Received 19 April 2004; received in revised form 9 August 2004; accepted 6 October First published online 23 November 2004
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1 FEMS Immunology and Medical Microbiology 43 (25) Induction of CD69 expression and Th1 cytokines release from human peripheral blood lymphocytes after in vitro stimulation with Alloiococcus otitidis and three middle ear pathogens Atsushi Harimaya a,, Tetsuo Himi a, Nobuhiro Fujii b, Jussi Tarkkanen c, Petteri Carlson d, Jukka Ylikoski e, Petri Mattila e a Department of Otolaryngology, Sapporo Medical University School of Medicine, S-1, W-16, Chuo-ku, Sapporo, Hokkaido , Japan b Department of Microbiology, Sapporo Medical University School of Medicine, Sapporo, Japan c Department of Pathology, Haartman Institute, University of Helsinki, Helsinki, Finland d Unit of Clinical Bacteriology, HD-laboratories, Helsinki University Central Hospital, Helsinki, Finland e Department of Otorhinolaryngology, Helsinki University Central Hospital, Helsinki, Finland Received 19 April 24; received in revised form 9 August 24; accepted 6 October 24 First published online 23 November 24 Abstract Alloiococcus otitidis is a recently discovered pathogen of otitis media. However, only a limited number of studies are available about the pathogenic and immunological role of A. otitidis. The aim of this study was to investigate the activation and the cytokine production of human peripheral blood lymphocytes at the early immune response after stimulation with A. otitidis. After stimulation of whole human peripheral blood lymphocytes for 18 h with whole killed A. otitidis or the three major middle ear pathogens (Streptococcus pneumoniae, Haemophilus influenzae and Moraxella catarrhalis), the expression of CD69 and the production of cytokines were analyzed. The expression of CD69 on T cells and B cells was dose-dependently enhanced after stimulation with A. otitidis. The release of interleukin (IL)-12 was induced after stimulation with A. otitidis, whereas the release of IL-4 was not induced after stimulation with A. otitidis. In addition, the release of interferon (IFN)-c was induced after stimulation with A. otitidis. Although the release of IFN-c started within 18 h after stimulation with A. otitidis, intracellular production of IFN-c was not observed in either CD4 + T cells or CD8 + T cells within 18 h upon stimulation. The patterns of CD69 expression and T helper-type 1 (Th1)-promoting cytokines production were similarly shown when human peripheral blood lymphocytes were stimulated with the other three major pathogens. Our results suggest that A. otitidis has sufficient immunogenic potential to modulate a host immune response, like the other three major middle ear pathogens, and also suggest that the immunogenicity of A. otitidis is very similar, at the early immune response, to that of the three major middle ear pathogens. Ó 24 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved. Keywords: Alloiococcus otitidis; CD69; Interleukin-12; Interferon-c; Th1 cytokines 1. Introduction Otitis media is the most common infectious disease and an important otological problem in childhood. Corresponding author. Tel.: x3491; fax: address: harimaya@sapmed.ac.jp (A. Harimaya). Streptococcus pneumoniae, nontypeable Haemophilus influenzae and Moraxella catarrhalis are the three major middle ear pathogens [1]. Recently, unknown Gram-positive cocci were recovered from middle ear effusions of children presenting otitis media [2]. The organism was determined as a new species of bacteria and named Alloiococcus otitis [3], and then the name /$22. Ó 24 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved. doi:1.116/j.femsim
