Published online before print March 27, 2008
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* Medical Microbiology, Department of Laboratory Medicine and
Laboratory of Clinical and Experimental Allergy Research, Department of Otorhinolaryngology, Malmö University Hospital, Lund University, Malmö, Sweden
1Correspondence: Medical Microbiology, Department of Laboratory Medicine, Malmö University Hospital, Lund University, SE-205 02 Malmö, Sweden. E-mail: kristian.riesbeck{at}med.lu.se
ABSTRACT
The respiratory pathogen Moraxella catarrhalis has a high affinity for human IgD and is mitogenic for peripheral blood B lymphocytes. Moraxella IgD-binding protein, which is a multifunctional outer membrane protein with adhesive properties, is responsible for the interaction. Previous experiments with the Ig-binding B cell superantigens protein A and protein L from Staphylococcus aureus and Peptostreptococcus magnus, respectively, have suggested that nonimmune BCR cross-linking induces B cell apoptosis through the intrinsic pathway. The goal of this study was to characterize early and late B cell events in the presence of M. catarrhalis in comparison with S. aureus. Despite an increased phosphatidyl serine translocation as revealed by Annexin V binding in flow cytometry analyses, neither M. catarrhalis nor S. aureus induced activation-associated apoptotic cell death in purified human tonsillar B cells. In contrast, a vigorous B cell proliferation, as quantified using thymidine incorporation and CFSE staining, was observed. An increased expression of an array of surface proteins (i.e., CD19, CD21, CD40, CD45, CD54, CD69, CD86, CD95, and HLA-DR) and IgM production was found upon activation with M. catarrhalis. In conclusion, M. catarrhalis-dependent B cell activation does not result in apoptosis but in cell division and nonspecific IgM synthesis, suggesting that the bacterial interaction with tonsillar B cells serves to redirect the early adaptive immune response.
Key Words: B lymphocyte • immunoglobulin D • Staphylococcus aureus • superantigen
INTRODUCTION
Moraxella catarrhalis is an uncapsulated, Gram-negative diplococcus, which for a long time has been considered to be a relatively harmless commensal inhabiting the respiratory tract [1 2 3 4 ]. Although M. catarrhalis can be found as a commensal in healthy children, it is also the third most frequently isolated bacterium in patients with otitis media. M. catarrhalis also causes lower respiratory tract infections in adults with predisposing conditions such as chronic obstructive pulmonary disease. Other infections associated with M. catarrhalis are sinusitis, acute laryngitis, suppurative keratitis, and in rare cases, septicaemia.
The colonization of M. catarrhalis is a complex process involving several adhesion molecules, biofilm formation, and subepithelial and intracellular evasion [5 6 7 8 ]. To sustain colonization, M. catarrhalis also hinders the host innate immune system by conferring serum resistance [9 10 11 ], which displayed by M. catarrhalis, is a result of the expression of the ubiquitous surface protein (Usp) A1 and A2 and the hybrid UspA2H. The Usp family inhibits the classical and alternative pathways by binding to C4BP and C3 [12 , 13 ]. M. catarrhalis also interferes directly with the membrane attack complex by binding vitronectin to UspA2 [11 ].
M. catarrhalis interacts with the adaptive immune system by binding to surface-expressed IgD [14 ]. The ability to interact with IgD at a cellular level accounts for its strong mitogenic effect on human lymphocytes [4 , 15 16 17 ]. We have previously isolated and characterized the high molecular weight surface protein Moraxella IgD-binding protein (MID), which displays high affinity for soluble and surface-bound IgD [18 , 19 ]. The presence of MID, also designated Hag (for hemagglutinin), has been confirmed by other laboratories [20 , 21 ]. The mid gene was detected in all M. catarrhalis strains investigated, and M. catarrhalis isolated from nasopharynx, blood, or sputum expressed MID at approximately the same frequency [22 ]. Furthermore, no variation in MID expression was observed between strains from different geographical origins. The ubiquitous presence of MID and its stable expression between isolates are indicative of an important role for MID in M. catarrhalis. The apparent molecular mass of monomeric MID is estimated to 200 kDa. We have narrowed down the IgD-binding part of MID to 238 aa (i.e., MID962–1200), which binds to the heavy chain constant 1 region of IgD [23 , 24 ]. Furthermore, aa 198–206 on the IgD molecule are crucial for the MID interaction. Full-length MID and the recombinant MID962–1200 fragment function as a B cell superantigen (SAg) and activate B cells in the presence of T cell cytokines [17 , 19 ].
