- Email: [email protected]
Chlamydia trachomatis pulmonary infection induces greater inflammatory pathology in immunoglobulin A deficient mice
ellular Immunology Cellular Immunology 230 (2004) 56–64 www.elsevier.com/locate/ycimm
Chlamydia trachomatis pulmonary infection induces greater inXammatory pathology in immunoglobulin A deWcient mice Ashlesh K. Murthya, Jyotika Sharmab, Jacqueline J. Coalsonc, Guangming Zhongb, Bernard P. Arulanandama,¤ b
a Department of Biology, University of Texas at San Antonio, San Antonio, TX 78249, USA Department of Microbiology and Immunology, University of Texas Health Science Center, San Antonio, TX 78229, USA c Department of Pathology, University of Texas Health Science Center, San Antonio, TX 78229, USA
Received 10 July 2004; accepted 10 September 2004
Abstract Chlamydia trachomatis is an intracellular bacterial pathogen that primarily infects via mucosal surfaces. Using mice with a targeted disruption in IgA gene expression (IgA¡/¡ mice), we have studied the contribution of IgA, the principal mucosal antibody isotype, in primary immune defenses against pulmonary C. trachomatis infection. Bacterial burden was comparable between IgA¡/¡ and IgA+/+ animals following C. trachomatis challenge. Serum and pulmonary anti-Chlamydia antibody levels were higher in IgA¡/¡ animals, with the exception of IgA. Lung sections of challenged IgA¡/¡ mice showed more extensive immunopathology than corresponding IgA+/+ animals. Real-time PCR analysis demonstrated signiWcantly greater IFN- and TGF- mRNA expression in IgA¡/¡ as compared to IgA+/+ animals. Together, these results suggest that IgA may not be necessary for clearance of primary C. trachomatis infection. However, IgA¡/¡ mice displayed exaggerated lung histopathology and altered cytokine production, indicating an important role for IgA in regulating C. trachomatis induced pulmonary inXammation and maintenance of mucosal homeostasis. 2004 Elsevier Inc. All rights reserved. Keywords: Chlamydia trachomatis; Immunoglobulin A; InXammation; Innate
1. Introduction Chlamydia trachomatis is an obligate intracellular gram-negative pathogen that is the leading cause of bacterial sexually transmitted disease [1]. C. trachomatis exhibits tropism for mucosal surfaces such as the conjunctiva and the genitourinary tract [1]. Chlamydial infections of the lower genital tract often ascend to the fallopian tubes [2] leading to complications such as pelvic inXammatory disease, ectopic pregnancy, and tubal infertility [2–4]. Despite considerable research, an eVective vaccine against chlamydia is still unavailable.
*
Corresponding author. Fax: +1 210 458 5523. E-mail address: [email protected] (B.P. Arulanandam).
0008-8749/$ - see front matter 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.cellimm.2004.09.002
Cell-mediated and humoral immune mechanisms have been shown to be important in immunity against C. trachomatis. SpeciWcally, T-helper (Th1) type immunity and IFN- production are required for the optimal clearance of chlamydial infection [5,6]. Although CD8+ T-lymphocytes are not required for protection [7], adoptive transfer of chlamydia-speciWc cytotoxic T-cells has been shown to contribute to bacterial clearance [8]. The role of humoral immunity in chlamydial infections has not been resolved. Antibodies were shown not to be important in clearance of chlamydial infections, using mice that were rendered antibody deWcient by anti- treatment [9,10]. Also, Su et al. [11] demonstrated that B-cell deWcient (MT) mice were capable of resolving primary and secondary genital infection with C. trachomatis. However, in the absence of speciWc antibody, mice were
A.K. Murthy et al. / Cellular Immunology 230 (2004) 56–64
shown to be more susceptible to reinfection. Additionally, in a lung C. trachomatis mouse pneumonitis (MoPn) infection model, MT mice not only displayed signiWcantly higher mortality and lung bacterial loads than wild-type mice, but also exhibited impaired T-cell responses to the infection [12]. Furthermore, Moore et al. [13] have demonstrated that the interaction of antibodies and Fc receptors is required for the generation of strong Th1 type immune responses against chlamydia. It has also been reported that immunization with anti-idiotypic antibody against chlamydial glycolipid exoantigen provided protection against genital C. trachomatis challenge [14]. Taken together, these studies suggest that humoral immune mechanisms do play a role in protection against chlamydial infection. IgA is the principal immunoglobulin isotype involved in inhibition of bacterial attachment and neutralization of viruses at mucosal surfaces [15]. In addition, IgA also has been shown to be directly involved in the immunoregulation of phagocytic cells such as monocytes and neutrophils [16–18]. Serum IgA and secretory IgA (sIgA) have individually been shown to suppress inXammation by reducing inXammatory cytokine production or the oxidative burst [17,19]. Furthermore, IgA has been shown to be required for eVective priming of T-cells and development of Th1 type responses [20,21]. In chlamydial infections, IgA has been shown to be eVective in resistance against reinfection in the genital tract [22,23]. However, the role of IgA in innate immunity against chlamydia is not clearly understood. In this study, we have evaluated the role of IgA in a pulmonary C. trachomatis infection, using mice with a targeted disruption in the switch region and -heavy chain locus (IgA¡/¡ mice). Our studies demonstrate that IgA may not be essential for clearance of this primary chlamydial infection. However, IgA deWcient mice exhibit exaggerated lung histopathology and altered inXammatory cytokine production, indicating the importance of IgA in regulating inXammation after bacterial challenge.
2. Materials and methods 2.1. Bacteria Chlamydia trachomatis MoPn strain was grown on conXuent HeLa cell monolayers. The cells were lysed using a sonicator (Fisher, Pittsburgh, PA) and the elementary bodies (EBs) were puriWed on RenograYn gradients as described [24]. Aliquots of bacteria were stored at ¡70 °C in sucrose–phosphate–glutamine buVer. Chlamydia genus-speciWc murine monoclonal antibody [25] was used to conWrm the strain of the puriWed bacterium.
57
2.2. Mice C57BL/6 £ 129 IgA deWcient (IgA¡/¡) mice were generated as described [26] and C57BL/6 £ 129 F2 (IgA+/+) mice were used as wild-type controls. C57BL/6 wild-type and B-cell deWcient (MT) mice were purchased from Jackson Laboratory (Bar Harbor, ME). All mice were 4– 8 weeks old at the time of infection. Mice were housed and bred at the University of Texas at San Antonio Animal Care Facility and provided food and water ad libitum. Animal care and experimental procedures were performed in compliance with the Institutional Animal Care and Use Committee (IACUC) guidelines. 2.3. Intranasal infection procedures and determination of lung bacterial loads Intranasal (i.n.) infections were performed as previously described [27]. BrieXy, mice were anesthetized i.n. with 3% isoXuorane using a rodent anesthesia system (Harvard Apparatus, Holliston, MA) and immediately inoculated i.n. with 200 inclusion forming units (IFU) of MoPn in 25 l of sterile PBS. Lungs from mice were removed 10 days after challenge and weighed. Tissues were homogenized with an electric stirrer (Arrow Junior, Vineland, NJ) and the homogenate centrifuged at 250g for 10 min at 4 °C. The collected supernatants were incubated for 24 h with HeLa cells grown on coverslips in 24well plates. The infected HeLa cells were Wxed with 2% paraformaldehyde and permeabilized with 2% saponin. Cells were washed using PBS and incubated with ModiWed Dulbecco’s Eagle’s Medium containing 10% fetal bovine serum for 1 h to block non-speciWc binding. Thereafter, cells were washed and incubated with antiChlamydia genus-speciWc murine monoclonal antibody and polyclonal rabbit anti-Chlamydia antibody for 1 h and then incubated for an additional 2 h with goat anti-mouse IgG conjugated to Cy3 (Jackson Immunoresearch, West Grove, PA) and goat anti-rabbit Ig conjugated to FITC (Sigma, St. Louis, MO) plus Hoescht nuclear stain. The treated coverslip cultures were then washed and mounted onto superfrost microscope slides (Fisher) using Fluorsave reagent (Calbiochem, La Jolla, CA). Images were acquired using an Axiocam digital camera (Zeiss, Thornwood, NY) connected to a Zeiss Axioskop 2 Plus research microscope. The bacterial loads were calculated and expressed as the number of inclusion forming units per gram of lung tissue. 2.4. Collection of bronchioalveolar lavage Xuid For collection of bronchioalveolar lavage (BAL) Xuid, mice were sacriWced and tracheae intubated using a 0.58 mm OD polyethylene catheter (Becton–Dickinson, Sparks, MD). The lungs were then lavaged two to three times with separate 0.5 ml vol of PBS. The recovered
58
A.K. Murthy et al. / Cellular Immunology 230 (2004) 56–64
BAL Xuids were centrifuged at 500g for 5 min at 4 °C and the supernatants were stored at ¡70 °C until use. 2.5. Detection of antibody and isotype levels by ELISA Microtiter plates were coated overnight with 104 IFU of UV-inactivated MoPn in sodium bicarbonate buVer (pH 9.5), washed with PBS containing 0.3% Brij-35 (Sigma) and blocked for 1 h at room temperature with PBS containing 2% bovine serum albumin (BSA, EM Science Gibbstown, NJ). Serial dilutions of serum or BAL Xuids were added to wells and incubated at room temperature for 2 h. The plates were then washed and incubated for an additional 1 h with goat anti-mouse total Ig, IgG1, IgG2a, IgG2b, IgM, or IgA conjugated to alkaline phosphatase (Southern Biotechnology Associates, Birmingham, AL). After incubation for 1 h, the plates were washed and p-nitrophenyl phosphate substrate was added for color development. Absorbance at 405 nm was measured using an ELISA microplate reader (Biotek Instruments, Winooski, VT). The reciprocal serum dilutions corresponding to 50% maximal binding were used to obtain titers. Because of the low amounts of antibodies in BAL Xuids and large dilution involved in the lavage procedure, titers could not be obtained for BAL, and all samples were tested undiluted. 2.6. Histology and immunoXuorescence staining Lungs from infected mice were removed 3, 5 or 10 days after challenge and embedded in optimal cutting temperature (OCT) resin and snap frozen. Serial horizontal cryosections of 5 m were prepared and placed on silane coated-slides (VWR International, West Chester, PA). All slides were dried overnight and Wxed in fresh acetone for 20 s at room temperature. Some sections also were Wxed with formalin for 10 min and stained by hematoxylin and eosin (H&E). For immunoXuorescent staining, slides were blocked with 3% BSA for 5 min, followed by incubation with 10% normal rat serum (Sigma) for 30 min. Tissue sections were subsequently incubated with R-phycoerythrin (R-PE) conjugated rat anti-mouse CD11b or CD3 (BD Biosciences, San Diego, CA) for 40 min. Sections were washed and mounted using Xuorsave reagent (Calbiochem, La Jolla, CA) containing Hoescht stain (blue for binding to DNA). Some sections were stained using anti-granulocyte (GR1) antibody according to the manufacturer’s instructions (BD Biosciences, San Jose, CA). Images were acquired using an Axiocam digital camera (Zeiss, Thornwood, NY) connected to a Zeiss Axioskop 2 Plus research microscope. 2.7. RNA isolation and real-time quantitative PCR Total RNA was obtained from snap frozen lungs using the Total RNA Isolation Kit (Austin, TX) accord-
