HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Wingren, A. G. Articles by Riesbeck, K. Search for Related Content PubMed PubMed Citation Articles by Wingren, A. G. Articles by Riesbeck, K.

The Novel IgD Binding Protein from Moraxella catarrhalis Induces Human B Lymphocyte Activation and Ig Secretion in the Presence of Th2 Cytokines¹

Department of Medical Microbiology, University Hospital Malmö, Lund University, Malmö, Sweden

	Abstract

Top Abstract Introduction Materials and Methods Results and Discussion References

 
Moraxella IgD binding protein (MID) is a novel bacterial outer membrane protein with IgD-binding properties. MID was purified from the respiratory pathogen Moraxella catarrhalis and is here shown to have B cell stimulatory properties. Purified MID in the range of 0.01–0.1 µg/ml was optimal to induce a proliferative response in human PBL. MID coupled to Sepharose and formalin-fixed M. catarrhalis preparations induced similar proliferative responses in PBL cultures. MID or MID-Sepharose stimulated purified human peripheral B cells as measured by proliferation. In contrast, MID or MID-Sepharose did not activate T cells. Preincubation of purified B cells with anti-IgD Abs inhibited MID-Sepharose-induced B cell proliferation. The addition of IL-4 specifically induced IL-6 production in MID-Sepharose-activated B cells. IgM secretion was detected in B cell cultures stimulated with MID or MID-Sepharose and IL-2 for 10 days. Secretion of IgG and IgA was efficiently induced in cultures from purified B cells stimulated with the combination of MID or MID-Sepharose and IL-4, IL-10, and soluble CD40 ligand, suggesting that Th2-derived cytokines were required for optimal plasma cell generation. Taken together, MID has properties that make it an important tool to study IgD-targeted activation of B cells.

	Introduction

Top Abstract Introduction Materials and Methods Results and Discussion References

 
The mature, naive B lymphocyte expresses properly rearranged IgM and IgD molecules on its surface. IgD has been suggested to be involved in peripheral B cell tolerance, B cell recruitment, and secondary Ig light chain rearrangement, i.e., receptor editing, but still much of the function of IgD remains to be solved (1, 2, 3). Only trace amounts of IgD are found in serum, but significant numbers of normal IgD-secreting plasma cells have been found in human bronchus-associated lymphoid tissue and tonsils (4, 5). This observation might be linked to the fact that the upper respiratory tract in humans are frequently colonized by strains of Moraxella catarrhalis and Haemophilus influenzae, which express outer membrane surface proteins that activate IgD⁺ B lymphocytes by cross-linking IgD (6, 7).

M. catarrhalis is a Gram-negative human mucosal pathogen causing middle ear infections in infants and children and lower respiratory tract infections in adults with chronic pulmonary disease (8). Formalin-fixed preparations of M. catarrhalis and Staphylococcus aureus Cowan strain I have earlier been shown to induce the proliferation of human peripheral blood B lymphocytes (9, 10).

Both M. catarrhalis and S. aureus were shown to be thymus-independent Ags for B cells. However, while S. aureus-induced B cell proliferation is the consequence of protein A binding to and cross-linking membrane Ig using V_H3 gene products, M. catarrhalis has been suggested to bind to the CH1 domain of IgD (7). Interestingly, M. catarrhalis, but not S. aureus, was able to stimulate purified B cells to induction of high quantities of secreted IgM in the absence of exogenous cytokines (10). This is in agreement with the ability of M. catarrhalis to provide an extensive cross-linking of surface IgD. It is well established that thymus-independent Ags such as bacterial polysaccharides, polymeric proteins, and LPS, can stimulate strong Ab responses in the absence of T cells. In contrast, thymus-dependent protein Ags require T cell help. The interaction between the CD40 molecule on B lymphocytes and the CD40 ligand (CD40L)³ on T lymphocytes as well as the B7/CD28 interaction seems to be required in most T cell-dependent B cell responses (11, 12, 13).

We have isolated an M. catarrhalis-derived 200-kDa outer membrane protein, Moraxella IgD binding protein (MID), with IgD-binding properties (14). We show here that MID or MID conjugated to cyanogen bromide (CNBr)-Sepharose (MID-Sepharose) induced a proliferative response in human peripheral B lymphocytes. MID-Sepharose together with IL-4 specifically induced a high level of IL-6 secretion from B cells. IL-2 efficiently induced enhanced IgM secretion by MID- or MID-Sepharose-activated B cells. Importantly, after exposure to CD40L, IL-4, and IL-10, MID- or MID-Sepharose-activated human peripheral B cells could be induced to promote the secretion of IgG and IgA. These results suggest that Th2-derived cytokines are necessary for efficient IgG and IgA production in this system. Thus, we have characterized a novel IgD-binding B cell stimulatory bacterial protein from the respiratory pathogen M. catarrhalis, which has the ability to serve as a tool for studies of B cell activation.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results and Discussion References

