- Email: [email protected]
Inhibition of lymphocyte CD3 expression by Chlamydophila pneumoniae infection
Microbial Pathogenesis 45 (2008) 290–296
Contents lists available at ScienceDirect
Microbial Pathogenesis journal homepage: www.elsevier.com/locate/micpath
Inhibition of lymphocyte CD3 expression by Chlamydophila pneumoniae infection Hiroyuki Yamaguchi a, b, *, Junji Matsuo b, Shigehiro Sugimoto a, Maki Utsumi a, Yoshimasa Yamamoto a a b
Laboratory of Molecular Microbiology, Department of Bioinformatics, Graduate School of Medicine, Osaka University, 1-7 Yamadaoka, Suita, Osaka 565-0871, Japan Department of Medical Laboratory Sciences, Graduate School of Health Sciences, Hokkaido University, Nishi-5 Kita-12 Jo, Kita-ku, Sapporo, Hokkaido 060-0812, Japan
a r t i c l e i n f o
a b s t r a c t
Article history: Received 10 January 2008 Received in revised form 16 June 2008 Accepted 26 June 2008 Available online 12 July 2008
Since lymphocytes are a major immune cell besides macrophages in the development of atherosclerosis, interaction between lymphocytes and Chlamydophila pneumoniae may contribute to the pathogenesis of chronic inflammatory diseases associated with C. pneumoniae. In this regard, we examined a possible alteration of CD3 expression of human lymphocyte Molt-4 cells by C. pneumoniae infection. The expression levels of CD3 molecules of lymphocyte Molt-4 cells were significantly decreased by C. pneumoniae infection. In contrast, heat-killed C. pneumoniae as well as mock (cell lysates) did not cause any alteration of CD3 expression of the cells. Treatment of the infected cells with NS-398 (cyclooxyganase-2 inhibitor) or AH-23848 (EP4 prostanoid receptor antagonist) abolished the inhibition of CD3 expression. The enhanced prostaglandin E2 (PGE2) productions in the culture supernatants of infected cells were confirmed by competitive enzyme-immunosorbent assay (ELISA). C. pneumoniae infection of enriched lymphocytes from human peripheral blood mononuclear cells also induced a decrease of CD3 expression. Thus, C. pneumoniae infection of lymphocytes induces a decrease of CD3 expression mediated by possibly PGE2 production. Ó 2008 Elsevier Ltd. All rights reserved.
Keywords: Chlamydophila pneumoniae lymphocytes Molt-4 cells CD3 prostaglandin E2
1. Introduction Chlamydophila pneumoniae is an obligate intracellular bacterium associated with human respiratory pathogen causing pneumonia [1–3]. Current studies indicate that this organism may be implicated in the induction of chronic inflammatory diseases such as asthma, arthritis, endocarditis, and atherosclerosis [4–7]. However, although accumulating studies indicated a possible linking C. pneumoniae infection and chronic inflammatory diseases, the mechanisms for development of such disease by this organism are not clear [8]. Lymphocytes are a major immune cell besides macrophages in the development of chronic inflammatory diseases, such as atherosclerosis [9, 10]. Therefore, a direct interaction between lymphocytes and this pathogen, if it occurs, may contribute to the pathogenesis of chronic inflammatory diseases associated with C. pneumoniae infection. Kaul et al. reported that C. pneumoniae DNA could be recovered from CD3þ peripheral blood leukocytes obtained from patients attending a cardiology [11]. In addition, our studies also demonstrated that C. pneumoniae infects and multiplies in lymphocytes in vitro [12, 13]. These findings
* Corresponding author. Department of Medical Laboratory Sciences, Graduate School of Health Sciences, Hokkaido University, Nishi-5 Kita-12 Jo, Kita-ku, Sapporo, Hokkaido 060-0812, Japan. Tel./fax: þ81 11 706 3326. E-mail address: [email protected] (H. Yamaguchi). 0882-4010/$ – see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.micpath.2008.06.005
indicate that lymphocytes are a potential host cell for C. pneumoniae infection. Therefore, it can be possible that immune functions of lymphocytes infected with C. pneumoniae may be altered and such alterations lead an inappropriate immune response to stimulation, which eventually contribute to the pathogenesis of the disease. CD3 receptor is a crucial molecule in T cell signal transduction [14–16]. Although its expression of cell is constitutive, dynamic regulation of T-cell receptor (TCR)/CD3 complex expression level is probably the most important mechanism allowing T cells to calibrate their response to different levels of stimuli or to sense harmful pathogen in cells. Therefore, a possible alteration of CD3 expression of lymphocytes by C. pneumoniae infection, if it occurs, may be a critical in the pathogenesis of the diseases associated with this pathogen. In this regard, in the present study, we examined a possible alteration of CD3 expression of lymphocytes by C. pneumoniae infection. 2. Results 2.1. C. pneumoniae infection resulted in a decrease of CD3 expression of Molt-4 cells Fig. 1 shows representative micrographs and dot plots of the cells with or without infection at 72 h after incubation. Flow data of double-labeled cells indicated a decrease of CD3 expression of
H. Yamaguchi et al. / Microbial Pathogenesis 45 (2008) 290–296
291
Fig. 1. Representative double-fluorescence staining micrographs (A) and dot-plot images (B) of C. pneumoniae-infected lymphocyte Molt-4 cells at 72 h after incubation. The cells were infected with bacteria at MOI of 10. The cells treated with the permeabilization kit (see Section 5) were stained with PE-conjugated anti-CD3 MoAb and FITC-conjugated antiChlamydia-MoAb. The specimens were then analyzed by fluorescence microscopy and flow cytometry. (A) Red and green colors are indicating CD3 expression and C. pneumoniae inclusion of lymphocyte Molt-4 cells, respectively. Magnification, 1000. (B) Gated cells (R1) are showing analyzed cell population excluding cell debris and dead cells (upper panels). The dot-plot intensity showing CD3 expression of infected cells was lower than that of uninfected cells (lower panels). The mean intensities of CD3 expression of uninfected and infected cells were 75.06 and 51.46, respectively. In this case, the reduction rate showing a decrease of CD3 expression of infected cells compared with that of uninfected cells was 23.6%. The infectious rate in culture was approximately 10%.
infected cells compared with CD3 expression of uninfected cells. There was no difference between dot-plot patterns with side scatter (SSC) and forward scatter (FSC), indicating the influence of cell debris and dead cells causing artificial staining results was minimal. The micrographs also showed that the double-labeled cells were morphologically normal and this staining method was certainly dyed in different colors. Kinetic change of CD3 expression of Molt-4 cells by C. pneumoniae infection was assessed as follows. The cells were infected at MOI of 1 or 10 and incubated for up to 72 h. At various time points, CD3 expression was measured by flow cytometry using anti-CD3 MoAb. As shown in Fig. 2, C. pneumoniae infection induced a decrease of CD3 expression depending on MOI. The decreasing rates of CD3 molecules of Molt-4 cells became the maximum at 72 h after infection. In contrast, a 4 b 1 integrin very late activation antigen-4 (VLA-4) expression was not altered by the infection for up to 72 h. Heat-killed bacteria and HEp-2 cell lysates (mock) did not cause any alteration of CD3 expression of Molt-4 cells (Fig. 3). 2.2. Inhibition of CD3 expression of human peripheral blood lymphocytes by C. pneumoniae infection In order to determine a possible alteration of CD3 expression of primary lymphocytes by infection as observed in established Molt-
4 cells, human lymphocytes enriched from peripheral blood mononuclear cells (PBMCs) were infected with or without viable bacteria in the presence or absence of cycloheximide for up to 48 h, and alteration of CD3 expression of primary lymphocytes were assessed at 24 and 48 h after infection. As shown in Fig. 4, the infected cells showed an obvious decrease of CD3 expression of primary lymphocytes at 48 h after infection. The inhibition of CD3 expression was completely diminished by the treatment with cycloheximide, which inhibits host cell protein synthesis. 2.3. Inhibition of CD3 expression mediated by soluble factors Alteration of CD3 expression of lymphocytes may be induced by several mechanisms, including an involvement of soluble factors produced by lymphocytes. In order to determine such a possibility in the inhibition induced by C. pneumoniae infection, transwell plates with a membrane were utilized for assessment of possible involvement of soluble factors. Both chambers of the transwell plates were cultured with Molt-4 cells. The cells in the upper chamber were infected with C. pneumoniae at MOI of 1–50, but the lower chamber contained only cells without infection. The percentage inhibition of CD3 expression of both chambers determined by flow cytometry was measured at 72 h after incubation. As shown in Fig. 5, the infected Molt-4 cells in the upper chamber
292
H. Yamaguchi et al. / Microbial Pathogenesis 45 (2008) 290–296
VLA-4 expression
100
*
90
*
80 70
*
* 60
Control MOI 1 MOI 10
50 40
0
24
48
72
VLA-4 expression ( of control at each time point)
CD3 expression ( of control at each time point)
CD3 expression 100 90 80 70 60 50 40
0
24
48
72
Time after the infection (h)
Time after the infection (h)
Fig. 2. Inhibition of CD3 expression by C. pneumoniae infection. Molt-4 cells infected with bacteria (MOI 1 or 10) were incubated for up to 72 h. Cells were then washed, stained, and analyzed by flow cytometry. Mean fluorescence intensity was measured at each time point. Results are given as a percentage of uninfected cells at the same time point. Each point represents the mean SD (error bars) for three experiments. *p < 0.05, Significantly different from the control at the same time point.
showed a decrease of CD3 expression depending on MOI. The cells in the lower chamber without infection also showed a decrease of CD3 expression of Molt-4 cells as observed in the upper chamber. Even though the cells without infection as control in both chambers were cultured for 72 h, no alteration of CD3 expression of Molt-4 cells was observed.