ing to the manufacturer’s instructions. First strand cDNA synthesis was carried out from total RNA as described [28]. RT-PCR of the cDNA products was performed with a DyNAmo SYBR Green qPCR kit (Finnzymes, Boston, MA) using a DNA Engine Opticon continuous Xuorescence detection system (MJ Research, Waltham, MA). Sense and antisense primers used were as follows; IFN-, 5⬘-TCAAGTGGCATAGATGTGG AAGAA-3⬘ and 5⬘-TGGCTCTGCAGGATTTTCA TG-3⬘; TGF-, 5⬘-TGACGTCACTGGAGTTGTACG G-3⬘ and 5⬘-GGTTCATGTCATGGATGGTGC-3⬘; and GAPDH, 5⬘-TTCACCACCATGGAGAAGGC-3⬘ and 5⬘-GGCATGGACTGTGGTCATGA-3⬘. Real-time PCR conditions were as follows: 1 s of denaturation at 95 °C, 10 s of primer annealing at 62.5 °C and 20 s of elongation at 72 °C for 40 cycles. QuantiWcation was carried out by monitoring the Xuorescent DNA binding dye SYBR green, at the end of each elongation cycle at 78 °C for IFN- and 81.5 °C for TGF-. Relative quantiWcation was carried out by normalizing levels of the gene of interest to the housekeeping gene GAPDH, and to baseline levels of cytokine expression in uninfected mice of corresponding groups. The values obtained were expressed as fold changes of the gene expression. 2.8. Statistical analyses Sigma Stat (Chicago, IL) was used to perform all the tests of signiWcance. The Mann–Whitney rank sum test was used to determine diVerences between experimental groups, and data were considered statistically signiWcant if p values were <0.05. All data shown are representative of at least 2–3 independent experiments and each experiment shown was analyzed independently.
3. Results 3.1. IgA may not be required for clearance of a primary C. trachomatis lung infection To determine the role of B-cells and antibodies against a primary infection, age matched MT (B-cell deWcient) and corresponding wild-type animals were challenged with 200 IFU of MoPn. Lung bacterial loads 10 days after challenge revealed a 6-log increase in the number of organisms recovered in the MT mice as compared to similarly infected wild-type animals (data not shown). Furthermore, the MT mice developed a progressive loss of body weight, whereas the average weight loss of infected wild-type animals was minimal, indicative of bacterial clearance (data not shown). These results are in agreement with Yang and Brunham [12] who demonstrated that B-cells play an important role in primary bacterial clearance of C. trachomatis infection from the lungs.
A.K. Murthy et al. / Cellular Immunology 230 (2004) 56–64
Fig. 1. Lungs of infected IgA¡/¡ and wild-type animals display equivalent numbers of chlamydia. (A) Wild-type and IgA¡/¡ deWcient mice were infected i.n. on day 0 with 200 IFU MoPn, and lung bacterial loads were examined on day 10 and expressed as IFU/g of lung. Each symbol represents an individual mouse (n D 6). (B) Body weight loss after bacterial challenge. Results are representative of three independent experiments.
59
Fig. 2. Serum anti-Chlamydia antibody titers are higher in the IgA¡/¡ animals. Mice were bled 10 days after challenge and serum analyzed by isotype-speciWc ELISA using UV-inactivated MoPn coated microtiter plates. The results are reported as 50% end point titers. DiVerences in binding between IgA¡/¡ and IgA+/+ mice (n D 4) were signiWcant at p < 0.05 for IgG1 and IgG2b. Results are representative of three independent experiments.
Since IgA is the predominant immunoglobulin in the respiratory mucosa, we extended our observation by examining the contribution of IgA against primary C. trachomatis lung infection in IgA deWcient mice using a similar challenge inoculum. As shown in Fig. 1A, equivalent numbers of bacteria were recovered from the lungs of infected IgA¡/¡ and wild-type animals. In addition, the average weight loss following challenge was similar in both groups of animals (Fig. 1B). These results suggest that although B-cells are required for clearance of a primary C. trachomatis lung infection, the contribution of IgA in this process is not crucial. 3.2. Antibody proWles after primary C. trachomatis pulmonary infection To examine possible reasons for the limited diVerences in bacterial clearance between IgA¡/¡ and IgA+/+ animals, analyses of sera and BAL Xuids were carried out for expression of anti-MoPn antibodies of deWned isotypes. Overall, serum IgM and IgG (IgG1, IgG2a, and IgG2b) titers tended to be higher in the IgA¡/¡ mice as compared to wild-type animals 10 days following bacterial challenge (Fig. 2). Anti-MoPn IgG1 and IgG2b antibody levels were signiWcantly higher in the IgA¡/¡ animals. Respiratory antibody production in BAL Xuids of infected animals also was analyzed 10 days after challenge (Fig. 3). It was found that levels of
Fig. 3. Respiratory anti-Chlamydia antibody titers are higher in the IgA¡/¡ animals. Mice were euthanized 10 days after challenge, BAL Xuids collected and analyzed by isotype-speciWc ELISA using UVinactivated MoPn coated microtiter plates. DiVerences in binding between IgA¡/¡ and IgA+/+ mice (n D 4) were signiWcant at p < 0.05 for IgG1 and IgA. Each symbol represents an individual mouse. Results are representative of two independent experiments.
all antibody isotypes tended to be greater in the IgA¡/¡ animals, with the obvious exception of IgA, which was detected only in the wild-type mice. In particular, levels of IgG1 were signiWcantly greater in the IgA deWcient
60
A.K. Murthy et al. / Cellular Immunology 230 (2004) 56–64
mice. These results suggest that the higher titers of other antibody isotypes present in IgA¡/¡ animals may compensate for the loss of IgA in the clearance of C. trachomatis from the lung. 3.3. IgA¡/¡ mice develop greater histopathological changes after pulmonary C. trachomatis infection Lung histopathology was examined in tissues obtained from IgA¡/¡ and corresponding wild-type control mice challenged with 200 IFU MoPn. Lungs were removed on days 3, 5 or 10 after challenge. Sections from lungs removed 3 days after challenge showed foci of pulmonary edema and scattered inXammatory cells in the peribronchiolar alveoli of both types of mice (Figs. 4A, arrow and B). The distribution of the alveolar exudates was within and around the airways, indicating
Fig. 4. IgA¡/¡ mice exhibit extensive histopathological changes after pulmonary C. trachomatis challenge. (A) IgA+/+ lung: day 3, (B) IgA¡/¡ lung: day 3, (C) IgA+/+ lung: day 5, (D) IgA¡/¡ lung: day 5, (E) IgA+/+ lung: day 10, (F) IgA¡/¡ lung: day 10, (G) mock-infected IgA+/+ lung, (H) mock-infected IgA¡/¡ lung. a: airway; b: bronchiole. MagniWcation 10£. Results are representative of three independent experiments.
the establishment of an early bronchopneumonia. However, at this time-point, lung sections of the IgA¡/¡ animals (Fig. 4B) showed more extensive inWltration in the lungs than the corresponding wild-type animals (Fig. 4A). Examination at higher magniWcation revealed the presence of more cellular debris and greater numbers of polymorphonucleur cells in sections from IgA¡/¡ lungs when compared to wild-type animals (data not shown). While the tissue sections from both groups displayed bronchopneumonia by day 5 after challenge, the lesions were more extensive in the IgA¡/¡ animals (Fig. 4D) when compared to wild-type animals (Fig. 4C). The size of the inXammatory inWltrates was small in the IgA+/+ animals (Fig. 4C) with sparing of alveolar spaces around them, as compared to the much larger inWltrates in IgA¡/¡ animals (Fig. 4D). Interestingly, it was also found that IgA¡/¡ animals also displayed increased numbers of inWltrating inXammatory cells around the bronchioles (peribronchiolar cuYng) and the vasculature (perivascular cuYng), which was not observed in lung sections from IgA+/+ animals (data not shown). The histopathology 10 days after challenge displayed progressive changes, except that the lungs of IgA¡/¡ animals (Fig. 4F) displayed complete alveolar Wlling of entire lobes of the infected lungs, while the wild-type lungs (Fig. 4E) still showed a bronchopneumonia pattern. There were no histopathological diVerences in the mock-infected lungs of both groups of animals (Figs. 4G and H). Since the histopathology on day 5 after challenge displayed considerable diVerences between the IgA ¡/¡ and wild-type animals, we carried out a detailed analysis of the immune response to infection at this timepoint, using in situ immunohistochemistry. Staining of lung sections for granulocytes with anti-Gr1 antibody demonstrated abundant neutrophils in the parenchymal inWltrates of IgA¡/¡ and IgA+/+ animals (data not shown). In addition, IgA¡/¡ mice displayed greater neutrophilic inWltrates around the bronchioles and vasculature and also inside the bronchiolar lumen of IgA¡/¡ mice, with noticable disruption of bronchiolar wall architecture, which was not observed in lung sections of IgA+/+ animals (data not shown). Patterns of T-lymphocyte staining revealed similar distribution of cells within parenchymal inWltrates of the IgA+/+ (Fig. 5A, arrow) and IgA¡/¡ animals. However, T-lymphocytes were observed around bronchioles (Fig. 5B, arrow) and vasculature (Fig. 5B, inset) of IgA¡/¡ animals alone. Similar patterns of macrophage distribution were observed, with no evidence of B-cells at this time-point after challenge (data not shown). There was no apparent pathology in lung sections from control uninfected IgA¡/¡ (Fig. 5D) and wild-type mice (Fig. 5C). Thus, infected IgA¡/¡ mice exhibited greater inXammatory lung histopathology than similarly infected wild-type animals.