 
Reagents

The purification of the M. catarrhalis outer membrane protein MID has recently been described (14). Abs to the following human Ags were used: CD19-RPE, CD3-RPE, rabbit anti-human IgD, and rabbit anti-human IgM (DAKO, Glostrup, Denmark). The mouse anti-human IgD mAb LN has been described previously (7). Human IgD was obtained from Dade- Behring (Paris, France). Human rIL-2 was purchased from Roche (Mannheim, Germany), human rIL-4 and IL-10 was obtained from PeproTech (London, U.K.), and human rIL-6 was obtained from BD PharMingen (San Diego, CA). Human soluble rCD40L and enhancer (anti-CD40L Ab) were used at the concentrations suggested by the manufacturer (Alexis, Läufelfingen, Switzerland). PHA was purchased from Sigma-Aldrich (St. Louis, MO). The MID protein was conjugated to FITC as previously described (14) and to CNBr-Sepharose according to the manufacturer’s instructions. In brief, 4.0 mg MID was diluted in 1 ml coupling buffer (0.1 M NaHCO₃ containing 0.5 M NaCl (pH 8.3)). CNBr-activated Sepharose 4B (1.5 ml; Amersham Biosciences, Uppsala, Sweden) was preswelled and washed in 1 mM HCl. MID and CNBr-Sepharose were mixed and rotated overnight at 4°C. Excess ligand was quantitated by the bicinchoninic acid protein assay kit (Pierce, Rockford, IL) and thereafter washed away. The remaining active groups were blocked with 0.1 M Tris-HCl (pH 8.0) and 1 M ethanolamine (pH 8.0) for 2 h. Finally, the conjugated protein was washed with three cycles of 0.1 M acetate buffer containing 0.5 M NaCl (pH 4.0) and 0.1 M Tris-HCl containing 0.5 M NaCl (pH 8.0). The amount of MID protein bound to CNBr-Sepharose was estimated to 2.0 mg by measuring unbound ligand. The final product was diluted in 5 ml 0.1 M NH₄CO₃ (pH 8.0).

Cell preparations

Human PBMC were isolated from healthy donors by Lymphoprep (Nycomed, Oslo, Norway) density gradient centrifugation. CD19⁺ B lymphocytes were isolated using anti-CD19-conjugated magnetic beads (positive selection) or a B cell isolation kit (negative selection) and a VarioMACS magnetic cell sorter (Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer’s instructions. CD19⁺ B cells isolated by positive selection were routinely >97% HLA-DR⁺ and B cells isolated by negative selection were routinely 90–95% CD19⁺ as assessed by fluorescence analysis using a FACSCaliber (BD Biosciences, San Jose, CA). All cultures were conducted in RPMI 1640 medium (Life Technologies, Paisley, U.K.) supplemented with 10% FCS, 2 mM glutamine, and 10 µg/ml gentamicin (complete medium). A total of 2 x 10⁵ cells were cultured in 96-well round-bottom plates (Nunc, Roskilde, Denmark) in triplicate in a final volume of 200 µl complete medium. Proliferation was measured routinely after 3 days (or as indicated) by [methyl-³H]thymidine incorporation (5 µCi/well; Amersham Biosciences) using an 18-h pulse period.

Detection of IL-6, IgA, IgD, IgE, IgG, and IgM by ELISA

IL-6 production was measured from supernatants harvested on day 3, and Ig production from supernatants harvested on days 10–12. In brief, ELISA plates (Maxisorp, Nunc) were coated with 50 µl of a solution containing rat anti-IL-6 Ab (2 µg/ml; BD PharMingen) or 100 µl rabbit anti-human IgA, IgD, IgE, IgG, or IgM (DAKO) in a 1/1500 dilution in 0.1 M Na₂HPO₄, pH 9.0. Standards and supernatants were diluted in PBS/Tween 20 (0.05%). To determine IL-6, biotinylated rat anti-IL-6 Ab (1 µg/ml; BD PharMingen) and thereafter HRP-conjugated avidin were added. To determine Ig, HRP-conjugated anti-human IgA, IgE, IgG (DAKO), IgD (BioSource, Camarillo, CA), and IgM (Sigma-Aldrich) were used. Tetramethylbenzidine and hydrogen peroxide were used as chromogen and substrate. Finally, the absorbance at 405 nm was determined.

Statistics

The statistical significance of differences was calculated using the nonparametric Mann-Whitney U test.

	Results and Discussion

Top Abstract Introduction Materials and Methods Results and Discussion References

 
MID binds to IgD on human B cells

We have recently isolated the novel IgD-binding outer membrane protein MID from the human mucosal pathogen M. catarrhalis (14). In the present paper, we demonstrate that MID binds to the human IgD B cell receptor and is stimulatory for human IgD⁺ B cells. Human PBL were isolated and analyzed for the expression of B and T cell surface markers and for binding of FITC-conjugated MID protein by flow cytometric analysis. B cells make up 5–15% of the PBL population in a healthy donor. These cells coexpress CD19, IgM, and IgD. We show that FITC-conjugated MID bound to CD19⁺ B cells, but not to CD3⁺ T cells (Fig. 1) (14). In the experiment shown in Fig. 1, FITC-conjugated MID bound 50% of the CD19⁺ cells. The interaction of MID with CD19⁺ cells was efficiently inhibited by preincubation of the cells with a rabbit anti-IgD Ab (Fig. 1, D and F), but not with a rabbit anti-IgM Ab (Fig. 1, E and F). This is in line with earlier results demonstrating that MID does not bind to any of the soluble Ig isotypes IgG, IgM, IgA, or IgE (14). We also incubated PBL with the MID protein, followed by a rabbit anti-MID polyclonal Ab at different dilutions. The rabbit anti-MID Ab bound to PBL incubated with the MID protein, but not to cells with no MID added (data not shown). Moreover, the binding of FITC-conjugated MID to PBL could be inhibited by preincubation of the cells with unlabeled MID or preincubation with human IgD (not shown).