2.4. Decrease of CD3 expression mediated by PGE2 in infected cells As shown in Figs. 6 and 7, on treatment both drugs similarly diminished C. pneumoniae induced inhibition of CD3 expression of Molt-4 cells. The percentages of Chlamydia-positive cells in infected cultures did not significantly differ between NS-398 treatment and untreatment for up to 72 h after infection (data not shown). These results indicate a possible involvement of PGE2 on the inhibition of CD3 expression of Molt-4 cells by C. pneumoniae infection. Therefore, to confirm this possible involvement of PGE2, the amount of PEG2 in culture supernatants of Molt-4 cells infected with C. pneumoniae was measured. As shown in Fig. 8, the production of PGE2 in culture supernatant of Molt-4 cells was significantly enhanced by C. pneumoniae infection. No enhanced production of PGE2 was observed by stimulation with either mock or heat-killed
bacteria. In the presence of drugs, C. pneumoniae infection did not cause any increase of PGE2 production of Molt-4 cells. 3. Discussion Molt-4, a well-characterized human lymphocyte cell line [17], has been used in our previous studies of C. pneumoniae infection model [12,13]. It was demonstrated that C. pneumoniae could infect and grow in Molt-4 cells as well as human peripheral blood lymphocytes [12,13]. We also confirmed typical inclusions formed in infected cells, indicating the infection and growth of C. pneumoniae in Molt-4 cells and this system is suitable for analyzing details of C. pneumoniae–lymphocyte interaction. The inhibition of CD3 expression of Molt-4 cells was induced by C. pneumoniae infection. However, mock or heat-killed bacteria treatment did not show any alteration of CD3 expression of the cells. Thus, the results clearly indicate that the inhibition of CD3
CD3 expression ( of control at each time point)
48h 72h 100
90
*
80
* 70
60
Control
Mock
Heat killed
Viable (MOI 10)
Fig. 3. Alteration of CD3 expression of Molt-4 cells treated with either mock or heatkilled bacteria at 42 and 72 h after incubation. Sonicated HEp-2 cells were used for mock control. The data shown represent the mean þ SD (error bars) for three experiments. *p < 0.05, Significantly different from the control at the same time point.
Fig. 4. Levels of CD3 expression of human lymphocytes enriched from peripheral blood with or without cycloheximide at 24 and 48 h after infection. Mean fluorescence intensity was measured at each time point. Results are given as a percentage of uninfected cells at the same time point. The infectious rate in culture was approximately 2%. Each bar represents the mean þ SD (error bars) for three experiments. *p < 0.05, Significantly different from the control at the same time point.
72h
90
* *
* *
70
60
50 Control
48h
1
10
of control)
80
*
*
293
Upper chamber Lower chamber
CD3 expression (
of control)
100
CD3 expression (
H. Yamaguchi et al. / Microbial Pathogenesis 45 (2008) 290–296
100
*
90
*
* *
*
* *
80
*
*
70
50
MOI Fig. 5. Involvement of soluble factors in inhibition of CD3 expression of Molt-4 cells infected with C. pneumoniae. Molt-4 cells were added to both upper and lower chambers in transwell plates, and the cells in the upper chamber were infected with C. pneumoniae. At 72 h after infection, mean fluorescence intensities for CD3 expressions of Molt-4 cells in both chambers were assessed by flow cytometry. Results are given as a percentage of uninfected cells at the same time point. Each bar represents the mean þ SD (error bars) for three experiments. *p < 0.05, Significantly different from the control at the same side of chamber at the same time point.
expression of lymphocytes is induced by infection, not by antigen stimulation. The inhibition of CD3 expression of lymphocytes enriched from human PBMCs by C. pneumoniae infection was also observed as seen in infected Molt-4 cells. However, the inhibition level of CD3 expression of human primary lymphocytes was lower than that of Molt-4 cells infected with bacteria. Although it is not clear the precise mechanisms of inhibition in the primary cells and the cell line cells, the inhibition potency by infection may be reflected by a difference in host cell proliferation and/or in bacterial growth in host cells.
60
Control Control (+DMSO)
(+DMSO) 6.25
12.5
25
50
AH-23846 (µM) C.pneumoniae infected
Fig. 7. Effect of AH-23846 (EP4 prostanoid receptor antagonist) on the inhibition of CD3 expression of Molt-4 cells infected with C. pneumoniae. The infected cells (MOI 50) were incubated with or without a selective EP4 receptor antagonist for up to 72 h. At 48 and 72 h after infection, mean fluorescence intensities for CD3 expressions of the cells were assessed by flow cytometry. Results are given as a percentage of uninfected cells at the same time point. Each bar represents the mean þ SD (error bars) for three experiments. *p < 0.05, Significantly different from infected cells without an antagonist at the same time point.
Signaling via the TCR/CD3 complex has been extensively studied using anti-TCR or anti-CD3 antibodies, which bind to and activate the receptor similar to MHC-presented antigen [18,19]. Activation of the TCR/CD3 complex leads to the tyrosine phosphorylation of several signaling components that are responsible for activation of MAP kinase and production of IL-2. Ultimately, this signal results in an activation of T-cell [18,19]. Thus, the stimulation
48h
of control)
72h
100
CD3 expression (
* 90
*
* *
80
*
70
60
Control
Control (+DMSO)
(+DMSO)
1µM
4µM
NS-398 C.pneumoniae infected
Fig. 6. Effect of NS-398 (Cox-2 inhibitor) on the inhibition of CD3 expression of Molt-4 cells infected with C. pneumoniae. The infected cells (MOI 25) were incubated with or without a selective Cox-2 inhibitor for up to 72 h. At 48 and 72 h after infection, mean fluorescence intensities for CD3 expressions of the cells were assessed by flow cytometry. Results are given as a percentage of uninfected cells at the same time point. Each bar represents the mean þ SD (error bars) for three experiments. *p < 0.05, Significantly different from the infected cells without the inhibitor at the same time point.
Fig. 8. PGE2 production in the culture supernatant of Molt-4 cells infected with C. pneumoniae. The infected cells (MOI 50) were incubated for up to 72 h. At 48 and 72 h after infection, the amounts of PGE2 in each culture supernatant were measured by ELISA. The data shown represents the mean þ SD (error bars) for three experiments. *p < 0.05, Significantly different from control at the same time point. At 72 h after the infection, the percentage values of alteration of CD3 expression of Molt-4 cells treated with none (control), mock, viable, heat-killed bacteria, NS-398 alone, and viable plus NS-398 are 100 3.1, 105 7.1, 74.4 3.2*, 98.8 4.1, 106.6 4.3, and 102.7 5.1, respectively (*p < 0.05 vs. control).