A.K. Murthy et al. / Cellular Immunology 230 (2004) 56–64
61
Fig. 6. IFN- and TGF- mRNA are expressed at higher levels in the lungs of C. trachomatis infected IgA¡/¡ mice. RNA was prepared from infected tissues (n D 3) followed by examination of IFN- and TGF- levels by real-time PCR. (A) levels of IFN- and TGF- on day 3 and (B) on day 5 after challenge. IFN- and TGF- levels at day 5 in the lungs of IgA¡/¡ mice were signiWcantly diVerent from similarly infected IgA+/+ mice (p < 0.05). Results are representative of three independent experiments.
Fig. 5. Infected IgA¡/¡ animals exhibit peribronchiolar and perivascular cuYng. Lung sections were stained with an anti CD3 antibody conjugated with phycoerythrin (PE) following challenge. (A) IgA+/+ lung: day 5, (B) IgA¡/¡ lung: day 5, (C) mock-infected IgA+/+ lung, (D) mock-infected IgA¡/¡ lung. AS: alveolar space; B: bronchiole; A: arteriole. MagniWcation 10£. Results are representative of two independent experiments.
3.4. Altered expression of IFN- and TGF- in the lungs of C. trachomatis infected IgA¡/¡ mice Since IFN- is known to be important in clearance of primary C. trachomatis infection [29], we determined the level of IFN- expression in the lungs of infected IgA¡/¡ and wild-type animals, using real time PCR (Fig. 6). As shown in panel A, IFN- expression was relatively low in both groups of animals 3 days after challenge. However, 5 days after challenge, IgA¡/¡ mice exhibited an approximately 12-fold greater increase in IFN- levels than wildtype animals (panel B). Transforming growth factor- (TGF-) is a known Ig isotype switch factor for IgA [30], and is thought to play an important role in chlamydial infections by modulating cellular immune responses, enhancing host defenses via production of IgA, and potentially contributing to scar tissue formation [31]. Therefore, we also examined the levels of TGF- in the lungs of infected IgA¡/¡ and IgA+/+ animals (Fig. 6). Similar levels of this cytokine were seen in both the IgA¡/¡ and wild-type animals on day 3 following challenge (panel A). However, IgA¡/¡ mice displayed a 28-fold greater production of TGF- than wild-type animals on day 5 (panel B).
4. Discussion We have evaluated the role of IgA against primary pulmonary Chlamydia trachomatis infection. The absence of IgA did not adversely aVect bacterial clearance upon challenge, possibly due to compensatory mechanisms, but was associated with greater pulmonary histopathological changes than in wild-type animals. Therefore, IgA may play an important role, apart from simple neutralization, in regulating inXammation and in maintenance of mucosal homeostasis after chlamydial infection. Although IgA is the principal immunoglobulin isotype involved in host defense against pathogens at mucosal surfaces [15], our results suggest that IgA may not be necessary for clearance of a primary C. trachomatis lung infection. IgA¡/¡ mice exhibit higher titers of other anti-chlamydial antibody isotypes, which probably compensate for the lack of IgA. IgA¡/¡ mice also have been shown to produce higher titers of serum [26] and pulmonary antibodies [20], when compared to wild-type animals. Because of the high degree of inXammation during chlamydial infection, there is a likelihood of serum antibody diVusion into the respiratory compartment, apart from local antibody production. In addition, the levels of IFN-, known to be important in clearance of chlamydial infections [29], were signiWcantly elevated in the lungs of IgA¡/¡ as compared to wild-type animals in our studies. Therefore, compensatory mechanisms involving other antibody isotypes and elevated IFN- may promote bacterial clearance in IgA¡/¡ mice with
62
A.K. Murthy et al. / Cellular Immunology 230 (2004) 56–64
similar eYciency to the wild-type animals. To this end, monoclonal anti-chlamydial IgA treatment has been shown to eVectively reduce the incidence of genital tract infection, but has a marginal eVect on clearance of organisms [22,23]. IgA, however, does play a major role in regulating inXammatory cytokine responses to bacterial infections. In our study, the absence of IgA resulted in greater pulmonary histopathology following i.n. C. trachomatis challenge. Although evidence of inXammation was found in both types of animals, a more extensive lung involvement was observed in the IgA¡/¡ animals, associated with signs of severe inXammation such as perivascular and peribronchiolar cuYng, which were not present in the lung sections from IgA+/+ animals. In particular, the level of IFN- in the lungs of mice was found to be 12-fold greater in the absence of IgA. Likewise, the frequency of CD3+/CD4+ T-cells secreting IFN- is increased in children with selective IgA deWciency [32]. IFN- has been shown to activate polymorphonuclear cells leading to enhancement of oxidative responses [33] and also to prime macrophages to release proteinases that degrade the host tissue [34]. Lung sections from C. trachomatis infected IgA¡/¡ mice, but not IgA+/+ mice, demonstrated macrophage and neutrophilic inWltrates around vasculature and bronchioles. These cells, in the presence of IFN-, may cause severe inXammatory damage to these structures, as evidenced by the enhanced immunopathological changes observed in the absence of IgA. Human serum IgA has been reported to down-regulate the release of TNF- and IL-6 in H. inXuenzae type b-activated monocytes via induction of IL-1 receptor antagonist [17,18]. In addition, secretory IgA has been shown to bind colostral neutrophils and reduce the release of superoxide anions [19]. IgA has been shown to mediate many of its biological activities in humans by interacting with CD89 (Fc receptor), found on a variety of cell types including neutrophils, eosinophils, and macrophages [35]. However, it is important to note that the murine CD89 homologue has yet to be described. Infected IgA¡/¡ mice also produced signiWcantly greater amounts of TGF- transcripts in the lungs than wild-type animals 5 days after MoPn challenge. TGF- is an important immunoregulatory cytokine that has eVects on various cell types [36] and is an isotype switch factor for IgA [30]. Since TGF- occurs in both latent and active forms, we analyzed the levels of active protein in BAL Xuids collected on day 5 after challenge, using ELISA. However, active TGF- protein could not be detected at this time-point (Murthy and Arulanandam, unpublished observations). This suggests a lack of correlation between TGF- gene expression and active protein in the lung at this time-point, which supports similar observations by Williams et al. [31], indicating the likelihood of post-transcriptional regulation of TGF- during chlamydial infection.
Although TGF- is a potent inducer of matrix formation and is involved in wound healing, excessive amounts of this cytokine may be responsible for scarring and Wbrosis [37,38]. In addition, latent TGF- released into host tissues can be activated by various factors including IFN- [39]. It has been shown recently that endogenous IFN- can negatively regulate the TGF- 1 signaling pathway, resulting in retardation of wound healing [40]. Therefore, in genital chlamydial infections, repeated episodes of severe inXammation in the fallopian tubes due to elevated levels of IFN-, plus potential excessive Wbrosis and scarring due to elevated levels of TGF- in the absence of IgA, may lead to serious complications such as infertility. To this end, it has been shown that anti-chlamydial MOMP IgA secreting B-cells are reduced during periods of intense ocular inXammation in human trachoma [41], suggesting that IgA may be involved in regulating inXammatory processes. In fact, IgA immunodeWciency in humans is a heterologous condition that can be associated with inXammatory conditions such as Coeliac and Crohn’s disease [42,43]. The altered IFN-/TGF- axis may account for the increased pathology and eVective bacterial clearance in the absence of IgA, observed in our studies. The signiWcant increases in the levels of IFN- are synchronous with the greater severity of inXammatory lung histopathology in IgA¡/¡ mice. In the absence of IgA, the production of increased levels of TGF- upon C. trachomatis infection may be an important factor in the development of complications arising from chronic inXammation. In summary, the results of this study demonstrate that IgA plays an important role in innate C. trachomatis pulmonary infection. It appears that apart from providing epithelial barrier protection, IgA is pivotally involved in regulating cytokine responses and inXammatory pathology with this infection.
Acknowledgments This work was supported by National Institutes of Health Grants AR048973-02 and SO6GM008194-24. The authors gratefully acknowledge Dr. Neal Guentzel for review of the manuscript and Ms. Yanqing Huang for technical expertise.