View larger version (27K): [in this window] [in a new window]   FIGURE 1. Fluorescence flow cytometric analysis of the binding of FITC-conjugated MID to human PBL. PBL were labeled with anti-CD19-PE or anti-CD3-PE and MID-FITC with or without preincubation with 10 µg/ml of rabbit anti-human IgD Ab or rabbit anti-human IgM Ab as indicated. F, Results are presented as the mean ± SEM of three independent experiments. Statistical analysis revealed that the difference between the two samples with or without preincubation with rabbit anti-human IgD Ab was significant (p 0.05).

To test the possibility that purified MID could activate lymphocytes, PBL were isolated and cultured with different concentrations of MID. The proliferative response was analyzed between days 2 and 6 of culture. The optimal concentration of MID to activate PBL was 0.1 µg/ml (Fig. 2A). Concentrations >1.0 µg/ml had an inhibitory or possibly toxic effect on the cell proliferation (Fig. 2B). To mimic endogenous MID bound to the bacterial surface of M. catarrhalis, the purified MID protein was conjugated to CNBr-Sepharose. The MID-Sepharose preparation was thereafter tested in different dilutions for its ability to induce a proliferative response in human PBL cultures. A formalin-fixed preparation of M. catarrhalis was also titrated and used as a control to the MID- and MID-Sepharose-induced response. Indeed, we found that optimal dilutions of MID-Sepharose had the capacity to induce a more efficient proliferative response compared with unconjugated MID (Fig. 2B). Interestingly, the response to MID-Sepharose was very similar to that to M. catarrhalis. The addition of high concentrations of MID-Sepharose inhibited the proliferative response (data not shown). As a control, BSA was conjugated to CNBr-Sepharose using the same protocol as that for MID-Sepharose conjugation. BSA-Sepharose did not induce a proliferative response above background levels.

View larger version (21K): [in this window] [in a new window]   FIGURE 2. Human PBL proliferate in response to MID and MID-Sepharose. A, PBL (1 x 106/ml) were cultured in complete medium for 2–6 days with or without MID (0.01–0.1 µg/ml). B, PBL (1 x 106/ml) were cultured in complete medium for 3 days in the absence or presence of MID (0.01–10.0 µg/ml), MID-Sepharose (0.01–1.0 µg/ml), or M. catarrhalis (MCat; 1/2000 dilution of formalin-fixed preparation). [Methyl-3H]thymidine (5 µCi/well) was added 18 h before the end of the culture period. Results are presented as the mean ± SEM of four independent experiments. Cell proliferation in cultures stimulated with MID differed significantly from controls without MID (p 0.05; A). The difference between samples with no addition (no add) and those with addition of MID (0.1–1 µg/ml) or MID-Sepharose (0.01–1 µg/ml) was statistically significant (p 0.05; B).

MID binds to cell surface IgD, as shown by flow cytometry (Fig. 1), and to soluble IgD, as shown by Western and dot blots (14). Therefore, the next step was to analyze whether MID could activate purified naive, peripheral IgD⁺ human B cells. PBL were isolated, and B cells were purified by either positive or negative selection. The isolated B cells were cultured for 3 days with MID or MID-Sepharose. For optimal stimulation of purified B cells, MID was added at a concentration of 0.1–0.5 µg/ml, and MID-Sepharose was added at the same dilution as that used for stimulation of PBL. The purified CD19⁺ B cells responded to MID and MID-Sepharose, but not to the T cell mitogen PHA (Fig. 3A). A fraction of cells containing mainly T cells, NK cells, and monocytes, but no B cells as analyzed by flow cytometry, was cultured for 3 days with MID or MID-Sepharose. The CD19^- cell fraction showed a high proliferative response to PHA, but a very poor response to MID and MID-Sepharose, indicating that B cells were the target cells for MID. Nonpurified PBL from the same donor responded to MID, MID-Sepharose, and PHA (data not shown). Furthermore, purified B cells were preincubated with anti-IgD Abs and cultured with MID-Sepharose for 3 days. The proliferative response was clearly inhibited in the presence of a mouse anti-human IgD mAb, but not in the presence of an irrelevant mouse control Ab (Fig. 3B). A similar inhibitory pattern could be seen with another mouse anti-human IgD mAb (not shown). An inhibition experiment was also performed by neutralizing MID-Sepharose with 10 µg human IgD. The proliferative response analyzed after 3 days showed a 30–40% inhibition compared with cells stimulated with MID-Sepharose alone (not shown).