2 386 A. Harimaya et al. / FEMS Immunology and Medical Microbiology 43 (25) was revised as Alloiococcus otitidis [4]. Although this bacterium was difficult to detect in middle ear effusions in conventional cultures, it was detected in about 5% of otitis media cases using a polymerase chain reaction (PCR)-based method; a higher rate than for the three major pathogens [5,6]. These findings suggest that A. otitidis may be indeed one of the major pathogens of otitis media. However, only a limited number of studies are available about the pathogenic and immunological role of A. otitidis. To investigate the immunogenicity of A. otitidis, we have previously studied the immune response of human cells after stimulation with A. otitidis. A. otitidis could enhance the activation of human lymphocytes [7]. In addition, A. otitidis, as well as the other three most common pathogens, could induce the release of interleukin (IL)-8 from human epithelial and monocytic cell lines [8], and could also induce IL-12 from the human monocytic cell line, THP-1 [9]. Our studies suggest that A. otitidis has a potential to induce an inflammatory reaction as a result of the host immune response. To date, no other studies apart from ours have been performed on the immune responses against A. otitidis. The CD69 antigen, also designated as the activation inducer molecule (AIM) and early activation antigen (EA-1), is an 85-kDa disulfide-linked homodimer of differentially glycosylated subunits [1 12]. CD69 is one of the earliest activation markers expressed on activated lymphocytes (T, B and NK cells) and neutrophils, following stimulation by a variety of mitogenic agents [13]. Upon one to two hoursõ activation, CD69 expression can be found on the cell surface and persists for at least three days [13]. Cytokines, which are released from activated lymphocytes, are regulators of the immune response, and are implicated in inflammatory reactions in many diseases. T-lymphocytes produce two distinct patterns of cytokines, T helper-type 1 (Th1) cytokines such as interferon-c (IFN-c), IL-12 and IL-2, and Th2 cytokines such as IL-4 and IL-5 [14 16]. These patterns of cytokines select a type of immune response. Th1 cytokines shift lymphocytes to cell-mediated immunity and Th2 cytokines shift lymphocytes to humoral immunity, inducing the antibody production [14 16]. Th1 cytokines inhibit the actions of Th2 cells and vice versa [14 16]. Among the cytokines, IL-12 and IL-4 are the key ones to lead lymphocytes to produce Th1 and Th2 cytokines, respectively [17]. In order to elucidate whether A. otitidis has a potential to activate lymphocytes and to induce cytokines, i.e., which Th1 or Th2 cytokines would be induced by A. otitidis at the early immune response, we investigated the expression of CD69 and the release of Th1/Th2 cytokines upon stimulation with A. otitidis, and compared with the stimulation observed in the three major middle ear pathogens. 2. Materials and methods 2.1. Preparation and stimulation of lymphocytes Human peripheral blood lymphocytes (PBL) were obtained from six-healthy adult volunteers (2 females and 4 males). The average age was 31.5 years old, presenting a range from 27 to 42 years old and they did not have a medical history of otitis media or other infectious diseases for the past six months. Mononuclear cells were separated by Ficoll-Hypaque gradient centrifugation to remove dead cells and granulocytes. Subsequently, cells were washed and resuspended in RPMI 164 medium (Whittaker MA, Bioproducts, Walkersville, MD) supplemented with 1 mm glutamine, 1 IU ml 1 penicillin, 1 lg ml 1 streptomycin (Gibco, Chagrin Falls, OH) and 2.5% human blood group AB serum (Finnish Red Cross Blood Service, Helsinki, Finland) at a concentration of cells ml 1. Streptococcus pneumoniae serotype 19 (KK29), nontypeable H. influenzae (HA543), M. catarrhalis (LK293) and A. otitidis (NCFB289) were used for stimulation of lymphocytes. Bacteria were grown at 37 C in a humidified 5% CO 2 /95% air incubator to late logarithmic phase of growth and fixed with.25% glutaraldehyde. Lymphocytes ( cells ml 1 ) were incubated with bacterial cells in RPMI 164. After incubation and centrifugation, the pellets were analyzed using flow cytometry and the supernatants were assayed using an