Earlier in vivo experiments with the Ig-binding B cell SAg protein A (SpA) and protein L (PpL) from Staphylococcus aureus and Peptostreptococcus magnus, respectively, have suggested that nonimmune BCR-SAg cross-linking leads to activation-induced cell death (AICD) through the intrinsic pathway [25
26
27
]. SpA and PpL bind to conserved sites of the variable region on heavy (SpA) or light (PpL) chains of the BCR [28
, 29
]. These sites are independent from the normal antigen-binding site, and therefore, SAgs can interact with a larger part of the B cell pool than conventional antigens. This large polyclonal activation leads to an intense competition for secondary rescuing signals required for the avoidance of BCR-induced apoptosis. It has been shown that 30–50% of human B cells express the heavy chain variable region 3 (VH3) family required for SpA interaction [30
]. IgD is expressed in conjunction with IgM on all mature, nonstimulated B lymphocytes, thus accounting for 
75% of the total B cell pool [31
, 32
]. As there are more B cells susceptible to MID binding than to SpA binding, the starvation-induced stress stimuli leading to apoptosis should therefore theoretically be higher in M. catarrhalis than in S. aureus infections.
The aim of this study was to investigate the interactions between M. catarrhalis and human tonsillar B lymphocytes in vitro to increase the understanding of the MID/IgD interaction in bacterial pathogenesis. SAg expression in S. aureus and M. catarrhalis induced a vigorous proliferation in human tonsillar B cells. Interestingly, the SAg-stimulated cells also translocated phosphatidyl serine (PS), as revealed with the apoptosis marker Annexin V, albeit at a considerably lower level as compared with apoptosis induced by staurosporine. Finally, we conclude that apoptosis is not the fate of B lymphocytes after SAg stimulation but a prominent cell division and polyclonal IgM production.
MATERIALS AND METHODS
Antibodies and reagents
RPE-conjugated mouse anti-human IgD, CD3, CD19, CD40, CD45, and CD95 mAb and FITC-conjugated mouse anti-human IgM, IgA, IgE, IgG, CD19, CD21, CD69, CD86, and HLA-DR mAb were purchased from Dakopatts (Glostrup, Denmark). RPE-conjugated anti-human CD54 mAb and Annexin V-FITC Apoptosis Detection Kit I were supplied by BD Biosciences (Becton Dickinson, Franklin Lakes, NJ, USA). CFSE was purchased from Molecular Probes (Invitrogen, Carlsbad, CA, USA). Pokeweed mitogen (PWM) and staurosporine were obtained from Sigma-Aldrich (St. Louis, MO, USA).
Bacteria and culture conditions
M. catarrhalis strains RH4 and BBH18 were clinical isolates described previously [22
]. For comparison studies with S. aureus, the high-expressing SpA strain Cowan I was chosen [33
]. All bacterial strains were grown on blood agar base solid medium or in brain heart infusion liquid medium. MID-deficient M. catarrhalis 
mid was constructed as described previously [19
]. For the preparation of formalin-killed bacteria, M. catarrhalis or S. aureus was grown overnight in nutrient broth (Oxoid, Basingstoke Hampshire, UK), with or without addition of kanamycin (50 µg/ml). Bacteria were harvested and washed in PBS, pH 7.2. Thereafter, they were killed in 0.5% formalin for 2 h at room temperature. After four washes in PBS, bacteria were resuspended in RPMI-1640 medium (Life Technologies, Paisley, Scotland) and tested for IgD binding in a FACS Calibur flow cytometer (Becton Dickinson). The formalin-killed bacteria were stored in aliquots at –20°C.