View larger version (21K): [in this window] [in a new window]   FIGURE 3. Human peripheral B cells proliferate in response to MID and MID-Sepharose. A, Purified CD19+ B cells (1 x 106/ml) or CD19- cells (1 x 106/ml) cultured for 3 days in complete medium alone (no additive) or with PHA (1 µg/ml), MID (0.1 µg/ml), or MID-Sepharose (1.0 µg/ml). B, Purified CD19+ B cells (0.75 x 106/ml) were preincubated with 15–30 µg/ml of the indicated Abs in PBS/1% BSA for 30 min on ice. The cells were washed twice in PBS/1% BSA and thereafter cultured for 3 days in complete medium alone (no additive) or with MID-Sepharose (1.0 µg/ml). MID-Sepharose (1.0 µg/ml) without Ab addition and M. catarrhalis (1/2000 dilution of formalin-fixed preparation) served as controls. [Methyl-3H]thymidine (5 µCi/well) was added 18 h before the end of the culture period. Results are representative of one of two (A) or three (B) experiments performed.

Enhancement of proliferation and IL-6 production by the addition of costimulatory molecules to MID-stimulated B cells

We next wanted to analyze whether the addition of T cell-derived costimulatory molecules could enhance the MID-induced B cell response. Human peripheral B cells were purified by either positive or negative selection and cultured for 3 days with MID or MID-Sepharose together with soluble rCD40L, IL-2, or IL-4 or the combination of all three additives (Fig. 4). In all experiments shown CD19⁺ B cells were purified by positive selection, but similar results were obtained with CD19⁺ B cells purified by negative selection. The addition of soluble CD40L enhanced the MID- or MID-Sepharose induced proliferative B cell response, whereas IL-2 had no effect (Fig. 4A). The addition of IL-4 did not increase the proliferative response, but had an enhancing effect on IL-6 induction. Indeed, high amounts of secreted IL-6 could be measured in supernatants from B cells cultured with IL-4 and MID or MID-Sepharose (Fig. 4C), but not in supernatants from B cells cultured together with IL-4 but without MID (not shown). In contrast, IL-2 added at a concentration of 25–50 U/ml did not have any effect on MID-induced IL-6 production. The addition of CD40L, IL-2, and IL-4 without MID or MID-Sepharose had a minor effect on the B cell-specific proliferation and IL-6 production (Fig. 4, B and D).

View larger version (30K): [in this window] [in a new window]   FIGURE 4. Optimal B cell activation by MID requires costimulatory molecules. Purified CD19+ B cells (0.75 x 106/ml) from three individual donors were cultured for 3 days in complete medium alone (no add) or with or without MID (0.5 µg/ml) or MID-Sepharose (1.0 µg/ml) and rCD40L (10 ng/ml) plus enhancer (anti-CD40L; 1 µg/ml), rIL-2 (25–50 U/ml), or rIL-4 (10 ng/ml) as indicated. PHA (1 µg/ml) and M. catarrhalis (MCat; 1/2000 dilution of formalin-fixed preparation) were included as controls. Supernatants were collected after 3 days of culture. A and B, [Methyl-3H]thymidine (5 µCi/well) was added 18 h before the end of the culture period. C and D, IL-6 was quantified by ELISA. Results in A and C are presented as the mean ± SEM of three independent experiments. Results in B and D are presented as the mean of the maximal response ± SEM of three independent experiments. The difference between the samples with no addition (no add) and with MID-Sepharose plus all additives were statistically significant (p 0.05; A and C). In addition, the difference between the samples with all additives alone and those with MID or MID-Sepharose plus all additives were statistically significant (p 0.05; B and D).

IL-2 enhances IgM secretion by MID-activated B cells

Our next aim was to analyze whether the MID protein could induce production of soluble IgM and secretion of IgG or IgA. Human peripheral B cells were purified by positive selection and thereafter cultured for 10–12 days with MID together with CD40L, IL-2, or IL-4. We could not demonstrate any Ig secretion in B cell cultures that had been activated with MID or MID-Sepharose alone. We found that the addition of IL-2 or CD40L together with MID to B cells enhanced IgM secretion, whereas IL-4 had no such effect (Fig. 5). Similar results were obtained with MID conjugated to Sepharose. The combination of all additives together with MID or MID-Sepharose did not enhance the IgM response (not shown). Earlier studies have shown that IL-2 synergizes with CD40-mediated signals to drive both B cell proliferation and IgM secretion (10, 22). Indeed, our data show that MID-induced B cell activation results in enhanced IgM secretion in the presence of IL-2.

View larger version (18K): [in this window] [in a new window]   FIGURE 5. MID in combination with IL-2 induces IgM secretion. Purified CD19+ B cells (0.75 x 106/ml) were cultured for 10–12 days in complete medium alone (no add) or with MID (1.0 µg/ml) and rCD40L (10 ng/ml) plus enhancer (anti-CD40L; 1 µg/ml), rIL-2 (25–50 U/ml), or rIL-4 (10 ng/ml) as indicated. PHA (1 µg/ml) and M. catarrhalis (MCat; 1/2000 dilution of formalin-fixed preparation) were included as controls. Supernatants were collected after 10–12 days of culture, and IgM was quantified by ELISA. Results are presented as the mean ± SEM of three independent experiments. The difference between the samples with no addition (no add) and MID-Sepharose plus CD40L or IL-2 were statistically significant (p 0.05).