enzyme-linked immunosorbent assay (ELISA). Cells were analyzed in duplicate for each assay, and each experiment was independently repeated three or five times. As positive controls, stimulation of PBL was performed by incubation with phorbol 12-myristate-13-acetate (PMA, Sigma) and ionomycin (Calbiochem, La Jolla, CA), both dissolved in dimethylsulfoxide at a final concentration of 32 nm and 2 lm, respectively. As negative controls, PBL were incubated with medium alone Monoclonal antibodies and flow cytometry analysis of cell surface molecules and intracellular cytokines For flow cytometric analysis, fluorescent-conjugated monoclonal antibodies (mabs) were used. Anti-CD3- FITC (Becton Dickinson, San Jose, CA), anti-cd3- PerCP (Becton Dickinson), anti-cd4-pe (Pharmingen, San Diego, CA), anti-cd4-percp (Pharmingen), anti- CD8-PE (Pharmingen), anti-cd8-percp (Pharmingen), anti-cd19-percp (Pharmingen) and anti-cd69-pe (Immunotech, Marseille, France) were used for staining the cell surface molecules of lymphocytes. Anti-IFN-c- FITC (Becton Dickinson) and anti-il-4-pe (R & D Systems, Minneapolis, MN) were used for staining the intracellular cytokines of lymphocytes. After stimulation, lymphocytes were incubated with 1 lg ml 1 Brefeldin A (Sigma) for 4 h before harvest-
3 A. Harimaya et al. / FEMS Immunology and Medical Microbiology 43 (25) ing. The harvested lymphocytes were washed twice and anti-cell surface molecule mabs were added. After incubation at 4 C for 3 min, cells were fixed with 4% paraformaldehyde (PFA), washed twice and analyzed using a flow cytometer. For intracellular cytokine detection, the following procedure was also followed: lymphocytes were incubated in permeabilization buffer (PBS with.5% Saponin and 1% heat-inactivated newborn calf serum) for 1 min at cells ml 1. Subsequently, anti-intracellular cytokine mabs were added and cells were further incubated at 4 C for 3 min. Cells were washed once with permeabilization buffer, fixed in 4% PFA and analyzed using a flow cytometer. To examine cell surface molecules and intracellular cytokines, two- or three-colour analyses were performed using a fluorescence-activated cell sorting (FACScan) flow cytometer (Becton Dickinson) and CellQuest software (Becton Dickinson) Enzyme-linked immunosorbent assay Cytokine assays in the culture supernatants were performed using a commercially available ELISA kit. ELISA kits for IL-12 p7 or IL-2 were purchased from Immunotech (Marseille, France) and the kit for IFN-c was purchased from BioSource (Camarillo, CA). The absorbance of each sample was measured using a microplate reader (ERA 4, SLT Inc., Vienna, Austria) Statistical analysis All results were compared among the groups using unpaired StudentÕs t test. 3. Results 3.1. CD69 expression of lymphocytes after bacterial stimulation To investigate whether whole killed bacteria have a potential to stimulate lymphocytes, we measured CD69 expression of lymphocytes using flow cytometry after stimulation with the whole killed bacteria for 18 h. As shown in Fig. 1A, A. otitidis could enhance CD69 expression of CD3 + cells (T cells) in a dose-dependent manner. In addition, it was shown that A. otitidis could enhance CD69 expression of CD3 cells in a dose-dependent manner (Fig. 1A). Because most CD3 cells consist of B cells, we also investigated CD69 expression of CD19 + cells (B cells). As shown in Fig. 1B, A. otitidis could induce CD69 expression of B cells in a dose-dependent manner, similarly to CD69 expression of T cells. The three major pathogens also similarly enhanced CD69 expression of T cells or B cells in a dose-dependent manner (Fig. 1B). Compared with the three major pathogens, CD69 expression of B cells was low after stimulation with A. otitidis. However, CD69 expression of T cells after stimulation with A. otitidis was as high as that after stimulation with M. catarrhalis or S. pneumoniae (Fig. 1B) Cytokine production of lymphocytes after bacterial stimulation Next, we investigated which cytokines, Th1 