294
H. Yamaguchi et al. / Microbial Pathogenesis 45 (2008) 290–296
of TCR/CD3 complex has evolved to combat a wide variety of pathogens and may provide the specificity of the adaptive immune system. Although it is not clear whether the inhibition of CD3 by C. pneumoniae infection functionally affects CD3 signaling, the modulation of CD3 expression on lymphocytes may contribute to C. pneumoniae persistent infection via escape from immune network. In contrast to the inhibition of CD3 expression, no significant change of VLA-4 expression was observed in infected cells. VLA-4, which belongs to integrin family associating with cell–cell and cell– matrix adhesion, has a central role for T cell costimulation [20]. However, it is not known what is direct association between VLA-4 molecules and TCR/CD3 complex. The receptor complex of T cells is functionally and physically linked to other molecules such as CD2, CD4, CD5, and CD45 [10, 21]. These molecules directly cooperate with the TCR during antigen recognition and participate in signal transduction [10,20]. Although these findings indicate that C. pneumoniae infection may selectively cause an inhibition of CD3 expression, further study needs to determine whether downregulation of CD3 is selective or not by monitoring other molecules such as CD2, CD4 or CD8. The inhibition of CD3 expression of the cells induced by C. pneumoniae infection was slowly occurred and was depending on MOI. This may be depending on the growth of C. pneumoniae in cells. However, by flow cytometry analysis, some CD3 downregulated cells observed did not have detectable bacterium in the cells. This finding suggests that soluble factors produced by the cells infected with C. pneumoniae might be involved in inhibition of CD3 expression of the cells. In fact, the results of transwell experiments indicated that uninfected cells in the lower chambers showed a decrease of CD3 expression as seen in the upper chambers, which contained infected cells. Cyclooxygenase-2 (Cox-2), known as prostaglandin-endoperoxide synthase, is the key enzyme in biosynthesis of prostanoids such as prostaglandins, prostacyclin and thromboxanase [22–25]. Since the expression of Cox-2, which is eicosanoid precursor, is induced by LPS, growth factors and cytokines [26–28], this increasing may be involved in the modulation of inflammatory responses. It has also been shown that there is the augmented expression of eicosanoid precursor Cox-2 in monocyte-drived macrophages of atheromatous lesions but not in healthy arteries and enhanced expression of Cox-2 may be importance for atherogenesis [22]. Also, eicosanoid such as prostaglandin E2 (PGE2) may be a central mediator in inflammatory vascular diseases [23–25]. Moreover, it is well known that PGE2-mediated suppression of T cell functions could result in modulation of T cell receptor through the activation of G protein and adenylate cyclase [29–32]. Therefore, in order to define a possible involvement of Cox-2 expression and PGE2 in inhibition of CD3 expression of C. pneumoniae-infected lymphocytes, Molt-4 cells infected with bacteria were treated with NS-398 (cyclooxygenease-2 inhibitor) and AH-23848 (EP4 prostanoid receptor antagonist). Cox-2 inhibitor and EP4 antagonist obviously diminished the inhibition of CD3 expression of Molt-4 cells induced by C. pneumoniae infection. In addition, the enhancement of PGE2 production in culture supernatant of the cells by the infection was demonstrated. It is known that the enhanced expression of Cox-2 is observed in the monocyte-drived macrophages of atheromatous but not in healthy arteries [23–25,29] and enhanced expression of Cox-2 and PGE2 via EP4 receptor in monocytic cells is associated with increased plaque instability in human atheromas [33]. Several studies also have suggested that PGE2-mediated alteration in the transcriptional regulation of IL-2 and IL-2 receptor results in attenuated T-cell proliferation and activation in inflammatory vascular [34,35]. Moreover, current studies demonstrated that human peripheral blood cells or human epithelial cells by
C. pneumoniae infection could induce the enhancements of Cox-2 expression and PGE2 production [36–38]. These reports and the results obtained in this study support that eicosanoid such as PGE2 produced by C. pneumoniae infection may be a central soluble factor in the development of vascular diseases associated with C. pneumoniae infection. However, the presence of Cox-2 expression in lymphocytes is not yet totally accepted, and the finding obtained from the experiment with inhibitor may be barely adequate to conclude Cox-2 expression of lymphocytes. Thus, although it is possible that PGE2 induced by C. pneumoniae infection is involved in the inhibition of CD3 expression of lymphocytes caused by C. pneumoniae infection, further study needs to determine the mechanism by which C. pneumoniae stimulates lymphocytes to produce eicosanoid such as PGE2 via Cox-2. A current report indicates that C. pneumoniae in epithelial cells and endothelial cells could persistently escape from host immune detection system by secreting a proteolytically active molecule (CPAFct) into host cell cytosole, which can selectively degrade a host transcription factor, such as RFX5, that is required for major histcompatibility complex [39]. Because the association between the functional modulation by CPAFct and the inhibition of CD3 expression observed in this study is not clear, the details on the matter should be investigated as further study. 4. Conclusions The results of this study indicate that C. pneumoniae infection to lymphocytes induces a decrease of CD3 expression mediated by possibly PGE2 production using the quantitative method based on flow cytometric analysis. Our present observation of CD3 downexpression on lymphocytes by the infection is useful for understanding the mechanisms contribute to the escape of this pathogen from host immune detection system. 5. Materials and methods 5.1. Bacteria C. pneumoniae TW183 strain was kindly provided by G. Byrne, University of Tennessee, Memphis, TN. The bacteria were propagated in the HEp-2 cell culture system according to the methods described previously [40]. In brief, the infected cells were harvested on day 3 and disrupted by freezing–thawing and ultrasonication. After centrifugation to remove cell debris, bacteria were concentrated by a high-speed centrifugation. The bacterial pellets were resuspended in sucrose–phosphate–glutamic acid buffer (0.2 M sucrose, 3.8 mM KH2PO4, 6.7 mM Na2HPO4, 5 mM L-glutamic acid, pH7.4) and then stored at 80 C until used. The organisms resuspended in RPMI 1640 medium were used for experiments. The control inoculums were prepared from uninfected HEp-2 cells (mock). The number of infectious C. pneumoniae was determined as inclusion forming units (IFUs) by counting chlamydial inclusions formed in HEp-2 cells as previously described [40]. Heat-killed bacteria were prepared by heating at 70 C for 45 min as previously reported [41]. The viability of the heat-killed bacteria determined by specific inclusion formation showed that no viable bacteria remained. 5.2. Cells The human lymphocyte cell line Molt-4 (mature T-cell) [17] was cultured in RPMI 1640 medium containing 10% heat-inactivated fetal calf serum and antibiotics (gentamicin 10 mg/ml; vancomycin 10 mg/ml; amphotericin B 1 mg/ml) (Sigma, St. Louis, MO) at 37 C in 5% CO2. Human lymphocytes enriched from PBMCs were prepared by the method described previously [12]. The resulting lymphocyte
H. Yamaguchi et al. / Microbial Pathogenesis 45 (2008) 290–296
fractions stained with Giemsa showed that lymphocytes were greater than 95% by morphology. 5.3. C. pneumoniae infection The cells at a concentration of 5 105 cells/well (24-well plates) were infected with bacteria at multiplicities of infection (MOI) of 1– 50 by centrifugation for 1 h at room temperature. After washing to remove non-infected bacteria with Hank’s balanced solution (HBSS) (Sigma), the cells at a concentration of 1.5 105 cells/well (24-well plates or upper chamber of 24-well transwell plates with uninfected cells in the lower chamber [polyester membrane’s pore size, 0.22 mm; Coster, Corning, NY]) were incubated with or without cycloheximide (Sigma) (1 mg/ml), NS-398 (cyclooxygenease-2 [Cox2] inhibitor) (EMD Biosciences, San Diego, CA) (1 or 4 mM) or AH23848 (EP4 prostanoid receptor antagonist) (Sigma) (6.25–50 mM) for up to 72 h at 37 C in 5% CO2. The cell viabilities were assessed for up to 3 days after infection (MOI 10–50) with or without each drug by trypan blue dye exclusion method. C. pneumoniae infection and all concentrations of drugs tested did not show any significant cytotoxicity to the cells. 5.4. Flow cytometry To determine CD3 expression, staining of cells with phycoerythrin (PE)-labeled CD3 monoclonal antibody (MoAb) (BioLegend, San Diego, CA) was assessed by flow cytometry. VLA-4 staining with PE-labeled VLA-4 MoAb (BioLegend) was also performed as a control. C. pneumoniae infectious rate of lymphocytes was also confirmed by staining with fluorescein isothiocyanate (FITC)labeled Chlamydia lipopolysaccharide (LPS) MoAb (Progen Biotechniik, Deutschland, Germany). In brief, the cells were washed in cold phosphate-buffered saline (PBS) containing 1% (w/v) bovine serum albumin (BSA) and permeablized in Cytofix/Cytoperm solution (Becton Dickinson Biosciences, San Diego, CA) followed by washing in Perm/Wash buffer according to a manufacture’s manual. After washing, the cells were incubated with PE-labeled CD3 (or VLA-4) and/or FITC-labeled Chlamydia LPS MoAbs. The cells were then fixed in PBS containing 1% (w/v) paraformaldehyde. Ten thousand cells were analyzed immediately after labeling on a FACScan flow cytometer (Becton Dickinson Biosciences). The mean fluorescence intensity was obtained from the recorded data, and the results were expressed as the percentage of fluorescence intensity of control cells without bacterial infection at the same time point as follows: (fluorescence intensity of cells with bacterial infection/fluorescence intensity of control cells without bacterial infection) 100. The effect of heat-killed bacteria and HEp-2 cell lysates (mock) on these molecules expression of lymphocytes was also examined. 5.5. Detection of PGE2 The amounts of PGE2 in culture supernatants were determined by competition enzyme-linked immunosorbent assay (ELISA) (Prostaglandin E2 express EIA kit; Cayman chemical, Ann Arbor, MI) according to a manufacture’s manual. 5.6. Statistical analysis Statistical analysis was performed with the unpaired Student t test. Acknowledgments This work was supported by grants-in aid for scientific research from the Japan Society for the Promotion of Science (17590391).