The combination of IL-2, IL-4, and CD40L could not induce the secretion of IgG, IgA, or IgE from MID- or MID-Sepharose-stimulated B cells (data not shown). Thus, we speculated that an additional Th2 cytokine such as IL-10 might be required to induce Ig secretion in the presence of MID. We therefore undertook a more detailed analysis of the requirements for additives and cultured purified peripheral B cells for 10 days with MID and different combinations of additives as shown in Fig. 6. Interestingly, the addition of IL-4 and IL-10 together with CD40L increased IgG secretion in B cells stimulated with MID (Fig. 6A). Moreover, the combination of these three additives was necessary for the induction of optimal IgA secretion in MID-stimulated B cells (Fig. 6B). IL-4 or IL-10 did not differ in costimulatory properties when added together with CD40L. Similar results were obtained with the full combination of additives (IL-4, IL-10, and CD40L) and MID conjugated to Sepharose.

View larger version (22K): [in this window] [in a new window]   FIGURE 6. MID-activated B cells can be induced to secrete IgG and IgA in the presence of CD40L, IL-4, and IL-10. IgG (A) and IgA (B) production is shown. Purified CD19+ B cells (0.75 x 106/ml) were cultured for 10–12 days in complete medium alone (no add) or with MID (0.5 µg/ml) and rCD40L (10 ng/ml) plus enhancer (anti-CD40L; 1 µg/ml), rIL-4 (10 ng/ml), or rIL-10 (50 ng/ml) as indicated. PHA (1 µg/ml) and M. catarrhalis (MCat; 1/2000 dilution of formalin-fixed preparation) were included as controls. Supernatants were collected after 10–12 days of culture, and Ig concentrations were quantified by ELISA. Results are presented as the mean of the maximal response ± SEM of three independent experiments. Statistical analysis revealed that the difference between the samples with no addition (no add) and those with MID plus all additives were significant (p 0.05). The difference between the samples with all additives alone and those with MID plus all additives were also statistically significant (p 0.05).

The induction of IgG or IgA with CD40L, IL-4, and IL-10 without MID was less than that with the combination of MID plus the additives (Fig. 6). Our results indicate that the novel bacterial protein MID has properties similar to a potent B cell-specific Ag. It has been shown that a human monoclonal B cell line could be induced to switch and differentiate in vitro by the engagement of CD40 and the addition of IL-4 and IL-10 (20). In that study the clone CL-01 was induced to switching to all seven downstream isotypes. Whether cytokines such as TGF-, IL-5, and IL-13 are important in this system will be the focus of future studies. Indeed, TGF- has been shown to induce IgA secretion when added together with CD40L and IL-4 (23).

Concluding remarks

It has been known for several years that Moraxella (Branhamella) catarrhalis can activate B cells through IgD (7, 24, 25, 26). The IgD-binding protein that was not identified at the time was claimed to be a thymus-independent (TI) Ag. TI Ags, like bacterial polysaccharides, polymeric proteins, and LPS, stimulate proliferation and strong Ab responses in the absence of T cells. Ags belonging to the first group, the TI-1 Ags, contain an intrinsic activity that can directly induce the proliferation of B cells. The second group of TI Ags, TI-2 Ags, consists of molecules such as bacterial cell wall and capsular polysaccharides that have highly repetitive structures. In contrast, thymus-dependent Ags are classified as Ags that are unable to induce Ab responses in animals or humans in which the thymus fails to develop and generate peptide-specific T cells. Our data with MID support the earlier hypothesis that M. catarrhalis is a TI Ag. However, a stronger Ig production was found when T cells were substituted with soluble CD40L and cytokines compared with samples without costimulatory molecules.

M. catarrhalis is the third most frequent cause of bacterial otitis media and therefore is a major cause of disease. Significant numbers of normal IgD-secreting plasma cells have been found in human bronchus-associated lymphoid tissue and tonsils (4, 5). This has been suggested to be due to the fact that the upper respiratory tract in humans is frequently colonized by strains of H. influenzae and M. catarrhalis, which exposes membrane proteins that activate IgD⁺ B lymphocytes (7). Several studies have demonstrated that dextran- or BSA-conjugated anti-IgD Ab targeting enhances IgG1 secretion in vivo and in vitro (27, 28, 29, 30). Those studies were performed in the murine system with Ag conjugates that fall into the group of TI-2 Ags. Interestingly, in all in vitro systems the addition of T cells or T cell-derived signaling molecules was required for the induction of optimal Ig secretion of the anti-IgD conjugate-activated B cells (29, 30). The studies also demonstrated that cross-linking of membrane IgD is necessary for generating efficient B cell responses.

In conclusion, MID could be an important B cell stimulatory protein by its unique ability to target IgD. IL-2 that is mainly produced by inflammatory T cells of the Th1 phenotype significantly enhanced IgM secretion by MID-activated B cells. Moreover, the induction of IgA and IgG secretion in the presence of CD40L and the Th2 cytokines IL-4 and IL-10 makes MID a promising candidate for future investigations of immune functions.

	Acknowledgments

 
We thank Prof. Tomas Leanderson (Section of Immunology, Department of Cell and Molecular Biology, Lund University) for helpful comments.

