Med. 164:2229-2238.[Abstract/Free Full Text]
21
21. Kawakami, K., N. Yamamoto, Y. Kinjo, K. Miyagi, C. Nakasone, K. Uezu, T. Kinjo, T. Nakayama, M. Taniguchi, and A. Saito. 2003. Critical role of V14⁺ natural killer T cells in the innate phase of host protection against Streptococcus pneumoniae infection. Eur. J. Immunol. 33:3322-3330.[CrossRef][Medline]
22
22. Khelef, N., C.-M. Bachelet, B. B. Vargaftig, and N. Guiso. 1994. Characterization of murine lung inflammation after infection with parental Bordetella pertussis and mutants deficient in adhesions or toxins. Infect. Immun. 62:2893-2900.[Abstract/Free Full Text]
23
23. Kimura, T., Y. Ishii, Y. Morishima, A. Shibuya, K. Shibuya, M. Taniguchi, M. Mochizuki, A. E. Hegab, T. Sakamoto, A. Nomura, and K. Sekizawa. 2004. Treatment with -galactosylceramide attenuates the development of bleomycin-induced pulmonary fibrosis. J. Immunol. 172:5782-5789.[Abstract/Free Full Text]
24
24. King, D. P., D. M. Hyde, K. A. Jackson, D. M. Novosad, T. N. Ellis, L. Putney, M. Y. Stovall, L. S. Van Winkle, B. L. Beaman, and D. A. Ferrick. 1999. Protective response to pulmonary injury requires T lymphocytes. J. Immunol. 162:5033-5036.[Abstract/Free Full Text]
25
25. Ladel, C. H., C. Blum, and S. H. E. Kaufmann. 1996. Control of natural killer cell-mediated innate resistance against the intracellular pathogen Listeria monocytogenes by T lymphocytes. Infect. Immun. 64:1744-1749.[Abstract]
26
26. Leslie, D. D., M. S. Vincent, F. M. Spada, H. Das, M. Sugita, C. T. Morita, and M. B. Brenner. 2002. CD1-mediated gamma/delta T cell maturation of dendritic cells. J. Exp. Med. 196:1575-1584.[Abstract/Free Full Text]
27
27. Li, B., H. Bassiri, M. D. Rossman, P. Kramer, A. F. Eyuboglu, M. Torres, E. Sada, T. Imir, and S. R. Carding. 1998. Involvement of the Fas/Fas ligand pathway in activation-induced cell death of mycobacteria-reactive human gamma delta T cells: a mechanism for the loss of gamma delta T cells in patients with pulmonary tuberculosis. J. Immunol. 161:1558-1567.[Abstract/Free Full Text]
28
28. Mahon, B. P., B. J. Sheahan, F. Griffin, G. Murphy, and K. H. G. Mills. 1997. Atypical disease after Bordetella pertussis infection respiratory infection of mice with targeted disruptions of interferon- receptor or immunoglobulin m chain genes. J. Exp. Med. 186:1843-1851.[Abstract/Free Full Text]
29
29. Mantovani, A. 1999. The chemokine system: redundancy for robust outputs. Immunol. Today 20:254-257.[CrossRef][Medline]
30
30. McGuirk, P., B. P. Mahon, F. Griffin, and K. H. G. Mills. 1998. Compartmentalization of T cell responses following respiratory infection with Bordetella pertussis: hyporesponsiveness of lung T cells is associated with modulated expression of the costimulatory molecule CD28. Eur. J. Immunol. 28:153-163.[CrossRef][Medline]
31
31. McGuirk, P., C. McCann, and K. H. G. Mills. 2002. Pathogen-specific T regulatory 1 cells induced in the respiratory tract by a bacterial molecule that stimulates interleukin 10 production by dendritic cells: a novel strategy for evasion of protective T helper type 1 responses by Bordetella pertussis. J. Exp. Med. 195:221-231.[Abstract/Free Full Text]
32
32. Mills, K. H. G., A. Barnard, J. Watkins, and K. Redhead. 1993. Cell-mediated immunity to Bordetella pertussis: role of Th1 cells in bacterial clearance in a murine respiratory infection model. Infect. Immun. 61:399-410.[Abstract/Free Full Text]
33
33. Mills, K. H. G., M. Ryan, E. Ryan, and B. P. Mahon. 1998. A murine model in which protection correlates with pertussis vaccine efficacy in children reveals complementary roles for humoral and cell mediated immunity in protection against Bordetella pertussis. Infect. Immun. 66:594-602.[Abstract/Free Full Text]
34
34. Mombaerts, P., J. Arnoldi, F. Russ, S. Tonegawa, and S. H. E. Kaufmann. 1993. Different roles of ß and T cells in immunity against an intracellular bacterial pathogen. Nature 365:53-56.[CrossRef][Medline]
35
35. Moretta, L., E. Ciccone, M. C. Mingari, R. Biassoni, and A. Moretta. 1994. Human NK cells: origin, clonality, specificity and receptors. Adv. Immunol. 55:341-380.[Medline]
36
36. Nelson, D. J., C. McMenamin, A. S. McWilliam, M. Brenan, and P. G. Holt. 1994. Development of the airway intraepithelial dendritic cell network in the rat from class II major histocompatibility (Ia)-negative precursors: differential regulation of Ia expression at different levels of the respiratory tract. J. Exp. Med. 179:203-212.[Abstract/Free Full Text]