295
References [1] Grayston JT. Chlamydia pneumoniae, strain TWAR. Chest 1989;95:664–9. [2] Grayston JT, Wang SP, Kuo CC, Campbell LA. Current knowledge on Chlamydia pneumoniae, strain TWAR, an important cause of pneumonia and other acute respiratory diseases. Eur J Clin Microbiol Infect Dis 1989;8:191–202. [3] Kauppinen M, Saikku P. Pneumonia due to Chlamydia pneumoniae: prevalence, clinical features, diagnosis, and treatment. Clin Infect Dis 1995;21(Suppl. 3):S244–52. [4] Grayston JT. Chlamydia pneumoniae and atherosclerosis. Rev Med Interne 1996;17(Suppl. 1):S45–7. [5] Hahn DL, Dodge RW, Golubjatnikov R. Association of Chlamydia pneumoniae (strain TWAR) infection with wheezing, asthmatic bronchitis, and adult-onset asthma. JAMA 1991;266:225–30. [6] Norton R, Schepetiuk S, Kok TW. Chlamydia pneumoniae pneumonia with endocarditis. Lancet 1995;345:1376–7. [7] Saario R, Toivanen A. Chlamydia pneumoniae as a cause of reactive arthritis. Br J Rheumatol 1993;32:1112. [8] Ieven MM, Hoymans VY. Involvement of Chlamydia pneumoniae in atherosclerosis: more evidence for lack of evidence. J Clin Microbiol 2005; 43:19–24. [9] Jonasson L, Holm J, Skalli O, Bondjers G, Hansson GK. Regional accumulations of T cells, macrophages, and smooth muscle cells in the human atherosclerotic plaque. Arteriosclerosis 1986;6:131–8. [10] Sullivan GW, Sarembock IJ, Linden J. The role of inflammation in vascular diseases. J Leukoc Biol 2000;67:591–602. [11] Kaul R, Uphoff J, Wiedeman J, Yadlapalli S, Wenman WM. Detection of Chlamydia pneumoniae DNA in CD3þ lymphocytes from healthy blood donors and patients with coronary artery disease. Circulation 2000;102:2341–6. [12] Haranaga S, Yamaguchi H, Friedman H, Izumi S, Yamamoto Y. Chlamydia pneumoniae infects and multiplies in lymphocytes in vitro. Infect Immun 2001;69:7753–9. [13] Yamaguchi H, Haranaga S, Friedman H, Moor JA, Muffly KE, Yamamoto Y. A Chlamydia pneumoniae infection model using established human lymphocyte cell lines. FEMS Microbiol Lett 2002;216:229–34. [14] Allison JP, McIntyre BW, Bloch D. Tumor-specific antigen of murine Tlymphoma defined with monoclonal antibody. J Immunol 1982;129: 2293–300. [15] Haskins K, Kubo R, White J, Pigeon M, Kappler J, Marrack P. The major histocompatibility complex-restricted antigen receptor on T cells. I. Isolation with a monoclonal antibody. J Exp Med 1983;157:1149–69. [16] Meuer SC, Fitzgerald KA, Hussey RE, Hodgdon CJ, Schlossman SF, Reinherz EL. Clonotypic structures involved in antigen-specific human T cell function. Relationship to the T3 molecular complex. J Exp Med 1983;157:705–19. [17] Minowada J, Onuma T, Moore GE. Rosette-forming human lymphoid cell lines. I. Establishment and evidence for origin of thymus-derived lymphocytes. J Natl Cancer Inst 1972;49:891–5. [18] Abraham RT, Weiss A. Jurkat T cells and development of the T-cell receptor signaling paradigm. Nat Rev Immunol 2004;4:301–8. [19] Zhang XM, Berland R, Rosoff PM. Differential regulation of accessory mitogenic signaling receptors by the T cell antigen receptor. Mol Immunol 1995;32:323–32. [20] Berlin C, Berg EL, Briskin MJ, Andrew DP, Kilshaw PJ, Holzmann B, et al. Alpha 4 beta 7 integrin mediates lymphocyte binding to the mucosal vascular addressin MAdCAM-1. Cell 1993;74:185–95. [21] Beyers AD, Spruyt LL, Williams AF. Molecular associations between the Tlymphocyte antigen receptor complex and the surface antigens CD2, CD4, or CD8 and CD5. Proc Natl Acad Sci USA 1992;89:2945–9. [22] Schonbeck U, Sukhora GK, Graber P, Coulter S, Libby P. Augmented expression of cyclooxygenase-2 in human atherosclerotic lesions. Am J Pathol 1999;155:1281–91. [23] Beloqui O, Paramo JA, Orbe J, Benito A, Colina I, Monasterio A, et al. Monocyte cyclooxygenase-2 overactivity: a new marker of subclinical atherosclerosis in asymptomatic subjects with cardiovascular risk factors? Eur Heart J 2005;26:153–8. [24] Cipollone F, Fazia M, Iezzi A, Ciabattoni G, Pini B, Cuccurullo C, et al. Balance between PGD synthase and PGE synthase is a major determinant of atherosclerotic plaque instability in humans. Arterioscler Thromb Vasc Biol 2004;24:1259–65. [25] Clement N, Glorian M, Raymondjean M, Andreani M, Limon I. PGE2 amplifies the effects of IL-1beta on vascular smooth muscle cell de-differentiation: a consequence of the versatility of PGE2 receptors 3 due to the emerging expression of adenylyl cyclase 8. J Cell Physiol 2006;208:495–505. [26] D’Acquisto F, Iuvone T, Rombola L, Sautebin L, Rosa Di, Carnuccio R. Involvement of NF-kB in the regulation of cyclooxygenase-2 protein expression in LPS-stimulated J774 macrophages. FEBS Lett 1997;418:175–8. [27] Appleby SB, Ristimaki A, Neilson K, Narko K, Hla T. Structure of the human cyclo-oxygenase-2 gene. Biochem J 1994;302:723–7. [28] Pistritto G, Franzese O, Pozzoli G, Mancuso C, Tringli G, Preziosi P, et al. Bacterial lipopolysaccharide increases prostaglandin production by rat astrocytes via inducible cyclooxygenase: evidence for the involvement of nuclear factor kB. Biochem Biophys Res Commun 1999;263:570–4. [29] Bihoreau C, Heurtier A, Enjalbert A, Corvaia N, Bensussan A, Degos L, et al. Activation of the CD3/T cell receptor (TcR) complex or of protein kinase C potentiate adenylyl cyclase stimulation in a tumoral T cell line: involvement of two distinct intracellular pathways. Eur J Immunol 1991;21:2877–82.
296
H. Yamaguchi et al. / Microbial Pathogenesis 45 (2008) 290–296
[30] Bismuth G, Theodorou I, Gouy H, Le Gouvello S, Bernard A, Debre P. Cyclic AMP-mediated alteration of the CD2 activation process in human T lymphocytes. Preferential inhibition of the phosphoinositide cycle-related transduction pathway. Eur J Immunol 1988;18:1351–7. [31] Rincon M, Tugores A, Lopez-Rivas A, Silva A, Alonso M, De Landazuri MO, et al. Prostaglandin E2 and the increase of intracellular cAMP inhibit the expression of interleukin 2 receptors in human T cells. Eur J Immunol 1988;18:1791–6. [32] Snijdewint FG, Kalinski P, Wierenga EA, Bos JD, Kapsenberg ML. Prostaglandin E2 differentially modulates cytokine secretion profiles of human T helper lymphocytes. J Immunol 1993;150:5321–9. [33] Cipollone F, Prontera C, Pini B, Marini M, Fazia M, De Cesare D, et al. Overexpression of functionally coupled cyclooxygenase-2 and prostaglandin E synthase in symptomatic atherosclerotic plaques as a basis of prostaglandin E2-dependent plaque instability. Circulation 2001;104:921–7. [34] Anastassiou ED, Paliogianni F, Balow JP, Yamada H, Boumpas DT. Prostaglandin E2 and other cyclic AMP-elevating agents modulate IL-2 and IL-2R alpha gene expression at multiple levels. J Immunol 1992;148:2845–52. [35] Chen D, Rothenberg EV. Interleukin 2 transcription factors as molecular targets of cAMP inhibition: delayed inhibition kinetics and combinatorial transcription roles. J Exp Med 1994;179:931–42.
[36] Rodel J, Prochnau D, Prager K, Baumert J, Schmidt KH, Straube E. Chlamydia pneumoniae decreases smooth muscle cell proliferation through induction of prostaglandin E2 synthesis. Infect Immun 2004;72:4900–4. [37] Rupp J, Berger M, Reiling N, Gieffers J, Lindschau C, Haller H, et al. Cox-2 inhibition abrogates Chlamydia pneumoniae-induced PGE2 and MMP-1 expression. Biochem Biophys Res Commun 2004;320:738–44. [38] Yoneda H, Miura K, Matsushima H, Sugi K, Murakami T, Ouchi K, et al. Aspirin inhibits Chlamydia pneumoniae-induced NF-kappa B activation, cyclooxygenase-2 expression and prostaglandin E2 synthesis and attenuates chlamydial growth. J Med Microbiol 2003;52:409–15. [39] Fan P, Dong F, Huang Y, Zhong G. Chlamydia pneumoniae secretion of a protease-like activity factor for degrading host cell transcription factors is required for major histocompatibility complex antigen expression. Infect Immun 2002;70:345–9. [40] Roblin PM, Dumornay W, Hammerschlag MR. Use of HEp-2 cells for improved isolation and passage of Chlamydia pneumoniae. J Clin Microbiol 1992;30:1968–71. [41] Geng Y, Shane RB, Berencsi K, Gonczol E, Zaki MH, Margolis DJ, et al. Chlamydia pneumoniae inhibits apoptosis in human peripheral blood mononuclear cells through induction of IL-10. J Immunol 2000;164:5522–9.