	Footnotes

 
¹ This work was supported by grants from The Alfred Österlund Foundation, The Anna and Edwin Berger Foundation, The Crafoord Foundation, The Greta and Johan Kock Foundation, The Inga Britt and Arne Lundberg Foundation, The Magnus Bergvall Foundation, the Swedish Medical Research Council, the Swedish Society of Medicine, The T. H. C. Bergh Foundation, The Åke Wiberg Foundation, and the Cancer Foundation at University Hospital Malmö.

² Address correspondence and reprint requests to Dr. Kristian Riesbeck, Department of Medical Microbiology, University Hospital Malmö, Lund University, S-205 02 Malmö, Sweden. E-mail address: kristian.riesbeck{at}mikrobiol.mas.lu.se

³ Abbreviations used in this paper: CD40L, CD40 ligand; CNBr, cyanogen bromide; MID, Moraxella IgD binding protein; TI, thymus-independent.

Received for publication August 3, 2001. Accepted for publication April 2, 2002.

	References

Top Abstract Introduction Materials and Methods Results and Discussion References

 

1. Cooke, M. P., A. W. Heath, K. M. Shokat, Y. Zeng, F. D. Finkelman, P. S. Linsley, M. Howard, C. C. Goodnow. 1994. Immunoglobulin signal transduction guides the specificity of B cell-T cell interactions and is blocked in tolerant self-reactive B cells. J. Exp. Med. 179:425.[Abstract/Free Full Text]
2. Goodnow, C. C., J. Crosbie, S. Adelstein, T. B. Lavoie, S. J. Smith-Gill, R. A. Brink, H. Pritchard-Briscoe, J. S. Wotherspoon, R. H. Loblay, K. Raphael. 1988. Altered immunoglobulin expression and functional silencing of self-reactive B lymphocytes in transgenic mice. Nature 334:676.[Medline]
3. Liu, Y. J., O. de Bouteiller, C. Arpin, F. Briere, L. Galibert, S. Ho, H. Martinez-Valdes, J. Banchereau, S. Lebecque. 1996. Normal human IgD⁺IgM^- germinal center B cells can express up to 80 mutations in the variable region of their IgD transcripts. Immunity 4:603.[Medline]
4. Brandtzaeg, P., L. Surjan, P. Berdal. 1978. Immunoglobulin systems of human tonsils. I. Control subjects of various ages: quantification of Ig-producing cells, tonsillar morphometry and serum Ig concentrations. Clin. Exp. Immunol. 31:367.[Medline]
5. Ferrarini, M., G. Corte, G. Viale, M. L. Durante, A. Bargellesi. 1976. Membrane Ig on human lymphocytes: rate of turnover of IgD and IgM on the surface of human tonsil cells. Eur. J. Immunol. 6:372.[Medline]
6. Forsgren, A., A. O. Grubb. 1979. Many bacterial species bind human IgD. J. Immunol. 122:1468.[Abstract/Free Full Text]
7. Forsgren, A., A. Penta, S. F. Schlossman, T. F. Tedder. 1988. Branhamella catarrhalis activates human B lymphocytes following interactions with surface IgD and class I major histocompatibility complex antigens. Cell. Immunol. 112:78.[Medline]
8. Karalus, R., A. Campagnari. 2000. Moraxella catarrhalis: a review of an important human mucosal pathogen. Microbes Infect. 2:547.[Medline]
9. Forsgren, A., A. Svedjelund, H. Wigzell. 1976. Lymphocyte stimulation by protein A of Staphylococcus aureus. Eur. J. Immunol. 6:207.[Medline]
10. Huston, M. M., J. P. Moore, H. J. Mettes, G. Tavana, D. P. Huston. 1996. Human B cells express IL-5 receptor messenger ribonucleic acid and respond to IL-5 with enhanced IgM production after mitogenic stimulation with Moraxella catarrhalis. J. Immunol. 156:1392.[Abstract]
11. Ferguson, S. E., S. Han, G. Kelsoe, C. B. Thompson. 1996. CD28 is required for germinal center formation. J. Immunol. 156:4576.[Abstract]
12. Foy, T. M., J. D. Laman, J. A. Ledbetter, A. Aruffo, E. Claassen, R. J. Noelle. 1994. Gp39-CD40 interactions are essential for germinal center formation and the development of B cell memory. J. Exp. Med. 180:157.[Abstract/Free Full Text]
13. Ronchese, F., B. Haussman, S. Hubele, P. J. L. Lane. 1994. Mice transgenic for a soluble form of murine CTLA-4 show enhanced expansion of antigen-specific CD4⁺ T cells and defective antibody production in vivo. J. Exp. Med. 179:809.[Abstract/Free Full Text]
14. Forsgren, A., M. Brant, A. Möllenkvist, A. Muyombwe, H. Janson, N. Woin, K. Riesbeck. 2001. Isolation and characterization of a novel IgD-binding protein from Moraxella catarrhalis. J. Immunol. 167:2112.[Abstract/Free Full Text]