Cell preparations
Tonsils were obtained from 24 patients under the age of 30 (age range: 2–30 years, median 5.5 years) undergoing tonsillectomy at Malmö University Hospital (Sweden). The Ethics Committee of Lund University (Sweden) approved the study (No. 877/2005), and written, informed consent was obtained from all patients. Surgery was performed as a result of recurrent β-hemolytic group A streptococci tonsillitis (at least four infections during the year prior to surgery) or as a result of tonsillar hyperplasia. None of the patients displayed symptoms of acute infection at the time of surgery, and none had received any antibiotic treatment for at least 1 month before surgery. Apart from the tonsillar symptoms, all patients were healthy and did not receive any medication. The tonsils were divided into two groups: infected (n=8, age range 2–25 years, median 5 years) and hyperplasia (n=16, age range 4–30 years, median 12 years) depending on the clinical diagnosis leading to surgery. Tonsils were dissected in RPMI-1640 medium supplemented with 10% FCS, 2 mM glutamine, 50 µg/ml gentamicin, and 100 U/ml penicillin (complete medium). The homogenized cell suspension was filtered through a 70-µm nylon cell strainer followed by isolation of lymphocytes on Lymphoprep (Nycomed, Oslo, Norway) density gradients as described [23
]. The mixed lymphocytes were routinely screened for CD19, CD3, and IgD expression using flow cytometry. Untouched CD19+ B cells were isolated by an indirect magnetic labeling system (B Cell Isolation Kit II, Miltenyi Biotec, Bergisch Gladbach, Germany) with an additional purification step using magnetic-labeled antibodies directed against CD3 (Miltenyi Biotec), resulting in an ultrapure B cell preparation (CD3+ <1%). In all experiments with lymphocytes, 1 x 106 cells/ml were cultured in complete medium supplemented with various reagents in culture plates (Nunc, Roskilde, Denmark). Proliferation was quantified by [methyl-3H]-thymidine incorporation (5 µCi/well, Amersham Pharmacia Biotech, Little Chalfont, UK) using an 18-h pulse period or by CFSE. For proliferation assays using CFSE, 2–3 x 107 cells were preincubated with 2.5 µM CFSE in 500 ml PBS + 0.1% BSA for 10 min at 37°C. After repeated washes with ice-cold RPMI, cells were transferred to 24-well plates (1x106 cells/ml) and incubated in complete medium with different stimuli for 96 h in 37°C.
Flow cytometry analyses
The B cell surface phenotype changes after exposure to S. aureus, M. catarrhalis, or the MID mutant (compared with unstimulated cells) were investigated using flow cytometry analysis. Stimulated B cells were harvested in PBS-BSA at different time-points and labeled with antibodies directed to numerous surface markers for 45 min on ice. In experiments using FITC-labeled Annexin V/propidium iodine, cells were stained according to the manufacturer’s instructions (Apoptosis Detection Kit I, BD PharMingen) using 1 µM staurosporine as a positive control for apoptosis. Flow cytometry was also used when analyzing the specificity of the antibodies produced after M. catarrhalis-induced B cell stimulation. A panel of normal respiratory pathogens, i.e., M. catarrhalis strains BBH18 and RH4, Haemophilus influenzae strain NTHi772 [34
], and Streptococcus pneumoniae strain TIGR 4 [35
], was tested separately for antibody binding (IgM, IgD, IgA, IgG, and IgE) after 1 h incubation on ice with cell-free supernatants from B cells activated with M. catarrhalis.
ELISA
Antibody secretion post-M. catarrhalis stimulation was measured using cell-free supernatants harvested after 96 h. ELISA plates (Maxisorb, Nunc) were coated at 4°C overnight with polyclonal rabbit anti-human IgD, IgE, IgA, IgM, or IgG (Dakopatts), respectively, in 0.1 M Na2HPO4, pH 9.0. The plates were washed four times with PBS containing 0.05% Tween 20 (PBS-Tween), followed by 2 h blocking at room temperature using PBS-Tween supplemented with 1.5% OVA as blocking solution. Plates were washed four times with PBS-Tween before the addition of supernatants. The secreted Igs were detected with HRP-conjugated anti-human IgD, IgE, IgA, IgM, and IgG, respectively (Dakopatts). Finally, the plates were developed and measured at OD450. A standard serum was included for calculation of IgM concentrations.
Statistical analysis
Differences in proliferation and changes in surface markers were tested using Student’s t-test for paired data. Significant values were defined as P 
0.05 (*); 0.01 (**); 0.001 (***).