Contents lists available at ScienceDirect
Microbial Pathogenesis journal homepage: www.elsevier.com/locate/micpath
Inhibition of lymphocyte CD3 expression by Chlamydophila pneumoniae infection Hiroyuki Yamaguchi a, b, *, Junji Matsuo b, Shigehiro Sugimoto a, Maki Utsumi a, Yoshimasa Yamamoto a a b
Laboratory of Molecular Microbiology, Department of Bioinformatics, Graduate School of Medicine, Osaka University, 1-7 Yamadaoka, Suita, Osaka 565-0871, Japan Department of Medical Laboratory Sciences, Graduate School of Health Sciences, Hokkaido University, Nishi-5 Kita-12 Jo, Kita-ku, Sapporo, Hokkaido 060-0812, Japan
a r t i c l e i n f o
a b s t r a c t
Article history: Received 10 January 2008 Received in revised form 16 June 2008 Accepted 26 June 2008 Available online 12 July 2008
Since lymphocytes are a major immune cell besides macrophages in the development of atherosclerosis, interaction between lymphocytes and Chlamydophila pneumoniae may contribute to the pathogenesis of chronic inflammatory diseases associated with C. pneumoniae. In this regard, we examined a possible alteration of CD3 expression of human lymphocyte Molt-4 cells by C. pneumoniae infection. The expression levels of CD3 molecules of lymphocyte Molt-4 cells were significantly decreased by C. pneumoniae infection. In contrast, heat-killed C. pneumoniae as well as mock (cell lysates) did not cause any alteration of CD3 expression of the cells. Treatment of the infected cells with NS-398 (cyclooxyganase-2 inhibitor) or AH-23848 (EP4 prostanoid receptor antagonist) abolished the inhibition of CD3 expression. The enhanced prostaglandin E2 (PGE2) productions in the culture supernatants of infected cells were confirmed by competitive enzyme-immunosorbent assay (ELISA). C. pneumoniae infection of enriched lymphocytes from human peripheral blood mononuclear cells also induced a decrease of CD3 expression. Thus, C. pneumoniae infection of lymphocytes induces a decrease of CD3 expression mediated by possibly PGE2 production. Ó 2008 Elsevier Ltd. All rights reserved.
Keywords: Chlamydophila pneumoniae lymphocytes Molt-4 cells CD3 prostaglandin E2
1. Introduction Chlamydophila pneumoniae is an obligate intracellular bacterium associated with human respiratory pathogen causing pneumonia [1–3]. Current studies indicate that this organism may be implicated in the induction of chronic inflammatory diseases such as asthma, arthritis, endocarditis, and atherosclerosis [4–7]. However, although accumulating studies indicated a possible linking C. pneumoniae infection and chronic inflammatory diseases, the mechanisms for development of such disease by this organism are not clear [8]. Lymphocytes are a major immune cell besides macrophages in the development of chronic inflammatory diseases, such as atherosclerosis [9, 10]. Therefore, a direct interaction between lymphocytes and this pathogen, if it occurs, may contribute to the pathogenesis of chronic inflammatory diseases associated with C. pneumoniae infection. Kaul et al. reported that C. pneumoniae DNA could be recovered from CD3þ peripheral blood leukocytes obtained from patients attending a cardiology [11]. In addition, our studies also demonstrated that C. pneumoniae infects and multiplies in lymphocytes in vitro [12, 13]. These findings
* Corresponding author. Department of Medical Laboratory Sciences, Graduate School of Health Sciences, Hokkaido University, Nishi-5 Kita-12 Jo, Kita-ku, Sapporo, Hokkaido 060-0812, Japan. Tel./fax: þ81 11 706 3326. E-mail address: [email protected] (H. Yamaguchi). 0882-4010/$ – see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.micpath.2008.06.005
indicate that lymphocytes are a potential host cell for C. pneumoniae infection. Therefore, it can be possible that immune functions of lymphocytes infected with C. pneumoniae may be altered and such alterations lead an inappropriate immune response to stimulation, which eventually contribute to the pathogenesis of the disease. CD3 receptor is a crucial molecule in T cell signal transduction [14–16]. Although its expression of cell is constitutive, dynamic regulation of T-cell receptor (TCR)/CD3 complex expression level is probably the most important mechanism allowing T cells to calibrate their response to different levels of stimuli or to sense harmful pathogen in cells. Therefore, a possible alteration of CD3 expression of lymphocytes by C. pneumoniae infection, if it occurs, may be a critical in the pathogenesis of the diseases associated with this pathogen. In this regard, in the present study, we examined a possible alteration of CD3 expression of lymphocytes by C. pneumoniae infection. 2. Results 2.1. C. pneumoniae infection resulted in a decrease of CD3 expression of Molt-4 cells Fig. 1 shows representative micrographs and dot plots of the cells with or without infection at 72 h after incubation. Flow data of double-labeled cells indicated a decrease of CD3 expression of
H. Yamaguchi et al. / Microbial Pathogenesis 45 (2008) 290–296
291
Fig. 1. Representative double-fluorescence staining micrographs (A) and dot-plot images (B) of C. pneumoniae-infected lymphocyte Molt-4 cells at 72 h after incubation. The cells were infected with bacteria at MOI of 10. The cells treated with the permeabilization kit (see Section 5) were stained with PE-conjugated anti-CD3 MoAb and FITC-conjugated antiChlamydia-MoAb. The specimens were then analyzed by fluorescence microscopy and flow cytometry. (A) Red and green colors are indicating CD3 expression and C. pneumoniae inclusion of lymphocyte Molt-4 cells, respectively. Magnification, 1000. (B) Gated cells (R1) are showing analyzed cell population excluding cell debris and dead cells (upper panels). The dot-plot intensity showing CD3 expression of infected cells was lower than that of uninfected cells (lower panels). The mean intensities of CD3 expression of uninfected and infected cells were 75.06 and 51.46, respectively. In this case, the reduction rate showing a decrease of CD3 expression of infected cells compared with that of uninfected cells was 23.6%. The infectious rate in culture was approximately 10%.
infected cells compared with CD3 expression of uninfected cells. There was no difference between dot-plot patterns with side scatter (SSC) and forward scatter (FSC), indicating the influence of cell debris and dead cells causing artificial staining results was minimal. The micrographs also showed that the double-labeled cells were morphologically normal and this staining method was certainly dyed in different colors. Kinetic change of CD3 expression of Molt-4 cells by C. pneumoniae infection was assessed as follows. The cells were infected at MOI of 1 or 10 and incubated for up to 72 h. At various time points, CD3 expression was measured by flow cytometry using anti-CD3 MoAb. As shown in Fig. 2, C. pneumoniae infection induced a decrease of CD3 expression depending on MOI. The decreasing rates of CD3 molecules of Molt-4 cells became the maximum at 72 h after infection. In contrast, a 4 b 1 integrin very late activation antigen-4 (VLA-4) expression was not altered by the infection for up to 72 h. Heat-killed bacteria and HEp-2 cell lysates (mock) did not cause any alteration of CD3 expression of Molt-4 cells (Fig. 3). 2.2. Inhibition of CD3 expression of human peripheral blood lymphocytes by C. pneumoniae infection In order to determine a possible alteration of CD3 expression of primary lymphocytes by infection as observed in established Molt-
4 cells, human lymphocytes enriched from peripheral blood mononuclear cells (PBMCs) were infected with or without viable bacteria in the presence or absence of cycloheximide for up to 48 h, and alteration of CD3 expression of primary lymphocytes were assessed at 24 and 48 h after infection. As shown in Fig. 4, the infected cells showed an obvious decrease of CD3 expression of primary lymphocytes at 48 h after infection. The inhibition of CD3 expression was completely diminished by the treatment with cycloheximide, which inhibits host cell protein synthesis. 2.3. Inhibition of CD3 expression mediated by soluble factors Alteration of CD3 expression of lymphocytes may be induced by several mechanisms, including an involvement of soluble factors produced by lymphocytes. In order to determine such a possibility in the inhibition induced by C. pneumoniae infection, transwell plates with a membrane were utilized for assessment of possible involvement of soluble factors. Both chambers of the transwell plates were cultured with Molt-4 cells. The cells in the upper chamber were infected with C. pneumoniae at MOI of 1–50, but the lower chamber contained only cells without infection. The percentage inhibition of CD3 expression of both chambers determined by flow cytometry was measured at 72 h after incubation. As shown in Fig. 5, the infected Molt-4 cells in the upper chamber
292
H. Yamaguchi et al. / Microbial Pathogenesis 45 (2008) 290–296
VLA-4 expression
100
*
90
*
80 70
*
* 60
Control MOI 1 MOI 10
50 40
0
24
48
72
VLA-4 expression ( of control at each time point)
CD3 expression ( of control at each time point)
CD3 expression 100 90 80 70 60 50 40
0
24
48
72
Time after the infection (h)
Time after the infection (h)
Fig. 2. Inhibition of CD3 expression by C. pneumoniae infection. Molt-4 cells infected with bacteria (MOI 1 or 10) were incubated for up to 72 h. Cells were then washed, stained, and analyzed by flow cytometry. Mean fluorescence intensity was measured at each time point. Results are given as a percentage of uninfected cells at the same time point. Each point represents the mean SD (error bars) for three experiments. *p < 0.05, Significantly different from the control at the same time point.
showed a decrease of CD3 expression depending on MOI. The cells in the lower chamber without infection also showed a decrease of CD3 expression of Molt-4 cells as observed in the upper chamber. Even though the cells without infection as control in both chambers were cultured for 72 h, no alteration of CD3 expression of Molt-4 cells was observed.