or Th2, could be induced at the early immune response after stimulation PBL with the bacteria. We measured the level of IL-12 and IL-4, the key cytokines of Th1- and Th2-type responses, respectively, in the supernatant of PBL after stimulation. While the release of IL-12 p7 was enhanced by A. otitidis and the three major pathogens (Fig. 2A), the release of IL-4 was not enhanced by any of these pathogens (Fig. 2B). The release of IFN-c, as well as that of IL-12 p7, was also enhanced by A. otitidis and the three major pathogens (Fig. 3A). The release of IFN-c after stimulation with A. otitidis or the three major pathogens was of a similar extent, in a time-dependent manner (Fig. 3B). With all of the four pathogens, the release of IFN-c was rapidly enhanced at 18 h upon stimulation (Fig. 3B). To investigate the source of IFN-c, we next analyzed the intracellular IFN-c production of CD4 + and CD8 + T cells using flow cytometry analysis. Unexpectedly, the production of intracellular IFN-c could be observed in neither CD4 + nor CD8 + T cells, even upon 18 h stimulation with any of the pathogens (Fig. 4A and B). Similarly, the production of intracellular IFN-c was observed in neither CD4 + nor CD8 + T cells, also upon 6 or 12 h stimulation with any of the pathogens (data not shown). 4. Discussion In this study, in order to examine the capacity of A. otitidis to induce activation of lymphocytes at the early immune response, we used multiparameter flow cytometry analysis with immunofluorescent staining of the activation antigen, CD69, to identify, within bulk lymphocytes cultures, the lymphocytes subpopulations involved in the activation process. To evaluate the activated state of lymphocytes in an immune response, bulk methods such as 3 H-thymidine incorporation and 3-(4,5- dimethyl-2-thiazolyl)-2,5-diphenyl-2h-tetrazolium bromide (MTT) dye uptake, or flow cytometry analysis of the expression of cell-surface activation markers have been the standard approach [18,19]. Flow cytometry analysis can identify the subpopulations of lymphocytes within bulk lymphocytes cultures, whereas the bulk methods cannot [18]. There are several activation
4 388 A. Harimaya et al. / FEMS Immunology and Medical Microbiology 43 (25) Fig. 1. CD69 expression of human PBL after stimulation with middle ear pathogens. (A) Representative flow cytometry analysis of CD69 expression of CD3 + cells after stimulation with A. otitidis. After incubation of PBL for 18 h with whole killed A. otitidis (bacteria/lymphocyte ratios of :1,.1:1,.1:1, 1:1 and 1:1), the cells were stained with anti-cd69 mab and anti-cd3 mab. Then, 1, events were collected with CellQuest ä software, using a fluorescence or forward scatter (FSC) threshold. Acquired data were displayed as two-color dot plots as shown, and were analyzed using CellQuest ä. The values represent percentages of CD69 + CD3 + cells in PBL. Data are representative of three independent experiments. (B) CD69 expression of T and B cells in human PBL after stimulation with whole killed A. otitidis, S. pneumoniae, H. influenzae or M. catarrhalis (bacteria/lymphocyte ratios of :1,.1:1, 1:1 and 1:1). After the stimulation, PBL was collected and stained with anti-cd69 mab and anti-cd3 mab (for T cells) or anti-cd19 mab (for B cells). Then, data was acquired and analyzed using a flow cytometer and CellQuest ä. Values represent means ± standard deviation (SD) of three independent experiments. antigens of lymphocytes, such as CD69, CD25 (IL-2 receptor), CD71 (transferrin receptor) and HLA-DR [18,19]. While CD69 reaches maximum level within several hours to a few days after stimulation, CD25, CD71 and HLA-DR require 3 1 days after stimulation to reach maximum expression [13,18,19]. CD69 is undetectable on the plasma membrane of resting lymphocytes, but is rapidly expressed on antigen- or mitogen-stimulated lymphocytes [13,18]. Expression of CD69 in response to stimuli requires new RNA transcription and protein synthesis and is believed to be