RESULTS
M. catarrhalis and S. aureus induce tonsillar B cell proliferation
According to the AICD model, SAg-mediated BCR cross-linking leads to a phenotype change, limited rounds of proliferation, and finally, apoptosis. We have previously demonstrated that whole cell M. catarrhalis, purified MID, and the IgD-binding fragment MID962–1200 stimulate human PBLs to proliferate [15
, 17
, 19
]. Lymphocytes from infected and hyperplasia tonsils were routinely screened in flow cytometry for CD19, CD3, and IgD expression (Table 1
). The B and T cell ratio differed significantly between the two groups, with 55.5% CD19+ cells in the hyperplasia group compared with only 40.6% in the infected group. No significant difference was seen when comparing IgD expression in purified B cells from the two groups: infected, 57.2%; and hyperplasia, 59.2%. To investigate the mitogenic effect of M. catarrhalis as compared with S. aureus on the two separate groups of tonsillar lymphocytes, formalin-treated bacteria were incubated with a mixture of tonsillar B and T lymphocytes or with purified B cells only. M. catarrhalis strain BBH18 and S. aureus Cowan I strongly activated the mixed lymphocytes as revealed with [3H]-thymidine incorporation at 96 h (Fig. 1A
). The importance of BCR signaling in M. catarrhalis-dependent proliferation was illustrated by the finding that the M. catarrhalis mid mutant did not activate the lymphocytes. Interestingly, experiments with ultrapure B cells (<1% T cells) clearly showed that the proliferative response to M. catarrhalis and S. aureus could be T cell-independent (Fig. 1B)
. Only mixed lymphocytes were able to respond to PWM, which was included as a positive control. Moreover, purified B cells incubated with M. catarrhalis mid did not incorporate [3H]-thymidine above background values. Similar results were obtained with another clinical isolate M. catarrhalis RH4 and its corresponding MID mutant (not shown). As can be seen in Figure 1
, lymphocytes isolated from infected tonsils showed a higher proliferation with prominent background activation but also a higher level of variation between separate experiments. However, the only significant difference between the groups was between purified B cells incubated with M. catarrhalis mid (P=0.0332). Therefore, all further experiments were performed with cells from the hyperplasia group to minimize the variation. To characterize the kinetics of proliferation, [3H]-thymidine incorporation was measured every 24 h over a period of 8 days. [3H]-Thymidine uptake during the first 120 h in a purified B lymphocyte population incubated with M. catarrhalis or M. catarrhalis mid is demonstrated in Figure 2A
. MID-induced [3H]-thymidine incorporation was detected as early as 48 h with a peak at 96 h. The purified B cells stimulated with S. aureus follow similar kinetics, albeit showing a lower level of proliferation after 5 days in culture (Fig. 2B)
.
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Table 1. Comparison of CD19 and IgD Expression in Tonsillar Lymphocytes from Patients with Hyperplasia or Recurrent Tonsillitis
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Figure 1. Tonsillar B cell proliferation was induced by MID expressing M. catarrhalis and SpA expressing S. aureus independently of T cells. (A) Mixed tonsillar lymphocytes and (B) purified B cells from hyperplasia (open bars) and infected tonsils (shaded bars) were incubated with formalin-killed M. catarrhalis (BBH18), isogenic M. catarrhalis mid, S. aureus (Cowan I), and PWM for 96 h and analyzed for [3H]-thymidine uptake. Data are presented as mean + SD from nine (hyperplasia, n=6) (A) or 11 (hyperplasia, n=5; B) different donors. Significant values were calculated using Student’s t-test for paired data and defined as P 0.05 (*); 0.01 (**); 0.001 (***).
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Figure 2. A kinetic study of SAg-induced proliferation in vitro. (A) B cell activation with M. catarrhalis ( ) and the MID-deficient mutant ( ). (B) B cell proliferation with S. aureus () compared with unstimulated B cells (). Results represent mean values from five random donors diagnosed for tonsil hyperplasia and analyzed at different occasions. Error bars indicate SD. Significant values were calculated using Student’s t-test for paired data and defined as P 0.05 (*); 0.01 (**); 0.001 (***).
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mid showed no increased Annexin V binding compared with unstimulated B cells (Fig. 3D
3E
3F)
. Intriguingly, the SAg-activated B cells became positive for Annexin V at approximately the same time (48 h) as B cells started to proliferate, as revealed by thymidine incorporation (Fig. 2)
. No significant change in propidium iodide uptake was seen at this early time-point (48 h), indicating that necrosis was not induced (data not shown). To further monitor the level of PS, staurosporine was included as a positive control for apoptosis (Fig. 3J
3K
3L)
. After 48 h, B cells incubated with staurosporine bound Annexin V significantly higher than cells incubated with S. aureus or M. catarrhalis (M. catarrhalis, P0.0001; and S. aureus, P=0.0087). Taken together, the PS shift observed in SAg-activated B cells was significantly higher than the background (P0.01 for M. catarrhalis and S. aureus) and significantly lower than the positive control after 48 h and thus, did not represent true preapoptotic cells.