15. Kawabe, T., T. Naka, K. Yoshida, T. Tanaka, H. Fujiwara, S. Suematsu, N. Yoshida, T. Kishimoto, H. Kikutani. 1994. The immune responses in CD40-deficient mice: impaired immunoglobulin class switching and germinal center formation. Immunity 1:167.[Medline]
16. Renshaw, B. R., III W. C. Fanslow, R. J. Armitage, K. A. Campbell, D. Liggitt, B. Wright, B. L. Davison, C. R. Maliszewski. 1994. Humoral immune responses in CD40 ligand-deficient mice. J. Exp. Med. 180:1889.[Abstract/Free Full Text]
17. Xu, J., T. M. Foy, J. D. Laman, E. A. Elliott, J. J. Dunn, T. J. Waldschmidt, J. Elsemore, R. J. Noelle, R. A. Flavell. 1994. Mice deficient for the CD40 ligand. Immunity 1:423.[Medline]
18. Grewal, I. S., R. A. Flavell. 1998. CD40 and CD154 in cell-mediated immunity. Annu. Rev. Immunol. 16:111.[Medline]
19. Banchereau, J., F. Rousset. 1992. Human B lymphocytes: phenotype, proliferation and differentiation. Adv. Immunol. 52:125.[Medline]
20. Cerutti, A., H. Zan, A. Schaffer, L. Bergsagel, N. Harindranath, E. E. Max, P. Casali. 1998. CD40 ligand and appropriate cytokines induce switching to IgG, IgA, and IgE and coordinated germinal center and plasmacytoid phenotypic differentiation in a human monoclonal IgM⁺IgD⁺ B cell line. J. Immunol. 160:2145.[Abstract/Free Full Text]
21. Morse, L., D. Chen, D. Gray, D. Franklin, Y. Xiong, S. Chen-Kiang. 1997. Induction of cell cycle arrest and B cell terminal differentiation by CDK inhibitor p18^INK4c and IL-6. Immunity 6:47.[Medline]
22. Johnson-Léger, C., J. R. Christenson, M. Holman, G. G. Klaus. 1998. Evidence for a critical role for IL-2 in CD40-mediated activation of naive B cells by primary CD4 T cells. J. Immunol. 161:4618.[Abstract/Free Full Text]
23. Nakamura, M., S. Kondo, M. Sugai, M. Nazarea, S. Imamura, T. Honjo. 1996. High frequency class switching of an IgM⁺ B lymphoma clone CH12F3 to IgA⁺ cells. Int. Immunol. 8:193.[Abstract/Free Full Text]
24. Banck, G.. 1982. Staphylococcus aureus Cowan I and Branhamella catarrhalis as B lymphocyte mitogens: culture conditions for optimal DNA synthesis and selective stimulation of human B lymphocytes. J. Immunol. Methods. 51:279.[Medline]
25. Calvert, J. E., A. Calogeras. 1986. Characteristics of human B cells responsive to the T-independent mitogen Branhamella catarrhalis. Immunology 58:37.[Medline]
26. Tedder, T. F.. 1990. Immunoglobulin D-binding bacteria. M. D. P. Boyle, ed. Bacterial Immunoglobulin-Binding Proteins 235. Academic Press, San Diego.
27. Goroff, D. K., J. M. Holmes, H. Bazin, F. Nisol, F. D. Finkelman. 1991. Polyclonal activation of the murine immune system by an antibody to IgD. XI. Contribution of membrane IgD cross-linking to the generation of an in vivo polyclonal antibody response. J. Immunol. 146:18.[Abstract]
28. Lees, A., S. C. Morris, G. Thyphronitis, J. M. Holmes, J. K. Inman, F. D. Finkelman. 1990. Rapid stimulation of large specific antibody responses with conjugates of antigen and anti-IgD antibody. J. Immunol. 145:3594.[Abstract]
29. Pecanha, L. M. T., C. M. Snapper, F. D. Finkelman, J. J. Mond. 1991. Dextran-conjugated anti-Ig antibodies as a model for T cell-independent type 2 antigen-mediated stimulation of Ig secretion in vitro. I. Lymphokine dependence. J. Immunol. 146:833.[Abstract]
30. Pecanha, L. M. T., H. Yamaguchi, A. Lees, R. J. Noelle, J. J. Mond, C. M. Snapper. 1993. Dextran-conjugated anti-IgD antibodies inhibit T cell-mediated IgE production but augment synthesis of IgM and IgD. J. Immunol. 150:2160.[Abstract]

This article has been cited by other articles:

				J. Jendholm, M. Morgelin, M. L. A. Perez Vidakovics, M. Carlsson, H. Leffler, L.-O. Cardell, and K. Riesbeck Superantigen- and TLR-Dependent Activation of Tonsillar B Cells after Receptor-Mediated Endocytosis J. Immunol., April 15, 2009; 182(8): 4713 - 4720. [Abstract] [Full Text] [PDF]

				J. Jendholm, M. Samuelsson, L.-O. Cardell, A. Forsgren, and K. Riesbeck Moraxella catarrhalis-dependent tonsillar B cell activation does not lead to apoptosis but to vigorous proliferation resulting in nonspecific IgM production J. Leukoc. Biol., June 1, 2008; 83(6): 1370 - 1378. [Abstract] [Full Text] [PDF]