integral to the activation process [2 22]. Concerning the major middle ear pathogens, Arva and Andersson [23] reported that whole killed S. pneumoniae could induce CD69 expression of lymphocytes and induced release of cytokines, and Tarkkanen et al. [7] reported that whole killed A. otitidis could induce CD69 expression of human lymphocytes. However, as far as we know, there have been no studies dealing with the issue of whether H. influenzae or M. catarrhalis are capable of inducing CD69 expression. Our results showed that A. otitidis and the three major pathogens could enhance CD69 expression of both T and B cells within 18 h, and this enhancement took place in a dosedependent manner. The results of CD69 expression suggest that whole killed A. otitidis, and the three major pathogens, can modulate activation of immunocompetent cells such as T and B cells during the early immune response. After stimulation of PBL for 18 h with whole killed A. otitidis, the release of Th1-promoting cytokines, IL- 12 and IFN-c, was induced, while the release of the typical Th2-promoting cytokine, IL-4, was not. A similar induction of Th1-type response was also seen after stimulation with the three major pathogens. Our previous
5 A. Harimaya et al. / FEMS Immunology and Medical Microbiology 43 (25) B %CD3+ cells expressing CD CD69 expression of T cells Sp Hi Mc Ao Bacteria/Lymphocyte ratio IL-12 p7 (pg/ml) IL-12 p7 A medium Sp Hi Mc Ao PMA + Io %CD19+ cells expressing CD CD69 expression of B cells Sp Hi Mc Ao Bacteria/Lymphocyte ratio IL-4 (pg/ml) IL-4 B medium Sp Hi Mc Ao PMA + Io Fig. 1 (continued) study demonstrated that IL-12 release was also induced from the human monocytic cell line, THP-1, after 16 h stimulation with A. otitidis, and with the three major pathogens [9]. IL-12 is a heterodimeric pro-inflammatory cytokine produced by mononuclear phagocytes and dendritic cells, and induces the production of IFN-c, favors the differentiation of Th1 cells and forms a link between innate and adaptive immunity [24]. Concerning induction of Th1 cytokines by the other middle ear pathogens, Arva and Andersson reported that S. pneumoniae induced Th1 cytokines from PBL [25], and Agren et al. [26] reported that H. influenzae induced Th1 cytokines from human tonsillar lymphocytes. Microbial components such as lipoteichoic acid (LTA) [27,28], lipopolysaccharide (LPS) [29], and heat-shock proteins [3] have the ability to induce Th1 cytokines production. Like the other pathogens, A. otitidis also may have such inducers of Th1 cytokines in the cell surface components. The release of IFN-c from PBL was rapidly enhanced after 18 h stimulation with A. otitidis, as was the case with the three major pathogens. However, intracellular Fig. 2. The release of IL-12 p7 and IL-4 from human PBL after stimulation with middle ear pathogens. After incubation of PBL for 18 h with whole killed A. otitidis, S. pneumoniae, H. influenzae or M. catarrhalis (bacteria/lymphocyte ratio of 2:1), the supernatants were collected and the amounts of IL-12 p7 (A) and IL-4 (B) were measured using an IL-12 p7 or IL-4 ELISA kit. Values represent means ± SD of five independent experiments. p <.5 as compared with cells incubated with medium alone. p <.1 as compared with cells incubated with medium alone. cytokine analysis using flow cytometry revealed that neither CD4 + nor CD8 + T cells were the source of IFN-c upon 18 h stimulation with any of these pathogens. Although we additionally examined IFN-c release after stimulation for 6 or 12 h, neither CD4 + nor CD8 + T cells were the source of IFN-c. These results suggest that IFN-c release induced at such very early hours after the start of stimulation was mainly released from the other cells of PBL. With the exception of T cells, natural killer (NK) cells are another main producer of IFN-c [31]. As far as we know, no study has been reported concerning IFN-c induction in NK cells after stimulation with the above mentioned middle ear pathogens. However, our previous study of cytotoxic assay revealed that A. otitidis could induce activation of NK cells [7]. The