References [1] R.P. Morrison, H.D. Caldwell, Immunity to murine chlamydial genital infection, Infect. Immun. 70 (2002) 2741–2751. [2] K.A. Kelly, Cellular immunity and Chlamydia genital infection: induction, recruitment, and eVector mechanisms, Int. Rev. Immunol. 22 (2003) 3–41. [3] L. Westrom, R. Joesoef, G. Reynolds, A. Hagdu, S.E. Thompson, Pelvic inXammatory disease and fertility. A cohort study of 1,844
A.K. Murthy et al. / Cellular Immunology 230 (2004) 56–64
[4] [5]
[6]
[7]
[8]
[9]
[10]
[11]
[12]
[13]
[14]
[15] [16]
[17]
[18]
[19]
[20]
[21]
women with laparoscopically veriWed disease and 657 control women with normal laparoscopic results, Sex. Transm. Dis. 19 (1992) 185–192. L. Westrom, P.A. Mardh, Chlamydial salpingitis, Br. Med. Bull. 39 (1983) 145–150. L.L. Perry, K. Feilzer, H.D. Caldwell, Immunity to Chlamydia trachomatis is mediated by T helper 1 cells through IFN-gammadependent and -independent pathways, J. Immunol. 158 (1997) 3344–3352. R.G. Rank, K.H. Ramsey, E.A. Pack, D.M. Williams, EVect of gamma interferon on resolution of murine chlamydial genital infection, Infect. Immun. 60 (1992) 4427–4429. S.G. Morrison, H. Su, H.D. Caldwell, R.P. Morrison, Immunity to murine Chlamydia trachomatis genital tract reinfection involves B cells and CD4(+) T cells but not CD8(+) T cells, Infect. Immun. 68 (2000) 6979–6987. J.U. Igietseme, D.M. Magee, D.M. Williams, R.G. Rank, Role for CD8+ T cells in antichlamydial immunity deWned by Chlamydia-speciWc T-lymphocyte clones, Infect. Immun. 62 (1994) 5195–5197. D.M. Williams, B. Grubbs, J. Schachter, Primary murine Chlamydia trachomatis pneumonia in B-cell-deWcient mice, Infect. Immun. 55 (1987) 2387–2390. H. Ramsey, L.S. Soderberg, R.G. Rank, Resolution of chlamydial genital infection in B-cell-deWcient mice and immunity to reinfection, Infect. Immun. 56 (1988) 1320–1325. H. Su, K. Feilzer, H.D. Caldwell, R.P. Morrison, Chlamydia trachomatis genital tract infection of antibody-deWcient gene knockout mice, Infect. Immun. 65 (1997) 1993–1999. X. Yang, R.C. Brunham, Gene knockout B cell-deWcient mice demonstrate that B cells play an important role in the initiation of T cell responses to Chlamydia trachomatis (mouse pneumonitis) lung infection, J. Immunol. 161 (1998) 1439–1446. T. Moore, C.O. Ekworomadu, F.O. Eko, et al., Fc receptor-mediated antibody regulation of T cell immunity against intracellular pathogens, J. Infect. Dis. 188 (2003) 617–624. J.A. Whittum-Hudson, D. Rudy, H. Gerard, et al., The anti-idiotypic antibody to chlamydial glycolipid exoantigen (GLXA) protects mice against genital infection with a human biovar of Chlamydia trachomatis, Vaccine 19 (2001) 4061–4071. M.E. Lamm, Interaction of antigens and antibodies at mucosal surfaces, Annu. Rev. Microbiol. 51 (1997) 311–340. H.M. Wolf, E. Vogel, M.B. Fischer, H. Rengs, H.P. Schwarz, M.M. Eibl, Inhibition of receptor-dependent and receptor-independent generation of the respiratory burst in human neutrophils and monocytes by human serum IgA, Pediatr. Res. 36 (1994) 235–243. H.M. Wolf, M.B. Fischer, H. Puhringer, A. Samstag, E. Vogel, M.M. Eibl, Human serum IgA downregulates the release of inXammatory cytokines (tumor necrosis factor-alpha, interleukin6) in human monocytes, Blood 83 (1994) 1278–1288. H.M. Wolf, I. Hauber, H. Gulle, et al., Anti-inXammatory properties of human serum IgA: induction of IL-1 receptor antagonist and Fc alpha R (CD89)-mediated down-regulation of tumour necrosis factor-alpha (TNF-alpha) and IL-6 in human monocytes, Clin. Exp. Immunol. 105 (1996) 537–543. A.C. Honorio-Franca, P. Launay, M.M. Carneiro-Sampaio, R.C. Monteiro, Colostral neutrophils express Fc alpha receptors (CD89) lacking gamma chain association and mediate noninXammatory properties of secretory IgA, J. Leukoc. Biol. 69 (2001) 289–296. B.P. Arulanandam, R.H. Raeder, J.G. Nedrud, D.J. Bucher, J. Le, D.W. Metzger, IgA immunodeWciency leads to inadequate Th cell priming and increased susceptibility to inXuenza virus infection, J. Immunol. 166 (2001) 226–231. Y. Zhang, S. Pacheco, C.L. Acuna, et al., Immunoglobulin A-deWcient mice exhibit altered T helper 1-type immune responses but
[22]
[23]
[24]
[25]
[26]
[27]
[28]
[29]
[30]
[31]
[32]
[33]
[34]
[35] [36]
[37]
63
retain mucosal immunity to inXuenza virus, Immunology 105 (2002) 286–294. S. Pal, I. Theodor, E.M. Peterson, L.M. de la Maza, Monoclonal immunoglobulin A antibody to the major outer membrane protein of the Chlamydia trachomatis mouse pneumonitis biovar protects mice against a chlamydial genital challenge, Vaccine 15 (1997) 575–582. T.W. Cotter, Q. Meng, Z.L. Shen, Y.X. Zhang, H. Su, H.D. Caldwell, Protective eYcacy of major outer membrane protein-speciWc immunoglobulin A (IgA) and IgG monoclonal antibodies in a murine model of Chlamydia trachomatis genital tract infection, Infect. Immun. 63 (1995) 4704–4714. G.M. Zhong, R.E. Reid, R.C. Brunham, Mapping antigenic sites on the major outer membrane protein of Chlamydia trachomatis with synthetic peptides, Infect. Immun. 58 (1990) 1450–1455. D. Zhang, X. Yang, H. Lu, G. Zhong, R.C. Brunham, Immunity to Chlamydia trachomatis mouse pneumonitis induced by vaccination with live organisms correlates with early granulocyte–macrophage colony-stimulating factor and interleukin-12 production and with dendritic cell-like maturation, Infect. Immun. 67 (1999) 1606–1613. G.R. Harriman, M. Bogue, P. Rogers, et al., Targeted deletion of the IgA constant region in mice leads to IgA deWciency with alterations in expression of other Ig isotypes, J. Immunol. 162 (1999) 2521–2529. B.P. Arulanandam, J.M. Lynch, D.E. Briles, S. Hollingshead, D.W. Metzger, Intranasal vaccination with pneumococcal surface protein A and interleukin-12 augments antibody-mediated opsonization and protective immunity against Streptococcus pneumoniae infection, Infect. Immun. 69 (2001) 6718–6724. R.M. Buchanan, B.P. Arulanandam, D.W. Metzger, IL-12 enhances antibody responses to T-independent polysaccharide vaccines in the absence of T and NK cells, J. Immunol. 161 (1998) 5525–5533. S. Wang, Y. Fan, R.C. Brunham, X. Yang, IFN-gamma knockout mice show Th2-associated delayed-type hypersensitivity and the inXammatory cells fail to localize and control chlamydial infection, Eur. J. Immunol. 29 (1999) 3782–3792. F. Briere, T. Defrance, B. Vanbervliet, et al., Transforming growth factor beta (TGF beta) directs IgA1 and IgA2 switching in human naive B cells, Adv. Exp. Med. Biol. 371A (1995) 21–26. D.M. Williams, B.G. Grubbs, S. Park-Snyder, R.G. Rank, L.F. Bonewald, Activation of latent transforming growth factor beta during Chlamydia trachomatis-induced murine pneumonia, Res. Microbiol. 147 (1996) 251–262. D. Kowalczyk, J. Baran, A.D. Webster, M. Zembala, Intracellular cytokine production by Th1/Th2 lymphocytes and monocytes of children with symptomatic transient hypogammaglobulinaemia of infancy (THI) and selective IgA deWciency (SIgAD), Clin. Exp. Immunol. 127 (2002) 507–512. M.J. Lieser, R.A. Kozol, S.D. Tennenberg, Interferon-gamma primes neutrophil-mediated gastric surface cell cytotoxicity, Am. J. Physiol. 268 (1995) G843–G848. R. Kumar, Z. Dong, I.J. Fidler, DiVerential regulation of metalloelastase activity in murine peritoneal macrophages by granulocyte–macrophage colony-stimulating factor and macrophage colony-stimulating factor, J. Immunol. 157 (1996) 5104–5111. R.C. Monteiro, J.G. van de Winkel, IgA Fc receptors, Annu. Rev. Immunol. 21 (2003) 177–204. L.F. Bonewald, S.L. Dallas, Role of active and latent transforming growth factor beta in bone formation, J. Cell. Biochem. 55 (1994) 350–357. W.A. Border, N.A. Noble, T. Yamamoto, et al., Natural inhibitor of transforming growth factor-beta protects against scarring in experimental kidney disease, Nature 360 (1992) 361–364.
64
A.K. Murthy et al. / Cellular Immunology 230 (2004) 56–64
[38] W.A. Border, E. Ruoslahti, Transforming growth factor-beta in disease: the dark side of tissue repair, J. Clin. Invest. 90 (1992) 1–7. [39] D.R. Twardzik, J.A. Mikovits, J.E. Ranchalis, A.F. Purchio, L. Ellingsworth, F.W. Ruscetti, Gamma-interferon-induced activation of latent transforming growth factor-beta by human monocytes, Ann. N. Y. Acad. Sci. 593 (1990) 276–284. [40] Y. Ishida, T. Kondo, T. Takayasu, Y. Iwakura, N. Mukaida, The essential involvement of cross-talk between IFN-gamma and TGFbeta in the skin wound-healing process, J. Immunol. 172 (2004) 1848–1855.
[41] S. Ghaem-Maghami, R.L. Bailey, D.C. Mabey, et al., Characterization of B-cell responses to Chlamydia trachomatis antigens in humans with trachoma, Infect. Immun. 65 (1997) 4958– 4964. [42] M. Iizuka, H. Itou, M. Sato, et al., Crohn’s disease associated with selective immunoglobulin A deWciency, J. Gastroenterol. Hepatol. 16 (2001) 951–952. [43] F. Cataldo, V. Marino, G. Bottaro, P. Greco, A. Ventura, Celiac disease and selective immunoglobulin A deWciency, J. Pediatr. 131 (1997) 306–308.