37
37. Nelson, D. J., and P. G. Holt. 1995. Defective regional immunity in the respiratory tract of neonates is attributable to hyporesponsiveness of local dendritic cells to activation signals. J. Immunol. 155:3517-3524.[Abstract]
38
38. Nishimura, H., M. Emoto, K. Hiromatsu, S. Yamamoto, K. Matsuura, H. Gomi, T. Ikeda, S. Itohara, and Y. Yoshikai. 1995. The role of gamma delta T cells in priming macrophages to produce tumour necrosis factor alpha. Eur. J. Immunol. 25:1465-1468.[Medline]
39
39. Odyniec, A., M. Szczepanik, M. P. Mycko, M. Stasiolek, C. S. Raine, and K. W. Selmaj. 2004. Gamma-delta T cells enhance the expression of experimental autoimmune encephalomyelitis by promoting antigen presentation and IL-12 production. J. Immunol. 173:682-694.[Abstract/Free Full Text]
40
40. Pooboni, S., N. Roberts, C. Westrope, D. R. Jenkins, H. Killer, H. C. Pandya, and R. K. Firmin. 2003. Extracorporeal life support in pertussis. Pediatr. Pulmonol. 36:310-315.[CrossRef][Medline]
41
41. Sandberg, J. K., and H. G. Ljunggren. 2005. Development and function of CD1d restricted NKT cells: influence of sphingolipids, SAP and sex. Trends Immunol. 26:347-349.[CrossRef][Medline]
42
42. Shapiro, S. D., N. M. Goldstein, A. M. Houghton, D. K. Kobayashi, D. Kelley, and A. Belaaouaj. 2003. Neutrophil elastase contributes to cigarette smoke-induced emphysema in mice, Am. J. Pathol. 163:2329-2335.
43
43. Skeen, M. J., E. P. Rix, M. M. Freeman, and H. K. Ziegler. 2001. Exaggerated proinflammatory and Th1 responses in the absence of T cells after infection with Listeria monocytogenes. Infect. Immun. 69:7213-7223.[Abstract/Free Full Text]
44
44. Skeen, M. J., M. M. Freeman, and H. K. Ziegler. 2004. Changes in peritoneal myeloid populations and their proinflammatory cytokine expression during infection with Listeria monocytogenes are altered in the absence of gamma/delta T cells. J. Leukoc. Biol. 76:104-115.[Abstract/Free Full Text]
45
45. Spada, F. M., E. P. Grant, P. J. Peters, M. Sugita, A. Melián, D. S. Leslie, H. K. Lee, E. van Donselaar, D. A. Hanson, A. M. Krensky, O. Majdic, S. A. Porcelli, C. T. Morita, and M. B. Brenner. 2000. Self-recognition of CD1 by T cells: implications for innate immunity. J. Exp. Med. 191:937-948.[Abstract/Free Full Text]
46
46. Speyer, C. L., T. A. Neff, R. L. Warner, R.-F. Guo, J. V. Sarma, N. C. Riedemann, M. E. Murphy, H. S. Murphy, and P. A. Ward. 2003. Regulatory effects of iNOS on acute lung inflammatory responses in mice. Am. J. Pathol. 163:2319-2328.[Abstract/Free Full Text]
47
47. Tagawa, T., H. Nishimura, T. Yajima, H. Hara, K. Kishihara, G. Matsuzaki, I. Yoshino, Y. Maehara, and Y. Yoshikai. 2004. Vdelta1⁺ gammadelta T cells producing CC chemokines may bridge a gap between neutrophils and macrophages in innate immunity during Escherichia coli infection in mice. J. Immunol. 173:5156-5164.[Abstract/Free Full Text]
48
48. Trinchieri, G. 1989. Biology of natural killer cells. Adv. Immunol. 47:187-376.[Medline]
49
49. Vincent, M. S., K. Roessner, D. Lynch, D. Wilson, S. M. Cooper, J. Tschopp, L. H. Sigal, and R. C. Budd. 1996. Apoptosis of Fas high CD4⁺ synovial T cells by borrelia-reactive Fas-ligand (high) gamma delta T cells in Lyme arthritis. J. Exp. Med. 184:2109-2117.[Abstract/Free Full Text]
50
50. Widney, D. P., Y. Hu, A. K. Foreman-Wykert, K. C. Bui, T. T. Nguyen, B. Lu, C. Gerard, J. F. Miller, and J. B. Smith. 2005. CXCR3 and its ligands participate in the host response to Bordetella bronchiseptica infection of the mouse respiratory tract but are not required for clearance of bacteria from the lung. Infect. Immun. 73:485-493.[Abstract/Free Full Text]
51
51. Ye, Z., S. Haley, A. P. Gee, P. J. Henslee-Downey, and L. S. Lamb. 2002. In vitro interactions between gamma delta T cells, DC, and CD4⁺ T cells; implications for the immunotherapy of leukaemia. Cytotherapy 4:293-304.[CrossRef][Medline]
52
52. Yoshimoto, T., and W. E. Paul. 1994. CD4⁺ NK1.1⁺ T cells promptly produce interleukin 4 in response to in vivo challenge with anti-CD3. J. Exp. Med. 179:1285-1295.[Abstract/Free Full Text]

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Infection and Immunity, March 2006, p. 1837-1845, Vol. 74, No. 3
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