2.4. Decrease of CD3 expression mediated by PGE2 in infected cells As shown in Figs. 6 and 7, on treatment both drugs similarly diminished C. pneumoniae induced inhibition of CD3 expression of Molt-4 cells. The percentages of Chlamydia-positive cells in infected cultures did not significantly differ between NS-398 treatment and untreatment for up to 72 h after infection (data not shown). These results indicate a possible involvement of PGE2 on the inhibition of CD3 expression of Molt-4 cells by C. pneumoniae infection. Therefore, to confirm this possible involvement of PGE2, the amount of PEG2 in culture supernatants of Molt-4 cells infected with C. pneumoniae was measured. As shown in Fig. 8, the production of PGE2 in culture supernatant of Molt-4 cells was significantly enhanced by C. pneumoniae infection. No enhanced production of PGE2 was observed by stimulation with either mock or heat-killed
bacteria. In the presence of drugs, C. pneumoniae infection did not cause any increase of PGE2 production of Molt-4 cells. 3. Discussion Molt-4, a well-characterized human lymphocyte cell line [17], has been used in our previous studies of C. pneumoniae infection model [12,13]. It was demonstrated that C. pneumoniae could infect and grow in Molt-4 cells as well as human peripheral blood lymphocytes [12,13]. We also confirmed typical inclusions formed in infected cells, indicating the infection and growth of C. pneumoniae in Molt-4 cells and this system is suitable for analyzing details of C. pneumoniae–lymphocyte interaction. The inhibition of CD3 expression of Molt-4 cells was induced by C. pneumoniae infection. However, mock or heat-killed bacteria treatment did not show any alteration of CD3 expression of the cells. Thus, the results clearly indicate that the inhibition of CD3
CD3 expression ( of control at each time point)
48h 72h 100
90
*
80
* 70
60
Control
Mock
Heat killed
Viable (MOI 10)
Fig. 3. Alteration of CD3 expression of Molt-4 cells treated with either mock or heatkilled bacteria at 42 and 72 h after incubation. Sonicated HEp-2 cells were used for mock control. The data shown represent the mean þ SD (error bars) for three experiments. *p < 0.05, Significantly different from the control at the same time point.
Fig. 4. Levels of CD3 expression of human lymphocytes enriched from peripheral blood with or without cycloheximide at 24 and 48 h after infection. Mean fluorescence intensity was measured at each time point. Results are given as a percentage of uninfected cells at the same time point. The infectious rate in culture was approximately 2%. Each bar represents the mean þ SD (error bars) for three experiments. *p < 0.05, Significantly different from the control at the same time point.
72h
90
* *
* *
70
60
50 Control
48h
1
10
of control)
80
*
*
293
Upper chamber Lower chamber
CD3 expression (
of control)
100
CD3 expression (
H. Yamaguchi et al. / Microbial Pathogenesis 45 (2008) 290–296
100
*
90
*
* *
*
* *
80
*
*
70
50
MOI Fig. 5. Involvement of soluble factors in inhibition of CD3 expression of Molt-4 cells infected with C. pneumoniae. Molt-4 cells were added to both upper and lower chambers in transwell plates, and the cells in the upper chamber were infected with C. pneumoniae. At 72 h after infection, mean fluorescence intensities for CD3 expressions of Molt-4 cells in both chambers were assessed by flow cytometry. Results are given as a percentage of uninfected cells at the same time point. Each bar represents the mean þ SD (error bars) for three experiments. *p < 0.05, Significantly different from the control at the same side of chamber at the same time point.
expression of lymphocytes is induced by infection, not by antigen stimulation. The inhibition of CD3 expression of lymphocytes enriched from human PBMCs by C. pneumoniae infection was also observed as seen in infected Molt-4 cells. However, the inhibition level of CD3 expression of human primary lymphocytes was lower than that of Molt-4 cells infected with bacteria. Although it is not clear the precise mechanisms of inhibition in the primary cells and the cell line cells, the inhibition potency by infection may be reflected by a difference in host cell proliferation and/or in bacterial growth in host cells.
60
Control Control (+DMSO)
(+DMSO) 6.25
12.5
25
50
AH-23846 (µM) C.pneumoniae infected
Fig. 7. Effect of AH-23846 (EP4 prostanoid receptor antagonist) on the inhibition of CD3 expression of Molt-4 cells infected with C. pneumoniae. The infected cells (MOI 50) were incubated with or without a selective EP4 receptor antagonist for up to 72 h. At 48 and 72 h after infection, mean fluorescence intensities for CD3 expressions of the cells were assessed by flow cytometry. Results are given as a percentage of uninfected cells at the same time point. Each bar represents the mean þ SD (error bars) for three experiments. *p < 0.05, Significantly different from infected cells without an antagonist at the same time point.
Signaling via the TCR/CD3 complex has been extensively studied using anti-TCR or anti-CD3 antibodies, which bind to and activate the receptor similar to MHC-presented antigen [18,19]. Activation of the TCR/CD3 complex leads to the tyrosine phosphorylation of several signaling components that are responsible for activation of MAP kinase and production of IL-2. Ultimately, this signal results in an activation of T-cell [18,19]. Thus, the stimulation
48h
of control)
72h
100
CD3 expression (
* 90
*
* *
80
*
70
60
Control
Control (+DMSO)
(+DMSO)
1µM
4µM
NS-398 C.pneumoniae infected
Fig. 6. Effect of NS-398 (Cox-2 inhibitor) on the inhibition of CD3 expression of Molt-4 cells infected with C. pneumoniae. The infected cells (MOI 25) were incubated with or without a selective Cox-2 inhibitor for up to 72 h. At 48 and 72 h after infection, mean fluorescence intensities for CD3 expressions of the cells were assessed by flow cytometry. Results are given as a percentage of uninfected cells at the same time point. Each bar represents the mean þ SD (error bars) for three experiments. *p < 0.05, Significantly different from the infected cells without the inhibitor at the same time point.
Fig. 8. PGE2 production in the culture supernatant of Molt-4 cells infected with C. pneumoniae. The infected cells (MOI 50) were incubated for up to 72 h. At 48 and 72 h after infection, the amounts of PGE2 in each culture supernatant were measured by ELISA. The data shown represents the mean þ SD (error bars) for three experiments. *p < 0.05, Significantly different from control at the same time point. At 72 h after the infection, the percentage values of alteration of CD3 expression of Molt-4 cells treated with none (control), mock, viable, heat-killed bacteria, NS-398 alone, and viable plus NS-398 are 100 3.1, 105 7.1, 74.4 3.2*, 98.8 4.1, 106.6 4.3, and 102.7 5.1, respectively (*p < 0.05 vs. control).