Chlamydia trachomatis pulmonary infection induces greater inXammatory pathology in immunoglobulin A deWcient mice Ashlesh K. Murthya, Jyotika Sharmab, Jacqueline J. Coalsonc, Guangming Zhongb, Bernard P. Arulanandama,¤ b
a Department of Biology, University of Texas at San Antonio, San Antonio, TX 78249, USA Department of Microbiology and Immunology, University of Texas Health Science Center, San Antonio, TX 78229, USA c Department of Pathology, University of Texas Health Science Center, San Antonio, TX 78229, USA
Received 10 July 2004; accepted 10 September 2004
Abstract Chlamydia trachomatis is an intracellular bacterial pathogen that primarily infects via mucosal surfaces. Using mice with a targeted disruption in IgA gene expression (IgA¡/¡ mice), we have studied the contribution of IgA, the principal mucosal antibody isotype, in primary immune defenses against pulmonary C. trachomatis infection. Bacterial burden was comparable between IgA¡/¡ and IgA+/+ animals following C. trachomatis challenge. Serum and pulmonary anti-Chlamydia antibody levels were higher in IgA¡/¡ animals, with the exception of IgA. Lung sections of challenged IgA¡/¡ mice showed more extensive immunopathology than corresponding IgA+/+ animals. Real-time PCR analysis demonstrated signiWcantly greater IFN- and TGF- mRNA expression in IgA¡/¡ as compared to IgA+/+ animals. Together, these results suggest that IgA may not be necessary for clearance of primary C. trachomatis infection. However, IgA¡/¡ mice displayed exaggerated lung histopathology and altered cytokine production, indicating an important role for IgA in regulating C. trachomatis induced pulmonary inXammation and maintenance of mucosal homeostasis. 2004 Elsevier Inc. All rights reserved. Keywords: Chlamydia trachomatis; Immunoglobulin A; InXammation; Innate
1. Introduction Chlamydia trachomatis is an obligate intracellular gram-negative pathogen that is the leading cause of bacterial sexually transmitted disease [1]. C. trachomatis exhibits tropism for mucosal surfaces such as the conjunctiva and the genitourinary tract [1]. Chlamydial infections of the lower genital tract often ascend to the fallopian tubes [2] leading to complications such as pelvic inXammatory disease, ectopic pregnancy, and tubal infertility [2–4]. Despite considerable research, an eVective vaccine against chlamydia is still unavailable.
*
Corresponding author. Fax: +1 210 458 5523. E-mail address: [email protected] (B.P. Arulanandam).
0008-8749/$ - see front matter 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.cellimm.2004.09.002
Cell-mediated and humoral immune mechanisms have been shown to be important in immunity against C. trachomatis. SpeciWcally, T-helper (Th1) type immunity and IFN- production are required for the optimal clearance of chlamydial infection [5,6]. Although CD8+ T-lymphocytes are not required for protection [7], adoptive transfer of chlamydia-speciWc cytotoxic T-cells has been shown to contribute to bacterial clearance [8]. The role of humoral immunity in chlamydial infections has not been resolved. Antibodies were shown not to be important in clearance of chlamydial infections, using mice that were rendered antibody deWcient by anti- treatment [9,10]. Also, Su et al. [11] demonstrated that B-cell deWcient (MT) mice were capable of resolving primary and secondary genital infection with C. trachomatis. However, in the absence of speciWc antibody, mice were
A.K. Murthy et al. / Cellular Immunology 230 (2004) 56–64
shown to be more susceptible to reinfection. Additionally, in a lung C. trachomatis mouse pneumonitis (MoPn) infection model, MT mice not only displayed signiWcantly higher mortality and lung bacterial loads than wild-type mice, but also exhibited impaired T-cell responses to the infection [12]. Furthermore, Moore et al. [13] have demonstrated that the interaction of antibodies and Fc receptors is required for the generation of strong Th1 type immune responses against chlamydia. It has also been reported that immunization with anti-idiotypic antibody against chlamydial glycolipid exoantigen provided protection against genital C. trachomatis challenge [14]. Taken together, these studies suggest that humoral immune mechanisms do play a role in protection against chlamydial infection. IgA is the principal immunoglobulin isotype involved in inhibition of bacterial attachment and neutralization of viruses at mucosal surfaces [15]. In addition, IgA also has been shown to be directly involved in the immunoregulation of phagocytic cells such as monocytes and neutrophils [16–18]. Serum IgA and secretory IgA (sIgA) have individually been shown to suppress inXammation by reducing inXammatory cytokine production or the oxidative burst [17,19]. Furthermore, IgA has been shown to be required for eVective priming of T-cells and development of Th1 type responses [20,21]. In chlamydial infections, IgA has been shown to be eVective in resistance against reinfection in the genital tract [22,23]. However, the role of IgA in innate immunity against chlamydia is not clearly understood. In this study, we have evaluated the role of IgA in a pulmonary C. trachomatis infection, using mice with a targeted disruption in the switch region and -heavy chain locus (IgA¡/¡ mice). Our studies demonstrate that IgA may not be essential for clearance of this primary chlamydial infection. However, IgA deWcient mice exhibit exaggerated lung histopathology and altered inXammatory cytokine production, indicating the importance of IgA in regulating inXammation after bacterial challenge.
2. Materials and methods 2.1. Bacteria Chlamydia trachomatis MoPn strain was grown on conXuent HeLa cell monolayers. The cells were lysed using a sonicator (Fisher, Pittsburgh, PA) and the elementary bodies (EBs) were puriWed on RenograYn gradients as described [24]. Aliquots of bacteria were stored at ¡70 °C in sucrose–phosphate–glutamine buVer. Chlamydia genus-speciWc murine monoclonal antibody [25] was used to conWrm the strain of the puriWed bacterium.
57
2.2. Mice C57BL/6 £ 129 IgA deWcient (IgA¡/¡) mice were generated as described [26] and C57BL/6 £ 129 F2 (IgA+/+) mice were used as wild-type controls. C57BL/6 wild-type and B-cell deWcient (MT) mice were purchased from Jackson Laboratory (Bar Harbor, ME). All mice were 4– 8 weeks old at the time of infection. Mice were housed and bred at the University of Texas at San Antonio Animal Care Facility and provided food and water ad libitum. Animal care and experimental procedures were performed in compliance with the Institutional Animal Care and Use Committee (IACUC) guidelines. 2.3. Intranasal infection procedures and determination of lung bacterial loads Intranasal (i.n.) infections were performed as previously described [27]. BrieXy, mice were anesthetized i.n. with 3% isoXuorane using a rodent anesthesia system (Harvard Apparatus, Holliston, MA) and immediately inoculated i.n. with 200 inclusion forming units (IFU) of MoPn in 25 l of sterile PBS. Lungs from mice were removed 10 days after challenge and weighed. Tissues were homogenized with an electric stirrer (Arrow Junior, Vineland, NJ) and the homogenate centrifuged at 250g for 10 min at 4 °C. The collected supernatants were incubated for 24 h with HeLa cells grown on coverslips in 24well plates. The infected HeLa cells were Wxed with 2% paraformaldehyde and permeabilized with 2% saponin. Cells were washed using PBS and incubated with ModiWed Dulbecco’s Eagle’s Medium containing 10% fetal bovine serum for 1 h to block non-speciWc binding. Thereafter, cells were washed and incubated with antiChlamydia genus-speciWc murine monoclonal antibody and polyclonal rabbit anti-Chlamydia antibody for 1 h and then incubated for an additional 2 h with goat anti-mouse IgG conjugated to Cy3 (Jackson Immunoresearch, West Grove, PA) and goat anti-rabbit Ig conjugated to FITC (Sigma, St. Louis, MO) plus Hoescht nuclear stain. The treated coverslip cultures were then washed and mounted onto superfrost microscope slides (Fisher) using Fluorsave reagent (Calbiochem, La Jolla, CA). Images were acquired using an Axiocam digital camera (Zeiss, Thornwood, NY) connected to a Zeiss Axioskop 2 Plus research microscope. The bacterial loads were calculated and expressed as the number of inclusion forming units per gram of lung tissue. 2.4. Collection of bronchioalveolar lavage Xuid For collection of bronchioalveolar lavage (BAL) Xuid, mice were sacriWced and tracheae intubated using a 0.58 mm OD polyethylene catheter (Becton–Dickinson, Sparks, MD). The lungs were then lavaged two to three times with separate 0.5 ml vol of PBS. The recovered
58
A.K. Murthy et al. / Cellular Immunology 230 (2004) 56–64
BAL Xuids were centrifuged at 500g for 5 min at 4 °C and the supernatants were stored at ¡70 °C until use. 2.5. Detection of antibody and isotype levels by ELISA Microtiter plates were coated overnight with 104 IFU of UV-inactivated MoPn in sodium bicarbonate buVer (pH 9.5), washed with PBS containing 0.3% Brij-35 (Sigma) and blocked for 1 h at room temperature with PBS containing 2% bovine serum albumin (BSA, EM Science Gibbstown, NJ). Serial dilutions of serum or BAL Xuids were added to wells and incubated at room temperature for 2 h. The plates were then washed and incubated for an additional 1 h with goat anti-mouse total Ig, IgG1, IgG2a, IgG2b, IgM, or IgA conjugated to alkaline phosphatase (Southern Biotechnology Associates, Birmingham, AL). After incubation for 1 h, the plates were washed and p-nitrophenyl phosphate substrate was added for color development. Absorbance at 405 nm was measured using an ELISA microplate reader (Biotek Instruments, Winooski, VT). The reciprocal serum dilutions corresponding to 50% maximal binding were used to obtain titers. Because of the low amounts of antibodies in BAL Xuids and large dilution involved in the lavage procedure, titers could not be obtained for BAL, and all samples were tested undiluted. 2.6. Histology and immunoXuorescence staining Lungs from infected mice were removed 3, 5 or 10 days after challenge and embedded in optimal cutting temperature (OCT) resin and snap frozen. Serial horizontal cryosections of 5 m were prepared and placed on silane coated-slides (VWR International, West Chester, PA). All slides were dried overnight and Wxed in fresh acetone for 20 s at room temperature. Some sections also were Wxed with formalin for 10 min and stained by hematoxylin and eosin (H&E). For immunoXuorescent staining, slides were blocked with 3% BSA for 5 min, followed by incubation with 10% normal rat serum (Sigma) for 30 min. Tissue sections were subsequently incubated with R-phycoerythrin (R-PE) conjugated rat anti-mouse CD11b or CD3 (BD Biosciences, San Diego, CA) for 40 min. Sections were washed and mounted using Xuorsave reagent (Calbiochem, La Jolla, CA) containing Hoescht stain (blue for binding to DNA). Some sections were stained using anti-granulocyte (GR1) antibody according to the manufacturer’s instructions (BD Biosciences, San Jose, CA). Images were acquired using an Axiocam digital camera (Zeiss, Thornwood, NY) connected to a Zeiss Axioskop 2 Plus research microscope. 2.7. RNA isolation and real-time quantitative PCR Total RNA was obtained from snap frozen lungs using the Total RNA Isolation Kit (Austin, TX) accord-