6 39 A. Harimaya et al. / FEMS Immunology and Medical Microbiology 43 (25) IFN-gamma (pg/ml) A IFN-gamma A CD4+ T cells expressing intracellular IFN-gamma % intracellular IFN-gamma positive cells medium Sp Hi medium Sp Hi Mc Ao PMA + Io Kinetics of IFN-gamma release B medium Sp Hi Mc Ao Mc Ao PMA + Io IFN-gamma (pg/ml) B CD8+ T cells expressing intracellular IFN-gamma % intracellular IFN-gamma positive cells medium Sp hours Fig. 3. The release of IFN-c from human PBL after stimulation with middle ear pathogens. (A) The release of IFN-c from human PBL after stimulation. After incubation of PBL for 18 h with whole killed A. otitidis, S. pneumoniae, H. influenzae or M. catarrhalis (bacteria/ lymphocyte ratio of 2:1), the supernatants were collected and the amounts of IFN-c were measured using an IFN-c ELISA kit. Values represent means ± SD of five independent experiments. p <.1 as compared with cells incubated with medium alone. (B) Kinetics of IFN-c release from human PBL after stimulation with the pathogens for, 6, 12 or 18 h. After incubation of PBL with whole killed A. otitidis, S. pneumoniae, H. influenzae or M. catarrhalis (bacteria/ lymphocyte ratio of 2:1), the supernatants were collected and the amounts of IFN-c were measured using an IFN-c ELISA kit. Values represent means ± SD of three independent experiments. study of CD69 detection by Arva and Andersson [23] also revealed that S. pneumoniae could induce the activation of NK cell. The principal function of IFN-c is to activate macrophages in both innate immune responses and adaptive cell-mediated immune responses. NK cells secrete IFN-c in response to recognition of microbes, or in response to IL-12 [17,24,31]. In this setting, IFN-c functions as a mediator of innate immunity. In adaptive immunity, T cells produce IFN-c in response to antigen recognition, and this is enhanced by IL-12 [17,24,31]. Hi Mc Ao PMA + Io Fig. 4. Intracellular IFN-c production in CD4 + and CD8 + T cells after stimulation with middle ear pathogens. After incubation of PBL for 18 h with whole killed A. otitidis, S. pneumoniae, H. influenzae or M. catarrhalis (bacteria/lymphocyte ratio of 2:1), the cells were stained with anti-cd3 mab, anti-cd4 (or anti-cd8) mab and anti-intracellular IFN-c mab. Then, 1, events were collected with Cell- Quest ä software, using a fluorescence or forward scatter (FSC) threshold. The data were gated by CD3-positive cells and displayed as two-color dot plots. Then, the percentages of intracellular IFN-cpositive CD3 + CD4 + cells (A), or intracellular IFN-c-positive CD3 + CD8 + cells (B) were calculated. Values represent means ± SD of six independent experiments. p <.5 as compared with cells incubated with medium alone. The sequence of reactions involving IL-12 and IFN-c is central to cell-mediated immunity. Although our study did not show IFN-c release from T cells, a longer period of stimulation may have done so. Different cytokines play key roles in innate immunity to different classes of microbes. In infections by pyo-
7 A. Harimaya et al. / FEMS Immunology and Medical Microbiology 43 (25) genic extracellular bacteria, macrophages respond to bacterial endotoxins or other bacterial products by producing pro-inflammatory cytokines such as chemokines, tumor necrosis factor (TNF) and IL-1. Chemokines produced by macrophages and by endothelial cells stimulate the extravasation of leukocytes to the infection site, where the innate immune reaction is mounted to eliminate the infectious microbes [32]. In our previous study, we demonstrated that A. otitidis and the three major middle ear pathogens induced the release of chemokine (IL-8) from human epithelial cell lines and monocytic cell lines [8]. Our previous and present studies suggest that A. otitidis can modulate an immune response by inducing the release of chemokine and cytokines. 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