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Figure 3. SAg BCR cross-linking leads to an increased translocation of PS detected by Annexin V binding. Unstimulated tonsillar B cells (filled) are compared with M. catarrhalis (A–C), M. catarrhalis mid (D–F), S. aureus (G–I), or staurosporine (J–L). B cells were incubated with formalin-killed bacteria or 1 µM staurosporine as a positive control for apoptosis induction. A representative donor out of four analyzed is shown. Significant values were calculated using Student’s t-test for paired data and defined as P 0.05 (*); 0.01 (**); 0.001 (***).
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Figure 4. SAg stimulation induces strong cell division during the first 96 h of incubation, as revealed by CFSE staining. Human B lymphocytes were stained with CFSE and incubated with (A) M. catarrhalis, (B) S. aureus, (C), M. catarrhalis mid, and (D) B cells without any stimulus. Cells were analyzed by flow cytometry after 5 days of culture. A representative donor out of four analyzed is shown with numbers indicating new generations. The [methyl-3H]-thymidine incorporation for the CFSE profiles were for M. catarrhalis, 95,713 cpm; S. aureus, 70,423 cpm; M. catarrhalis mid, 1065 cpm; and finally, B cells, 364 cpm.
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Figure 5. MID-dependent IgD cross-linking causes a down-regulation of surface-expressed IgD and an up-regulation of early activation markers. Unstimulated B cells are shown as filled profiles (gray) and M. catarrhalis and the MID-deficient counterpart, as black profiles, indicating changes in expression of IgD (A), CD19 (B), CD40 (C), CD54 (D), CD69 (E), and CD86 (F). Representative results from one out of five donors are shown, and corresponding geometric mean fluorescence, SD, and P values are presented in Table 2
.
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Figure 6. A prolonged stimulation through IgD results in an up-regulation of surface molecules. Changes in the B cell phenotypes after SAg stimulation were monitored by flow cytometry after 72 h. Unstimulated B cells are shown as filled profiles (gray) and M. catarrhalis and the MID-deficient counterpart, as black profiles, indicating changes in expression of IgM (A), CD19 (B), CD21 (C), CD40 (D), CD45 (E), CD54 (F), CD69 (G) CD86 (H), CD95 (I), and HLA-DR (J). Representative results from one out of four donors are shown, and corresponding geometric mean fluorescence, SD, and P values are presented in Table 3
.
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Table 2. Changes in B Cell Phenotype after 24 h of Exposure to M. catarrhalis, M. catarrhalis mid, or S. aureus
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Table 3. Changes in B Cell Phenotype after 72 h of Exposure to M. catarrhalis, M. catarrhalis mid, or S. aureus
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Figure 7. MID activation via IgD induces IgM production independently of T cells. Cell-free supernatants were harvested after 96 h from B cells cultured with wild-type and MID-deficient M. catarrhalis as well as S. aureus and analyzed in ELISA for Ig production. Results are from four different donors diagnosed for tonsil hyperplasia. Error bars indicate +SD, and Pvalues are calculated, comparing M. catarrhalis-stimulated B cells with other stimuli using Student’s t-test.
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DISCUSSION
The effect of B cell SAgs on B cells has been a subject of discussion since the discovery of the first BCR-directed SAg. Early studies using whole SpA-expressing S. aureus showed an impressive mitogenic effect on the target cells [36 , 37 ]. These findings were revised when the same experiments were repeated with recombinant SpA without signs of B cell proliferation [38 ]. Furthermore, if the right secondary signals were provided, i.e., cytokines, CD154, or preactivated T cells, proliferation was also easily induced by recombinant SpA [39 ]. All of these early studies were done in vitro, and later, in vivo studies of mice injected with recombinant SpA or PpL showed a phenomenon, designated AICD [27 ]. Data from Silverman and Goodyear [27 ] indicated that the result of a SAg–B cell interaction was down-regulation of surface-expressed BCR, up-regulation of activation markers, limited rounds of proliferation, and finally, apoptotic cell death through the intrinsic pathway.