ing to the manufacturer’s instructions. First strand cDNA synthesis was carried out from total RNA as described [28]. RT-PCR of the cDNA products was performed with a DyNAmo SYBR Green qPCR kit (Finnzymes, Boston, MA) using a DNA Engine Opticon continuous Xuorescence detection system (MJ Research, Waltham, MA). Sense and antisense primers used were as follows; IFN-, 5⬘-TCAAGTGGCATAGATGTGG AAGAA-3⬘ and 5⬘-TGGCTCTGCAGGATTTTCA TG-3⬘; TGF-, 5⬘-TGACGTCACTGGAGTTGTACG G-3⬘ and 5⬘-GGTTCATGTCATGGATGGTGC-3⬘; and GAPDH, 5⬘-TTCACCACCATGGAGAAGGC-3⬘ and 5⬘-GGCATGGACTGTGGTCATGA-3⬘. Real-time PCR conditions were as follows: 1 s of denaturation at 95 °C, 10 s of primer annealing at 62.5 °C and 20 s of elongation at 72 °C for 40 cycles. QuantiWcation was carried out by monitoring the Xuorescent DNA binding dye SYBR green, at the end of each elongation cycle at 78 °C for IFN- and 81.5 °C for TGF-. Relative quantiWcation was carried out by normalizing levels of the gene of interest to the housekeeping gene GAPDH, and to baseline levels of cytokine expression in uninfected mice of corresponding groups. The values obtained were expressed as fold changes of the gene expression. 2.8. Statistical analyses Sigma Stat (Chicago, IL) was used to perform all the tests of signiWcance. The Mann–Whitney rank sum test was used to determine diVerences between experimental groups, and data were considered statistically signiWcant if p values were <0.05. All data shown are representative of at least 2–3 independent experiments and each experiment shown was analyzed independently.
3. Results 3.1. IgA may not be required for clearance of a primary C. trachomatis lung infection To determine the role of B-cells and antibodies against a primary infection, age matched MT (B-cell deWcient) and corresponding wild-type animals were challenged with 200 IFU of MoPn. Lung bacterial loads 10 days after challenge revealed a 6-log increase in the number of organisms recovered in the MT mice as compared to similarly infected wild-type animals (data not shown). Furthermore, the MT mice developed a progressive loss of body weight, whereas the average weight loss of infected wild-type animals was minimal, indicative of bacterial clearance (data not shown). These results are in agreement with Yang and Brunham [12] who demonstrated that B-cells play an important role in primary bacterial clearance of C. trachomatis infection from the lungs.
A.K. Murthy et al. / Cellular Immunology 230 (2004) 56–64
Fig. 1. Lungs of infected IgA¡/¡ and wild-type animals display equivalent numbers of chlamydia. (A) Wild-type and IgA¡/¡ deWcient mice were infected i.n. on day 0 with 200 IFU MoPn, and lung bacterial loads were examined on day 10 and expressed as IFU/g of lung. Each symbol represents an individual mouse (n D 6). (B) Body weight loss after bacterial challenge. Results are representative of three independent experiments.
59
Fig. 2. Serum anti-Chlamydia antibody titers are higher in the IgA¡/¡ animals. Mice were bled 10 days after challenge and serum analyzed by isotype-speciWc ELISA using UV-inactivated MoPn coated microtiter plates. The results are reported as 50% end point titers. DiVerences in binding between IgA¡/¡ and IgA+/+ mice (n D 4) were signiWcant at p < 0.05 for IgG1 and IgG2b. Results are representative of three independent experiments.
Since IgA is the predominant immunoglobulin in the respiratory mucosa, we extended our observation by examining the contribution of IgA against primary C. trachomatis lung infection in IgA deWcient mice using a similar challenge inoculum. As shown in Fig. 1A, equivalent numbers of bacteria were recovered from the lungs of infected IgA¡/¡ and wild-type animals. In addition, the average weight loss following challenge was similar in both groups of animals (Fig. 1B). These results suggest that although B-cells are required for clearance of a primary C. trachomatis lung infection, the contribution of IgA in this process is not crucial. 3.2. Antibody proWles after primary C. trachomatis pulmonary infection To examine possible reasons for the limited diVerences in bacterial clearance between IgA¡/¡ and IgA+/+ animals, analyses of sera and BAL Xuids were carried out for expression of anti-MoPn antibodies of deWned isotypes. Overall, serum IgM and IgG (IgG1, IgG2a, and IgG2b) titers tended to be higher in the IgA¡/¡ mice as compared to wild-type animals 10 days following bacterial challenge (Fig. 2). Anti-MoPn IgG1 and IgG2b antibody levels were signiWcantly higher in the IgA¡/¡ animals. Respiratory antibody production in BAL Xuids of infected animals also was analyzed 10 days after challenge (Fig. 3). It was found that levels of
Fig. 3. Respiratory anti-Chlamydia antibody titers are higher in the IgA¡/¡ animals. Mice were euthanized 10 days after challenge, BAL Xuids collected and analyzed by isotype-speciWc ELISA using UVinactivated MoPn coated microtiter plates. DiVerences in binding between IgA¡/¡ and IgA+/+ mice (n D 4) were signiWcant at p < 0.05 for IgG1 and IgA. Each symbol represents an individual mouse. Results are representative of two independent experiments.
all antibody isotypes tended to be greater in the IgA¡/¡ animals, with the obvious exception of IgA, which was detected only in the wild-type mice. In particular, levels of IgG1 were signiWcantly greater in the IgA deWcient
60
A.K. Murthy et al. / Cellular Immunology 230 (2004) 56–64
mice. These results suggest that the higher titers of other antibody isotypes present in IgA¡/¡ animals may compensate for the loss of IgA in the clearance of C. trachomatis from the lung. 3.3. IgA¡/¡ mice develop greater histopathological changes after pulmonary C. trachomatis infection Lung histopathology was examined in tissues obtained from IgA¡/¡ and corresponding wild-type control mice challenged with 200 IFU MoPn. Lungs were removed on days 3, 5 or 10 after challenge. Sections from lungs removed 3 days after challenge showed foci of pulmonary edema and scattered inXammatory cells in the peribronchiolar alveoli of both types of mice (Figs. 4A, arrow and B). The distribution of the alveolar exudates was within and around the airways, indicating
Fig. 4. IgA¡/¡ mice exhibit extensive histopathological changes after pulmonary C. trachomatis challenge. (A) IgA+/+ lung: day 3, (B) IgA¡/¡ lung: day 3, (C) IgA+/+ lung: day 5, (D) IgA¡/¡ lung: day 5, (E) IgA+/+ lung: day 10, (F) IgA¡/¡ lung: day 10, (G) mock-infected IgA+/+ lung, (H) mock-infected IgA¡/¡ lung. a: airway; b: bronchiole. MagniWcation 10£. Results are representative of three independent experiments.
the establishment of an early bronchopneumonia. However, at this time-point, lung sections of the IgA¡/¡ animals (Fig. 4B) showed more extensive inWltration in the lungs than the corresponding wild-type animals (Fig. 4A). Examination at higher magniWcation revealed the presence of more cellular debris and greater numbers of polymorphonucleur cells in sections from IgA¡/¡ lungs when compared to wild-type animals (data not shown). While the tissue sections from both groups displayed bronchopneumonia by day 5 after challenge, the lesions were more extensive in the IgA¡/¡ animals (Fig. 4D) when compared to wild-type animals (Fig. 4C). The size of the inXammatory inWltrates was small in the IgA+/+ animals (Fig. 4C) with sparing of alveolar spaces around them, as compared to the much larger inWltrates in IgA¡/¡ animals (Fig. 4D). Interestingly, it was also found that IgA¡/¡ animals also displayed increased numbers of inWltrating inXammatory cells around the bronchioles (peribronchiolar cuYng) and the vasculature (perivascular cuYng), which was not observed in lung sections from IgA+/+ animals (data not shown). The histopathology 10 days after challenge displayed progressive changes, except that the lungs of IgA¡/¡ animals (Fig. 4F) displayed complete alveolar Wlling of entire lobes of the infected lungs, while the wild-type lungs (Fig. 4E) still showed a bronchopneumonia pattern. There were no histopathological diVerences in the mock-infected lungs of both groups of animals (Figs. 4G and H). Since the histopathology on day 5 after challenge displayed considerable diVerences between the IgA ¡/¡ and wild-type animals, we carried out a detailed analysis of the immune response to infection at this timepoint, using in situ immunohistochemistry. Staining of lung sections for granulocytes with anti-Gr1 antibody demonstrated abundant neutrophils in the parenchymal inWltrates of IgA¡/¡ and IgA+/+ animals (data not shown). In addition, IgA¡/¡ mice displayed greater neutrophilic inWltrates around the bronchioles and vasculature and also inside the bronchiolar lumen of IgA¡/¡ mice, with noticable disruption of bronchiolar wall architecture, which was not observed in lung sections of IgA+/+ animals (data not shown). Patterns of T-lymphocyte staining revealed similar distribution of cells within parenchymal inWltrates of the IgA+/+ (Fig. 5A, arrow) and IgA¡/¡ animals. However, T-lymphocytes were observed around bronchioles (Fig. 5B, arrow) and vasculature (Fig. 5B, inset) of IgA¡/¡ animals alone. Similar patterns of macrophage distribution were observed, with no evidence of B-cells at this time-point after challenge (data not shown). There was no apparent pathology in lung sections from control uninfected IgA¡/¡ (Fig. 5D) and wild-type mice (Fig. 5C). Thus, infected IgA¡/¡ mice exhibited greater inXammatory lung histopathology than similarly infected wild-type animals.