				W. Wang, L. Reitzer, D. A. Rasko, M. M. Pearson, R. J. Blick, C. Laurence, and E. J. Hansen Metabolic Analysis of Moraxella catarrhalis and the Effect of Selected In Vitro Growth Conditions on Global Gene Expression Infect. Immun., October 1, 2007; 75(10): 4959 - 4971. [Abstract] [Full Text] [PDF]

				P. Plamondon, N. R. Luke, and A. A. Campagnari Identification of a Novel Two-Partner Secretion Locus in Moraxella catarrhalis Infect. Immun., June 1, 2007; 75(6): 2929 - 2936. [Abstract] [Full Text] [PDF]

				M. Samuelsson, T. Hallstrom, A. Forsgren, and K. Riesbeck Characterization of the IgD Binding Site of Encapsulated Haemophilus influenzae Serotype b J. Immunol., May 15, 2007; 178(10): 6316 - 6319. [Abstract] [Full Text] [PDF]

				T. Nordstrom, J. Jendholm, M. Samuelsson, A. Forsgren, and K. Riesbeck The IgD-binding domain of the Moraxella IgD-binding protein MID (MID962-1200) activates human B cells in the presence of T cell cytokines J. Leukoc. Biol., February 1, 2006; 79(2): 319 - 329. [Abstract] [Full Text] [PDF]

				T. Nordstrom, A. M. Blom, T. T. Tan, A. Forsgren, and K. Riesbeck Ionic Binding of C3 to the Human Pathogen Moraxella catarrhalis Is a Unique Mechanism for Combating Innate Immunity J. Immunol., September 15, 2005; 175(6): 3628 - 3636. [Abstract] [Full Text] [PDF]

				S. Rosseau, K. Wiechmann, S. Moderer, J. Selhorst, K. Mayer, M. Krull, A. Hocke, H. Slevogt, W. Seeger, N. Suttorp, et al. Moraxella catarrhalis-Infected Alveolar Epithelium Induced Monocyte Recruitment and Oxidative Burst Am. J. Respir. Cell Mol. Biol., February 1, 2005; 32(2): 157 - 166. [Abstract] [Full Text] [PDF]

				T. Nordstrom, A. M. Blom, A. Forsgren, and K. Riesbeck The Emerging Pathogen Moraxella catarrhalis Interacts with Complement Inhibitor C4b Binding Protein through Ubiquitous Surface Proteins A1 and A2 J. Immunol., October 1, 2004; 173(7): 4598 - 4606. [Abstract] [Full Text] [PDF]

				M. M. Holm, S. L. Vanlerberg, I. M. Foley, D. D. Sledjeski, and E. R. Lafontaine The Moraxella catarrhalis Porin-Like Outer Membrane Protein CD Is an Adhesin for Human Lung Cells Infect. Immun., April 1, 2004; 72(4): 1906 - 1913. [Abstract] [Full Text] [PDF]

				E. V. Acosta-Rodriguez, C. L. Montes, C. C. Motran, E. I. Zuniga, F.-T. Liu, G. A. Rabinovich, and A. Gruppi Galectin-3 Mediates IL-4-Induced Survival and Differentiation of B Cells: Functional Cross-Talk and Implications during Trypanosoma cruzi Infection J. Immunol., January 1, 2004; 172(1): 493 - 502. [Abstract] [Full Text] [PDF]

				M. M. Holm, S. L. Vanlerberg, D. D. Sledjeski, and E. R. Lafontaine The Hag Protein of Moraxella catarrhalis Strain O35E Is Associated with Adherence to Human Lung and Middle Ear Cells Infect. Immun., September 1, 2003; 71(9): 4977 - 4984. [Abstract] [Full Text] [PDF]

				J. M. Timpe, M. M. Holm, S. L. Vanlerberg, V. Basrur, and E. R. Lafontaine Identification of a Moraxella catarrhalis Outer Membrane Protein Exhibiting Both Adhesin and Lipolytic Activities Infect. Immun., August 1, 2003; 71(8): 4341 - 4350. [Abstract] [Full Text] [PDF]

				A. Forsgren, M. Brant, M. Karamehmedovic, and K. Riesbeck The Immunoglobulin D-Binding Protein MID from Moraxella catarrhalis Is Also an Adhesin Infect. Immun., June 1, 2003; 71(6): 3302 - 3309. [Abstract] [Full Text] [PDF]

				A. Mollenkvist, T. Nordstrom, C. Hallden, J. J. Christensen, A. Forsgren, and K. Riesbeck The Moraxella catarrhalis Immunoglobulin D-Binding Protein MID Has Conserved Sequences and Is Regulated by a Mechanism Corresponding to Phase Variation J. Bacteriol., April 1, 2003; 185(7): 2285 - 2295. [Abstract] [Full Text] [PDF]

				T. Nordstrom, A. Forsgren, and K. Riesbeck The Immunoglobulin D-binding Part of the Outer Membrane Protein MID from Moraxella catarrhalis Comprises 238 Amino Acids and a Tetrameric Structure J. Biol. Chem., September 13, 2002; 277(38): 34692 - 34699. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Wingren, A. G. Articles by Riesbeck, K. Search for Related Content PubMed PubMed Citation Articles by Wingren, A. G. Articles by Riesbeck, K.