The main goal of this study was to test the AICD phenomenon on tonsillar lymphocytes exposed to MID-expressing M. catarrhalis in comparison with SpA-expressing S. aureus. Our experiments showed great similarity with the early events in the AICD model with proliferation shown with [methyl-3H]-thymidine incorporation and CFSE staining. The phenotype change induced by MID- or SpA-expressing bacteria correlates well with previously published data from in vivo experiments with recombinant SpA and PpL, showing a B cell primed for T cell help. Furthermore, our proliferation experiments with purified B cells stained with CFSE show that the MID-induced cell division is clearly T-independent.
To measure apoptosis, we monitored changes in surface expression of PS after exposure to SAg-expressing and non-SAg-expressing bacteria. PS is normally located in the intracellular part of the plasma membrane but is translocated to the surface early in apoptosis [40 ]. Interestingly, a clear shift in PS exposure was seen in SAg-stimulated B cells as compared with unstimulated cells or cells incubated with the MID-deficient M. catarrhalis. The SAg-induced change in surface PS was detected as early as 48 h of incubation with bacteria. This early shift was unexpected and did not correlate with the kinetics of thymidine incorporation that could be measured for up to 8 days. The CFSE staining also clearly showed that the thymidine uptake represents cell division and not just DNA replication (Fig. 4) . When further comparing the surface translocation of PS induced by MID or SpA with staurosporine, the SAg-induced shift was significantly lower. Changes in PS expression in activated B cells without signs of apoptosis have been reported to be important in BCR-mediated signaling [41 ]. Dillon et al. [41 ] also showed that the change in PS translocation in activated B cells is lower than in true apoptotic cells and postulate that the lower PS density in combination with lacking additional apoptotic surface changes required for phagocytosis saves activated B cells from macrophage clearing. Our conclusion is that the AICD model in vitro does not fit with SAg expressed on bacteria but is restricted to purified, recombinant SpA and PpL in vivo. In contrast to apoptosis, our data show a T-independent B cell proliferation, resulting in unspecific IgM production, thus delaying and redirecting the adaptive humoral response away from M. catarrhalis. This survival strategy is probably crucial for M. catarrhalis, which can be found in the outer mantle zone of human tonsils, where mainly naïve B cells are located [8 ]. Hypothetically, the nonimmune-activated B cells that change from IgD+/IgM+ to IgD–/IgM+ will later be negatively selected during affinity maturation. B cells that keep the IgD receptor will be favored in the process, and this could explain the high number of IgD+ plasma cells in the nasopharynx and in the middle ear cavity, areas that are frequently colonized by two pathogens (M. catarrhalis and H. influenzae) capable of binding IgD [14 , 42 43 44 ].
Why does not the AICD model fit with B cells activated with whole bacteria? First, the experiments done by Silverman and Goodyear [27 ] have been performed in vivo using genetically modified mice to increase the number of SpA-sensitive B cells from 5% to 95%, whereas our experiments were done in vitro using nonmodified human tonsillar lymphocytes. The reason for not using mice in our experiments is that MID has a specific affinity for human IgD and does not bind murine IgD. Second, we choose to work with whole bacteria instead of recombinant SAg. SpA and PpL exist in membrane-bound and soluble forms, and MID is restricted to the bacterial surface. In addition, we could not induce apoptosis with the IgD-binding fragment MID962–1200 (data not shown), indicating significant differences in B cell signaling after SpA or MID cross-linking or that the AICD phenomenon is restricted to murine B cells in vivo. When using whole bacterial cells, the SAg-activated B lymphocytes are able to access activating factors such as bacterial surface proteins, additional binding of the coreceptor complex, complement receptors, and specific pathogen-associated molecular patterns recognized by TLRs, which all can deliver secondary rescuing signals. Recent data from Bekeredjian-Ding et al. [45 ] indicate that B cell activation via SpA sensitizes B cells for TLR2-active lipoproteins. However, SpA and TLR2 ligation is not enough to induce IgM production as seen when SpA is combined with TLR7 or -9 ligands, which is consistent with our results when whole S. aureus is used.
Further studies are required to investigate in detail the Moraxella-dependent interaction with B cells also in vivo to give deeper insights into the function of IgD in the immune defense as well as characterizing unique M. catarrhalis virulence mechanisms.
Received November 23, 2007; revised February 5, 2008; accepted February 20, 2008.
REFERENCES
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