A.K. Murthy et al. / Cellular Immunology 230 (2004) 56–64
61
Fig. 6. IFN- and TGF- mRNA are expressed at higher levels in the lungs of C. trachomatis infected IgA¡/¡ mice. RNA was prepared from infected tissues (n D 3) followed by examination of IFN- and TGF- levels by real-time PCR. (A) levels of IFN- and TGF- on day 3 and (B) on day 5 after challenge. IFN- and TGF- levels at day 5 in the lungs of IgA¡/¡ mice were signiWcantly diVerent from similarly infected IgA+/+ mice (p < 0.05). Results are representative of three independent experiments.
Fig. 5. Infected IgA¡/¡ animals exhibit peribronchiolar and perivascular cuYng. Lung sections were stained with an anti CD3 antibody conjugated with phycoerythrin (PE) following challenge. (A) IgA+/+ lung: day 5, (B) IgA¡/¡ lung: day 5, (C) mock-infected IgA+/+ lung, (D) mock-infected IgA¡/¡ lung. AS: alveolar space; B: bronchiole; A: arteriole. MagniWcation 10£. Results are representative of two independent experiments.
3.4. Altered expression of IFN- and TGF- in the lungs of C. trachomatis infected IgA¡/¡ mice Since IFN- is known to be important in clearance of primary C. trachomatis infection [29], we determined the level of IFN- expression in the lungs of infected IgA¡/¡ and wild-type animals, using real time PCR (Fig. 6). As shown in panel A, IFN- expression was relatively low in both groups of animals 3 days after challenge. However, 5 days after challenge, IgA¡/¡ mice exhibited an approximately 12-fold greater increase in IFN- levels than wildtype animals (panel B). Transforming growth factor- (TGF-) is a known Ig isotype switch factor for IgA [30], and is thought to play an important role in chlamydial infections by modulating cellular immune responses, enhancing host defenses via production of IgA, and potentially contributing to scar tissue formation [31]. Therefore, we also examined the levels of TGF- in the lungs of infected IgA¡/¡ and IgA+/+ animals (Fig. 6). Similar levels of this cytokine were seen in both the IgA¡/¡ and wild-type animals on day 3 following challenge (panel A). However, IgA¡/¡ mice displayed a 28-fold greater production of TGF- than wild-type animals on day 5 (panel B).
4. Discussion We have evaluated the role of IgA against primary pulmonary Chlamydia trachomatis infection. The absence of IgA did not adversely aVect bacterial clearance upon challenge, possibly due to compensatory mechanisms, but was associated with greater pulmonary histopathological changes than in wild-type animals. Therefore, IgA may play an important role, apart from simple neutralization, in regulating inXammation and in maintenance of mucosal homeostasis after chlamydial infection. Although IgA is the principal immunoglobulin isotype involved in host defense against pathogens at mucosal surfaces [15], our results suggest that IgA may not be necessary for clearance of a primary C. trachomatis lung infection. IgA¡/¡ mice exhibit higher titers of other anti-chlamydial antibody isotypes, which probably compensate for the lack of IgA. IgA¡/¡ mice also have been shown to produce higher titers of serum [26] and pulmonary antibodies [20], when compared to wild-type animals. Because of the high degree of inXammation during chlamydial infection, there is a likelihood of serum antibody diVusion into the respiratory compartment, apart from local antibody production. In addition, the levels of IFN-, known to be important in clearance of chlamydial infections [29], were signiWcantly elevated in the lungs of IgA¡/¡ as compared to wild-type animals in our studies. Therefore, compensatory mechanisms involving other antibody isotypes and elevated IFN- may promote bacterial clearance in IgA¡/¡ mice with
62
A.K. Murthy et al. / Cellular Immunology 230 (2004) 56–64
similar eYciency to the wild-type animals. To this end, monoclonal anti-chlamydial IgA treatment has been shown to eVectively reduce the incidence of genital tract infection, but has a marginal eVect on clearance of organisms [22,23]. IgA, however, does play a major role in regulating inXammatory cytokine responses to bacterial infections. In our study, the absence of IgA resulted in greater pulmonary histopathology following i.n. C. trachomatis challenge. Although evidence of inXammation was found in both types of animals, a more extensive lung involvement was observed in the IgA¡/¡ animals, associated with signs of severe inXammation such as perivascular and peribronchiolar cuYng, which were not present in the lung sections from IgA+/+ animals. In particular, the level of IFN- in the lungs of mice was found to be 12-fold greater in the absence of IgA. Likewise, the frequency of CD3+/CD4+ T-cells secreting IFN- is increased in children with selective IgA deWciency [32]. IFN- has been shown to activate polymorphonuclear cells leading to enhancement of oxidative responses [33] and also to prime macrophages to release proteinases that degrade the host tissue [34]. Lung sections from C. trachomatis infected IgA¡/¡ mice, but not IgA+/+ mice, demonstrated macrophage and neutrophilic inWltrates around vasculature and bronchioles. These cells, in the presence of IFN-, may cause severe inXammatory damage to these structures, as evidenced by the enhanced immunopathological changes observed in the absence of IgA. Human serum IgA has been reported to down-regulate the release of TNF- and IL-6 in H. inXuenzae type b-activated monocytes via induction of IL-1 receptor antagonist [17,18]. In addition, secretory IgA has been shown to bind colostral neutrophils and reduce the release of superoxide anions [19]. IgA has been shown to mediate many of its biological activities in humans by interacting with CD89 (Fc receptor), found on a variety of cell types including neutrophils, eosinophils, and macrophages [35]. However, it is important to note that the murine CD89 homologue has yet to be described. Infected IgA¡/¡ mice also produced signiWcantly greater amounts of TGF- transcripts in the lungs than wild-type animals 5 days after MoPn challenge. TGF- is an important immunoregulatory cytokine that has eVects on various cell types [36] and is an isotype switch factor for IgA [30]. Since TGF- occurs in both latent and active forms, we analyzed the levels of active protein in BAL Xuids collected on day 5 after challenge, using ELISA. However, active TGF- protein could not be detected at this time-point (Murthy and Arulanandam, unpublished observations). This suggests a lack of correlation between TGF- gene expression and active protein in the lung at this time-point, which supports similar observations by Williams et al. [31], indicating the likelihood of post-transcriptional regulation of TGF- during chlamydial infection.
Although TGF- is a potent inducer of matrix formation and is involved in wound healing, excessive amounts of this cytokine may be responsible for scarring and Wbrosis [37,38]. In addition, latent TGF- released into host tissues can be activated by various factors including IFN- [39]. It has been shown recently that endogenous IFN- can negatively regulate the TGF- 1 signaling pathway, resulting in retardation of wound healing [40]. Therefore, in genital chlamydial infections, repeated episodes of severe inXammation in the fallopian tubes due to elevated levels of IFN-, plus potential excessive Wbrosis and scarring due to elevated levels of TGF- in the absence of IgA, may lead to serious complications such as infertility. To this end, it has been shown that anti-chlamydial MOMP IgA secreting B-cells are reduced during periods of intense ocular inXammation in human trachoma [41], suggesting that IgA may be involved in regulating inXammatory processes. In fact, IgA immunodeWciency in humans is a heterologous condition that can be associated with inXammatory conditions such as Coeliac and Crohn’s disease [42,43]. The altered IFN-/TGF- axis may account for the increased pathology and eVective bacterial clearance in the absence of IgA, observed in our studies. The signiWcant increases in the levels of IFN- are synchronous with the greater severity of inXammatory lung histopathology in IgA¡/¡ mice. In the absence of IgA, the production of increased levels of TGF- upon C. trachomatis infection may be an important factor in the development of complications arising from chronic inXammation. In summary, the results of this study demonstrate that IgA plays an important role in innate C. trachomatis pulmonary infection. It appears that apart from providing epithelial barrier protection, IgA is pivotally involved in regulating cytokine responses and inXammatory pathology with this infection.
Acknowledgments This work was supported by National Institutes of Health Grants AR048973-02 and SO6GM008194-24. The authors gratefully acknowledge Dr. Neal Guentzel for review of the manuscript and Ms. Yanqing Huang for technical expertise.