294
H. Yamaguchi et al. / Microbial Pathogenesis 45 (2008) 290–296
of TCR/CD3 complex has evolved to combat a wide variety of pathogens and may provide the specificity of the adaptive immune system. Although it is not clear whether the inhibition of CD3 by C. pneumoniae infection functionally affects CD3 signaling, the modulation of CD3 expression on lymphocytes may contribute to C. pneumoniae persistent infection via escape from immune network. In contrast to the inhibition of CD3 expression, no significant change of VLA-4 expression was observed in infected cells. VLA-4, which belongs to integrin family associating with cell–cell and cell– matrix adhesion, has a central role for T cell costimulation [20]. However, it is not known what is direct association between VLA-4 molecules and TCR/CD3 complex. The receptor complex of T cells is functionally and physically linked to other molecules such as CD2, CD4, CD5, and CD45 [10, 21]. These molecules directly cooperate with the TCR during antigen recognition and participate in signal transduction [10,20]. Although these findings indicate that C. pneumoniae infection may selectively cause an inhibition of CD3 expression, further study needs to determine whether downregulation of CD3 is selective or not by monitoring other molecules such as CD2, CD4 or CD8. The inhibition of CD3 expression of the cells induced by C. pneumoniae infection was slowly occurred and was depending on MOI. This may be depending on the growth of C. pneumoniae in cells. However, by flow cytometry analysis, some CD3 downregulated cells observed did not have detectable bacterium in the cells. This finding suggests that soluble factors produced by the cells infected with C. pneumoniae might be involved in inhibition of CD3 expression of the cells. In fact, the results of transwell experiments indicated that uninfected cells in the lower chambers showed a decrease of CD3 expression as seen in the upper chambers, which contained infected cells. Cyclooxygenase-2 (Cox-2), known as prostaglandin-endoperoxide synthase, is the key enzyme in biosynthesis of prostanoids such as prostaglandins, prostacyclin and thromboxanase [22–25]. Since the expression of Cox-2, which is eicosanoid precursor, is induced by LPS, growth factors and cytokines [26–28], this increasing may be involved in the modulation of inflammatory responses. It has also been shown that there is the augmented expression of eicosanoid precursor Cox-2 in monocyte-drived macrophages of atheromatous lesions but not in healthy arteries and enhanced expression of Cox-2 may be importance for atherogenesis [22]. Also, eicosanoid such as prostaglandin E2 (PGE2) may be a central mediator in inflammatory vascular diseases [23–25]. Moreover, it is well known that PGE2-mediated suppression of T cell functions could result in modulation of T cell receptor through the activation of G protein and adenylate cyclase [29–32]. Therefore, in order to define a possible involvement of Cox-2 expression and PGE2 in inhibition of CD3 expression of C. pneumoniae-infected lymphocytes, Molt-4 cells infected with bacteria were treated with NS-398 (cyclooxygenease-2 inhibitor) and AH-23848 (EP4 prostanoid receptor antagonist). Cox-2 inhibitor and EP4 antagonist obviously diminished the inhibition of CD3 expression of Molt-4 cells induced by C. pneumoniae infection. In addition, the enhancement of PGE2 production in culture supernatant of the cells by the infection was demonstrated. It is known that the enhanced expression of Cox-2 is observed in the monocyte-drived macrophages of atheromatous but not in healthy arteries [23–25,29] and enhanced expression of Cox-2 and PGE2 via EP4 receptor in monocytic cells is associated with increased plaque instability in human atheromas [33]. Several studies also have suggested that PGE2-mediated alteration in the transcriptional regulation of IL-2 and IL-2 receptor results in attenuated T-cell proliferation and activation in inflammatory vascular [34,35]. Moreover, current studies demonstrated that human peripheral blood cells or human epithelial cells by
C. pneumoniae infection could induce the enhancements of Cox-2 expression and PGE2 production [36–38]. These reports and the results obtained in this study support that eicosanoid such as PGE2 produced by C. pneumoniae infection may be a central soluble factor in the development of vascular diseases associated with C. pneumoniae infection. However, the presence of Cox-2 expression in lymphocytes is not yet totally accepted, and the finding obtained from the experiment with inhibitor may be barely adequate to conclude Cox-2 expression of lymphocytes. Thus, although it is possible that PGE2 induced by C. pneumoniae infection is involved in the inhibition of CD3 expression of lymphocytes caused by C. pneumoniae infection, further study needs to determine the mechanism by which C. pneumoniae stimulates lymphocytes to produce eicosanoid such as PGE2 via Cox-2. A current report indicates that C. pneumoniae in epithelial cells and endothelial cells could persistently escape from host immune detection system by secreting a proteolytically active molecule (CPAFct) into host cell cytosole, which can selectively degrade a host transcription factor, such as RFX5, that is required for major histcompatibility complex [39]. Because the association between the functional modulation by CPAFct and the inhibition of CD3 expression observed in this study is not clear, the details on the matter should be investigated as further study. 4. Conclusions The results of this study indicate that C. pneumoniae infection to lymphocytes induces a decrease of CD3 expression mediated by possibly PGE2 production using the quantitative method based on flow cytometric analysis. Our present observation of CD3 downexpression on lymphocytes by the infection is useful for understanding the mechanisms contribute to the escape of this pathogen from host immune detection system. 5. Materials and methods 5.1. Bacteria C. pneumoniae TW183 strain was kindly provided by G. Byrne, University of Tennessee, Memphis, TN. The bacteria were propagated in the HEp-2 cell culture system according to the methods described previously [40]. In brief, the infected cells were harvested on day 3 and disrupted by freezing–thawing and ultrasonication. After centrifugation to remove cell debris, bacteria were concentrated by a high-speed centrifugation. The bacterial pellets were resuspended in sucrose–phosphate–glutamic acid buffer (0.2 M sucrose, 3.8 mM KH2PO4, 6.7 mM Na2HPO4, 5 mM L-glutamic acid, pH7.4) and then stored at 80 C until used. The organisms resuspended in RPMI 1640 medium were used for experiments. The control inoculums were prepared from uninfected HEp-2 cells (mock). The number of infectious C. pneumoniae was determined as inclusion forming units (IFUs) by counting chlamydial inclusions formed in HEp-2 cells as previously described [40]. Heat-killed bacteria were prepared by heating at 70 C for 45 min as previously reported [41]. The viability of the heat-killed bacteria determined by specific inclusion formation showed that no viable bacteria remained. 5.2. Cells The human lymphocyte cell line Molt-4 (mature T-cell) [17] was cultured in RPMI 1640 medium containing 10% heat-inactivated fetal calf serum and antibiotics (gentamicin 10 mg/ml; vancomycin 10 mg/ml; amphotericin B 1 mg/ml) (Sigma, St. Louis, MO) at 37 C in 5% CO2. Human lymphocytes enriched from PBMCs were prepared by the method described previously [12]. The resulting lymphocyte
H. Yamaguchi et al. / Microbial Pathogenesis 45 (2008) 290–296
fractions stained with Giemsa showed that lymphocytes were greater than 95% by morphology. 5.3. C. pneumoniae infection The cells at a concentration of 5 105 cells/well (24-well plates) were infected with bacteria at multiplicities of infection (MOI) of 1– 50 by centrifugation for 1 h at room temperature. After washing to remove non-infected bacteria with Hank’s balanced solution (HBSS) (Sigma), the cells at a concentration of 1.5 105 cells/well (24-well plates or upper chamber of 24-well transwell plates with uninfected cells in the lower chamber [polyester membrane’s pore size, 0.22 mm; Coster, Corning, NY]) were incubated with or without cycloheximide (Sigma) (1 mg/ml), NS-398 (cyclooxygenease-2 [Cox2] inhibitor) (EMD Biosciences, San Diego, CA) (1 or 4 mM) or AH23848 (EP4 prostanoid receptor antagonist) (Sigma) (6.25–50 mM) for up to 72 h at 37 C in 5% CO2. The cell viabilities were assessed for up to 3 days after infection (MOI 10–50) with or without each drug by trypan blue dye exclusion method. C. pneumoniae infection and all concentrations of drugs tested did not show any significant cytotoxicity to the cells. 5.4. Flow cytometry To determine CD3 expression, staining of cells with phycoerythrin (PE)-labeled CD3 monoclonal antibody (MoAb) (BioLegend, San Diego, CA) was assessed by flow cytometry. VLA-4 staining with PE-labeled VLA-4 MoAb (BioLegend) was also performed as a control. C. pneumoniae infectious rate of lymphocytes was also confirmed by staining with fluorescein isothiocyanate (FITC)labeled Chlamydia lipopolysaccharide (LPS) MoAb (Progen Biotechniik, Deutschland, Germany). In brief, the cells were washed in cold phosphate-buffered saline (PBS) containing 1% (w/v) bovine serum albumin (BSA) and permeablized in Cytofix/Cytoperm solution (Becton Dickinson Biosciences, San Diego, CA) followed by washing in Perm/Wash buffer according to a manufacture’s manual. After washing, the cells were incubated with PE-labeled CD3 (or VLA-4) and/or FITC-labeled Chlamydia LPS MoAbs. The cells were then fixed in PBS containing 1% (w/v) paraformaldehyde. Ten thousand cells were analyzed immediately after labeling on a FACScan flow cytometer (Becton Dickinson Biosciences). The mean fluorescence intensity was obtained from the recorded data, and the results were expressed as the percentage of fluorescence intensity of control cells without bacterial infection at the same time point as follows: (fluorescence intensity of cells with bacterial infection/fluorescence intensity of control cells without bacterial infection) 100. The effect of heat-killed bacteria and HEp-2 cell lysates (mock) on these molecules expression of lymphocytes was also examined. 5.5. Detection of PGE2 The amounts of PGE2 in culture supernatants were determined by competition enzyme-linked immunosorbent assay (ELISA) (Prostaglandin E2 express EIA kit; Cayman chemical, Ann Arbor, MI) according to a manufacture’s manual. 5.6. Statistical analysis Statistical analysis was performed with the unpaired Student t test. Acknowledgments This work was supported by grants-in aid for scientific research from the Japan Society for the Promotion of Science (17590391).