References [1] R.P. Morrison, H.D. Caldwell, Immunity to murine chlamydial genital infection, Infect. Immun. 70 (2002) 2741–2751. [2] K.A. Kelly, Cellular immunity and Chlamydia genital infection: induction, recruitment, and eVector mechanisms, Int. Rev. Immunol. 22 (2003) 3–41. [3] L. Westrom, R. Joesoef, G. Reynolds, A. Hagdu, S.E. Thompson, Pelvic inXammatory disease and fertility. A cohort study of 1,844
A.K. Murthy et al. / Cellular Immunology 230 (2004) 56–64
[4] [5]
[6]
[7]
[8]
[9]
[10]
[11]
[12]
[13]
[14]
[15] [16]
[17]
[18]
[19]
[20]
[21]
women with laparoscopically veriWed disease and 657 control women with normal laparoscopic results, Sex. Transm. Dis. 19 (1992) 185–192. L. Westrom, P.A. Mardh, Chlamydial salpingitis, Br. Med. Bull. 39 (1983) 145–150. L.L. Perry, K. Feilzer, H.D. Caldwell, Immunity to Chlamydia trachomatis is mediated by T helper 1 cells through IFN-gammadependent and -independent pathways, J. Immunol. 158 (1997) 3344–3352. R.G. Rank, K.H. Ramsey, E.A. Pack, D.M. Williams, EVect of gamma interferon on resolution of murine chlamydial genital infection, Infect. Immun. 60 (1992) 4427–4429. S.G. Morrison, H. Su, H.D. Caldwell, R.P. Morrison, Immunity to murine Chlamydia trachomatis genital tract reinfection involves B cells and CD4(+) T cells but not CD8(+) T cells, Infect. Immun. 68 (2000) 6979–6987. J.U. Igietseme, D.M. Magee, D.M. Williams, R.G. Rank, Role for CD8+ T cells in antichlamydial immunity deWned by Chlamydia-speciWc T-lymphocyte clones, Infect. Immun. 62 (1994) 5195–5197. D.M. Williams, B. Grubbs, J. Schachter, Primary murine Chlamydia trachomatis pneumonia in B-cell-deWcient mice, Infect. Immun. 55 (1987) 2387–2390. H. Ramsey, L.S. Soderberg, R.G. Rank, Resolution of chlamydial genital infection in B-cell-deWcient mice and immunity to reinfection, Infect. Immun. 56 (1988) 1320–1325. H. Su, K. Feilzer, H.D. Caldwell, R.P. Morrison, Chlamydia trachomatis genital tract infection of antibody-deWcient gene knockout mice, Infect. Immun. 65 (1997) 1993–1999. X. Yang, R.C. Brunham, Gene knockout B cell-deWcient mice demonstrate that B cells play an important role in the initiation of T cell responses to Chlamydia trachomatis (mouse pneumonitis) lung infection, J. Immunol. 161 (1998) 1439–1446. T. Moore, C.O. Ekworomadu, F.O. Eko, et al., Fc receptor-mediated antibody regulation of T cell immunity against intracellular pathogens, J. Infect. Dis. 188 (2003) 617–624. J.A. Whittum-Hudson, D. Rudy, H. Gerard, et al., The anti-idiotypic antibody to chlamydial glycolipid exoantigen (GLXA) protects mice against genital infection with a human biovar of Chlamydia trachomatis, Vaccine 19 (2001) 4061–4071. M.E. Lamm, Interaction of antigens and antibodies at mucosal surfaces, Annu. Rev. Microbiol. 51 (1997) 311–340. H.M. Wolf, E. Vogel, M.B. Fischer, H. Rengs, H.P. Schwarz, M.M. Eibl, Inhibition of receptor-dependent and receptor-independent generation of the respiratory burst in human neutrophils and monocytes by human serum IgA, Pediatr. Res. 36 (1994) 235–243. H.M. Wolf, M.B. Fischer, H. Puhringer, A. Samstag, E. Vogel, M.M. Eibl, Human serum IgA downregulates the release of inXammatory cytokines (tumor necrosis factor-alpha, interleukin6) in human monocytes, Blood 83 (1994) 1278–1288. H.M. Wolf, I. Hauber, H. Gulle, et al., Anti-inXammatory properties of human serum IgA: induction of IL-1 receptor antagonist and Fc alpha R (CD89)-mediated down-regulation of tumour necrosis factor-alpha (TNF-alpha) and IL-6 in human monocytes, Clin. Exp. Immunol. 105 (1996) 537–543. A.C. Honorio-Franca, P. Launay, M.M. Carneiro-Sampaio, R.C. Monteiro, Colostral neutrophils express Fc alpha receptors (CD89) lacking gamma chain association and mediate noninXammatory properties of secretory IgA, J. Leukoc. Biol. 69 (2001) 289–296. B.P. Arulanandam, R.H. Raeder, J.G. Nedrud, D.J. Bucher, J. Le, D.W. Metzger, IgA immunodeWciency leads to inadequate Th cell priming and increased susceptibility to inXuenza virus infection, J. Immunol. 166 (2001) 226–231. Y. Zhang, S. Pacheco, C.L. Acuna, et al., Immunoglobulin A-deWcient mice exhibit altered T helper 1-type immune responses but
[22]
[23]
[24]
[25]
[26]
[27]
[28]
[29]
[30]
[31]
[32]
[33]
[34]
[35] [36]
[37]
63
retain mucosal immunity to inXuenza virus, Immunology 105 (2002) 286–294. S. Pal, I. Theodor, E.M. Peterson, L.M. de la Maza, Monoclonal immunoglobulin A antibody to the major outer membrane protein of the Chlamydia trachomatis mouse pneumonitis biovar protects mice against a chlamydial genital challenge, Vaccine 15 (1997) 575–582. T.W. Cotter, Q. Meng, Z.L. Shen, Y.X. Zhang, H. Su, H.D. Caldwell, Protective eYcacy of major outer membrane protein-speciWc immunoglobulin A (IgA) and IgG monoclonal antibodies in a murine model of Chlamydia trachomatis genital tract infection, Infect. Immun. 63 (1995) 4704–4714. G.M. Zhong, R.E. Reid, R.C. Brunham, Mapping antigenic sites on the major outer membrane protein of Chlamydia trachomatis with synthetic peptides, Infect. Immun. 58 (1990) 1450–1455. D. Zhang, X. Yang, H. Lu, G. Zhong, R.C. Brunham, Immunity to Chlamydia trachomatis mouse pneumonitis induced by vaccination with live organisms correlates with early granulocyte–macrophage colony-stimulating factor and interleukin-12 production and with dendritic cell-like maturation, Infect. Immun. 67 (1999) 1606–1613. G.R. Harriman, M. Bogue, P. Rogers, et al., Targeted deletion of the IgA constant region in mice leads to IgA deWciency with alterations in expression of other Ig isotypes, J. Immunol. 162 (1999) 2521–2529. B.P. Arulanandam, J.M. Lynch, D.E. Briles, S. Hollingshead, D.W. Metzger, Intranasal vaccination with pneumococcal surface protein A and interleukin-12 augments antibody-mediated opsonization and protective immunity against Streptococcus pneumoniae infection, Infect. Immun. 69 (2001) 6718–6724. R.M. Buchanan, B.P. Arulanandam, D.W. Metzger, IL-12 enhances antibody responses to T-independent polysaccharide vaccines in the absence of T and NK cells, J. Immunol. 161 (1998) 5525–5533. S. Wang, Y. Fan, R.C. Brunham, X. Yang, IFN-gamma knockout mice show Th2-associated delayed-type hypersensitivity and the inXammatory cells fail to localize and control chlamydial infection, Eur. J. Immunol. 29 (1999) 3782–3792. F. Briere, T. Defrance, B. Vanbervliet, et al., Transforming growth factor beta (TGF beta) directs IgA1 and IgA2 switching in human naive B cells, Adv. Exp. Med. Biol. 371A (1995) 21–26. D.M. Williams, B.G. Grubbs, S. Park-Snyder, R.G. Rank, L.F. Bonewald, Activation of latent transforming growth factor beta during Chlamydia trachomatis-induced murine pneumonia, Res. Microbiol. 147 (1996) 251–262. D. Kowalczyk, J. Baran, A.D. Webster, M. Zembala, Intracellular cytokine production by Th1/Th2 lymphocytes and monocytes of children with symptomatic transient hypogammaglobulinaemia of infancy (THI) and selective IgA deWciency (SIgAD), Clin. Exp. Immunol. 127 (2002) 507–512. M.J. Lieser, R.A. Kozol, S.D. Tennenberg, Interferon-gamma primes neutrophil-mediated gastric surface cell cytotoxicity, Am. J. Physiol. 268 (1995) G843–G848. R. Kumar, Z. Dong, I.J. Fidler, DiVerential regulation of metalloelastase activity in murine peritoneal macrophages by granulocyte–macrophage colony-stimulating factor and macrophage colony-stimulating factor, J. Immunol. 157 (1996) 5104–5111. R.C. Monteiro, J.G. van de Winkel, IgA Fc receptors, Annu. Rev. Immunol. 21 (2003) 177–204. L.F. Bonewald, S.L. Dallas, Role of active and latent transforming growth factor beta in bone formation, J. Cell. Biochem. 55 (1994) 350–357. W.A. Border, N.A. Noble, T. Yamamoto, et al., Natural inhibitor of transforming growth factor-beta protects against scarring in experimental kidney disease, Nature 360 (1992) 361–364.
64
A.K. Murthy et al. / Cellular Immunology 230 (2004) 56–64
[38] W.A. Border, E. Ruoslahti, Transforming growth factor-beta in disease: the dark side of tissue repair, J. Clin. Invest. 90 (1992) 1–7. [39] D.R. Twardzik, J.A. Mikovits, J.E. Ranchalis, A.F. Purchio, L. Ellingsworth, F.W. Ruscetti, Gamma-interferon-induced activation of latent transforming growth factor-beta by human monocytes, Ann. N. Y. Acad. Sci. 593 (1990) 276–284. [40] Y. Ishida, T. Kondo, T. Takayasu, Y. Iwakura, N. Mukaida, The essential involvement of cross-talk between IFN-gamma and TGFbeta in the skin wound-healing process, J. Immunol. 172 (2004) 1848–1855.
[41] S. Ghaem-Maghami, R.L. Bailey, D.C. Mabey, et al., Characterization of B-cell responses to Chlamydia trachomatis antigens in humans with trachoma, Infect. Immun. 65 (1997) 4958– 4964. [42] M. Iizuka, H. Itou, M. Sato, et al., Crohn’s disease associated with selective immunoglobulin A deWciency, J. Gastroenterol. Hepatol. 16 (2001) 951–952. [43] F. Cataldo, V. Marino, G. Bottaro, P. Greco, A. Ventura, Celiac disease and selective immunoglobulin A deWciency, J. Pediatr. 131 (1997) 306–308.