295
References [1] Grayston JT. Chlamydia pneumoniae, strain TWAR. Chest 1989;95:664–9. [2] Grayston JT, Wang SP, Kuo CC, Campbell LA. Current knowledge on Chlamydia pneumoniae, strain TWAR, an important cause of pneumonia and other acute respiratory diseases. Eur J Clin Microbiol Infect Dis 1989;8:191–202. [3] Kauppinen M, Saikku P. Pneumonia due to Chlamydia pneumoniae: prevalence, clinical features, diagnosis, and treatment. Clin Infect Dis 1995;21(Suppl. 3):S244–52. [4] Grayston JT. Chlamydia pneumoniae and atherosclerosis. Rev Med Interne 1996;17(Suppl. 1):S45–7. [5] Hahn DL, Dodge RW, Golubjatnikov R. Association of Chlamydia pneumoniae (strain TWAR) infection with wheezing, asthmatic bronchitis, and adult-onset asthma. JAMA 1991;266:225–30. [6] Norton R, Schepetiuk S, Kok TW. Chlamydia pneumoniae pneumonia with endocarditis. Lancet 1995;345:1376–7. [7] Saario R, Toivanen A. Chlamydia pneumoniae as a cause of reactive arthritis. Br J Rheumatol 1993;32:1112. [8] Ieven MM, Hoymans VY. Involvement of Chlamydia pneumoniae in atherosclerosis: more evidence for lack of evidence. J Clin Microbiol 2005; 43:19–24. [9] Jonasson L, Holm J, Skalli O, Bondjers G, Hansson GK. Regional accumulations of T cells, macrophages, and smooth muscle cells in the human atherosclerotic plaque. Arteriosclerosis 1986;6:131–8. [10] Sullivan GW, Sarembock IJ, Linden J. The role of inflammation in vascular diseases. J Leukoc Biol 2000;67:591–602. [11] Kaul R, Uphoff J, Wiedeman J, Yadlapalli S, Wenman WM. Detection of Chlamydia pneumoniae DNA in CD3þ lymphocytes from healthy blood donors and patients with coronary artery disease. Circulation 2000;102:2341–6. [12] Haranaga S, Yamaguchi H, Friedman H, Izumi S, Yamamoto Y. Chlamydia pneumoniae infects and multiplies in lymphocytes in vitro. Infect Immun 2001;69:7753–9. [13] Yamaguchi H, Haranaga S, Friedman H, Moor JA, Muffly KE, Yamamoto Y. A Chlamydia pneumoniae infection model using established human lymphocyte cell lines. FEMS Microbiol Lett 2002;216:229–34. [14] Allison JP, McIntyre BW, Bloch D. Tumor-specific antigen of murine Tlymphoma defined with monoclonal antibody. J Immunol 1982;129: 2293–300. [15] Haskins K, Kubo R, White J, Pigeon M, Kappler J, Marrack P. The major histocompatibility complex-restricted antigen receptor on T cells. I. Isolation with a monoclonal antibody. J Exp Med 1983;157:1149–69. [16] Meuer SC, Fitzgerald KA, Hussey RE, Hodgdon CJ, Schlossman SF, Reinherz EL. Clonotypic structures involved in antigen-specific human T cell function. Relationship to the T3 molecular complex. J Exp Med 1983;157:705–19. [17] Minowada J, Onuma T, Moore GE. Rosette-forming human lymphoid cell lines. I. Establishment and evidence for origin of thymus-derived lymphocytes. J Natl Cancer Inst 1972;49:891–5. [18] Abraham RT, Weiss A. Jurkat T cells and development of the T-cell receptor signaling paradigm. Nat Rev Immunol 2004;4:301–8. [19] Zhang XM, Berland R, Rosoff PM. Differential regulation of accessory mitogenic signaling receptors by the T cell antigen receptor. Mol Immunol 1995;32:323–32. [20] Berlin C, Berg EL, Briskin MJ, Andrew DP, Kilshaw PJ, Holzmann B, et al. Alpha 4 beta 7 integrin mediates lymphocyte binding to the mucosal vascular addressin MAdCAM-1. Cell 1993;74:185–95. [21] Beyers AD, Spruyt LL, Williams AF. Molecular associations between the Tlymphocyte antigen receptor complex and the surface antigens CD2, CD4, or CD8 and CD5. Proc Natl Acad Sci USA 1992;89:2945–9. [22] Schonbeck U, Sukhora GK, Graber P, Coulter S, Libby P. Augmented expression of cyclooxygenase-2 in human atherosclerotic lesions. Am J Pathol 1999;155:1281–91. [23] Beloqui O, Paramo JA, Orbe J, Benito A, Colina I, Monasterio A, et al. Monocyte cyclooxygenase-2 overactivity: a new marker of subclinical atherosclerosis in asymptomatic subjects with cardiovascular risk factors? Eur Heart J 2005;26:153–8. [24] Cipollone F, Fazia M, Iezzi A, Ciabattoni G, Pini B, Cuccurullo C, et al. Balance between PGD synthase and PGE synthase is a major determinant of atherosclerotic plaque instability in humans. Arterioscler Thromb Vasc Biol 2004;24:1259–65. [25] Clement N, Glorian M, Raymondjean M, Andreani M, Limon I. PGE2 amplifies the effects of IL-1beta on vascular smooth muscle cell de-differentiation: a consequence of the versatility of PGE2 receptors 3 due to the emerging expression of adenylyl cyclase 8. J Cell Physiol 2006;208:495–505. [26] D’Acquisto F, Iuvone T, Rombola L, Sautebin L, Rosa Di, Carnuccio R. Involvement of NF-kB in the regulation of cyclooxygenase-2 protein expression in LPS-stimulated J774 macrophages. FEBS Lett 1997;418:175–8. [27] Appleby SB, Ristimaki A, Neilson K, Narko K, Hla T. Structure of the human cyclo-oxygenase-2 gene. Biochem J 1994;302:723–7. [28] Pistritto G, Franzese O, Pozzoli G, Mancuso C, Tringli G, Preziosi P, et al. Bacterial lipopolysaccharide increases prostaglandin production by rat astrocytes via inducible cyclooxygenase: evidence for the involvement of nuclear factor kB. Biochem Biophys Res Commun 1999;263:570–4. [29] Bihoreau C, Heurtier A, Enjalbert A, Corvaia N, Bensussan A, Degos L, et al. Activation of the CD3/T cell receptor (TcR) complex or of protein kinase C potentiate adenylyl cyclase stimulation in a tumoral T cell line: involvement of two distinct intracellular pathways. Eur J Immunol 1991;21:2877–82.
296
H. Yamaguchi et al. / Microbial Pathogenesis 45 (2008) 290–296
[30] Bismuth G, Theodorou I, Gouy H, Le Gouvello S, Bernard A, Debre P. Cyclic AMP-mediated alteration of the CD2 activation process in human T lymphocytes. Preferential inhibition of the phosphoinositide cycle-related transduction pathway. Eur J Immunol 1988;18:1351–7. [31] Rincon M, Tugores A, Lopez-Rivas A, Silva A, Alonso M, De Landazuri MO, et al. Prostaglandin E2 and the increase of intracellular cAMP inhibit the expression of interleukin 2 receptors in human T cells. Eur J Immunol 1988;18:1791–6. [32] Snijdewint FG, Kalinski P, Wierenga EA, Bos JD, Kapsenberg ML. Prostaglandin E2 differentially modulates cytokine secretion profiles of human T helper lymphocytes. J Immunol 1993;150:5321–9. [33] Cipollone F, Prontera C, Pini B, Marini M, Fazia M, De Cesare D, et al. Overexpression of functionally coupled cyclooxygenase-2 and prostaglandin E synthase in symptomatic atherosclerotic plaques as a basis of prostaglandin E2-dependent plaque instability. Circulation 2001;104:921–7. [34] Anastassiou ED, Paliogianni F, Balow JP, Yamada H, Boumpas DT. Prostaglandin E2 and other cyclic AMP-elevating agents modulate IL-2 and IL-2R alpha gene expression at multiple levels. J Immunol 1992;148:2845–52. [35] Chen D, Rothenberg EV. Interleukin 2 transcription factors as molecular targets of cAMP inhibition: delayed inhibition kinetics and combinatorial transcription roles. J Exp Med 1994;179:931–42.
[36] Rodel J, Prochnau D, Prager K, Baumert J, Schmidt KH, Straube E. Chlamydia pneumoniae decreases smooth muscle cell proliferation through induction of prostaglandin E2 synthesis. Infect Immun 2004;72:4900–4. [37] Rupp J, Berger M, Reiling N, Gieffers J, Lindschau C, Haller H, et al. Cox-2 inhibition abrogates Chlamydia pneumoniae-induced PGE2 and MMP-1 expression. Biochem Biophys Res Commun 2004;320:738–44. [38] Yoneda H, Miura K, Matsushima H, Sugi K, Murakami T, Ouchi K, et al. Aspirin inhibits Chlamydia pneumoniae-induced NF-kappa B activation, cyclooxygenase-2 expression and prostaglandin E2 synthesis and attenuates chlamydial growth. J Med Microbiol 2003;52:409–15. [39] Fan P, Dong F, Huang Y, Zhong G. Chlamydia pneumoniae secretion of a protease-like activity factor for degrading host cell transcription factors is required for major histocompatibility complex antigen expression. Infect Immun 2002;70:345–9. [40] Roblin PM, Dumornay W, Hammerschlag MR. Use of HEp-2 cells for improved isolation and passage of Chlamydia pneumoniae. J Clin Microbiol 1992;30:1968–71. [41] Geng Y, Shane RB, Berencsi K, Gonczol E, Zaki MH, Margolis DJ, et al. Chlamydia pneumoniae inhibits apoptosis in human peripheral blood mononuclear cells through induction of IL-10. J Immunol 2000;164:5522–9.