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Originally published online as doi:10.1189/jlb.0205065 on November 21, 2005

Published online before print November 21, 2005
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(Journal of Leukocyte Biology. 2006;79:319-329.)
© 2006 by Society for Leukocyte Biology

The IgD-binding domain of the Moraxella IgD-binding protein MID (MID962-1200) activates human B cells in the presence of T cell cytokines

Therése Nordström, Johan Jendholm, Martin Samuelsson, Arne Forsgren and Kristian Riesbeck1

Medical Microbiology, Department of Laboratory Medicine, 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
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Moraxella catarrhalis immunoglobulin D (IgD)-binding protein (MID) is an outer membrane protein with specific affinity for soluble and cell-bound human IgD. Here, we demonstrate that mutated M. catarrhalis strains devoid of MID show a 75% decreased activation of human B cells as compared with wild-type bacteria. In contrast to MID-expressing Moraxella, the MID-deficient Moraxella mutants did not bind to human CD19+ IgD+ B cells. The smallest MID fragment with preserved IgD-binding capacity comprises 238 amino acids (MID962-1200). To prove the specificity of MID962-1200 for IgD, a Chinese hamster ovary (CHO) cell line expressing membrane-anchored human IgD was manufactured. MID962-1200 bound strongly to the recombinant IgD on CHO cells. Moreover, MID962-1200 stimulated peripheral blood lymphocyte (PBL) proliferation 5- and 15-fold at 0.1 and 1.0 µg/ml, respectively. This activation could be blocked completely by antibodies directed against the CD40 ligand (CD154). MID962-1200 also activated purified B cells in the presence of interleukin (IL)-2 or IL-4. An increased IL-6 production was seen after stimulation with MID962-1200, as revealed by a human cytokine protein array. MID962-1200 fused to green fluorescent protein (GFP) bound to human B cells and activated PBL to the same degree as MID962-1200. Taken together, MID is the only IgD-binding protein in Moraxella. Furthermore, the novel T cell-independent antigen MID962-1200 may, together with MID962-1200–GFP, be considered as promising reagents in the study of IgD-dependent B cell activation.

Key Words: B lymphocyte • immunoglobulin D • GFP • Moraxella catarrhalis


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Moraxella catarrhalis is an uncapsulated, Gram-negative diplococcus, which can be detected in nasopharyngeal cultures in 66% of children during the first year of life and in ~4% of adults at any given time. Despite M. catarrhalis, often considered as a commensal, the bacterium plays an important role in respiratory tract infections in children and adults [1 2 3 4 ]. More than 80% of children under the age of three years will be diagnosed with acute otitis media. After Haemophilus influenzae and pneumococci, M. catarrhalis is the third most common bacterial species causing acute otitis media. In adults and the elderly, M. catarrhalis is a common cause of lower respiratory tract infections, particularly in those with predisposing conditions, e.g., chronic obstructive pulmonary disease. Moreover, M. catarrhalis is often implicated as a cause of sinusitis in children and adults.

Despite M. catarrhalis being acknowledged as a human pathogen, only a few reports exist about its specific virulence factors. It is interesting that M. catarrhalis hampers the host innate immune system by conferring serum resistance [5 , 6 ]. We have recently shown that M. catarrhalis interferes with the complement activation pathway by binding C4b-binding protein (C4BP) and C3 to UspA1 and UspA2 [7 , 8 ].

In addition to the interaction with C4BP, M. catarrhalis displays a strong affinity for soluble immunoglobulin D (IgD) [9 ], where its binding at the cellular level explains the strong mitogenic effects on human lymphocytes by M. catarrhalis [10 11 12 13 ]. We have previously isolated and characterized the high molecular weight surface protein Moraxella IgD-binding protein (MID), which displays a high affinity for soluble and surface-bound IgD [14 ]. The presence of MID, also designated Hag, has been confirmed by two other laboratories [15 , 16 ]. The apparent molecular mass of monomeric MID is estimated to ~200 kDa. The mid gene was detected in 98 different strains, and Moraxella isolates from nasopharynx, blood, and sputum express MID at approximately the same frequency [17 ]. Moreover, no variation was observed between strains from different geographical origins.

In addition to the IgD-binding properties of MID, the outer membrane protein is an important adhesin of M. catarrhalis [18 , 19 ]. MID-expressing M. catarrhalis strains agglutinate human erythrocytes and bind to type II alveolar epithelial cells. In contrast, M. catarrhalis isolates with low MID expression levels do not agglutinate erythrocytes and have a 50% lower adhesive capacity [18 ]. We have shown that the hemagglutinating and adhesive part of MID is localized within the 150 amino acid residues MID764-913. In addition, antibodies against full-length MID, MID764-913, or a 30 amino acid-long consensus sequence (MID775-804) inhibited adhesion to alveolar epithelial cells. Furthermore, in a pulmonary clearance model, mice immunized with MID764-913 more strongly cleared M. catarrhalis wild-type bacteria compared with control mice [20 ].

Downstream of the adhesive MID764-913 sequence, we have defined a fragment comprising 238 amino acid residues (MID962-1200) with an essentially preserved IgD binding when compared with full-length MID1-2139 [21 ]. Shorter recombinant proteins gradually lose their IgD-binding capacity, and the shortest IgD-binding fragment, comprising 157 amino acids (MID985-1142), displays a 1000-fold, reduced IgD binding compared with the full-length molecule. The truncated MID962-1200 is efficiently attracted to a standard IgD serum and purified myeloma IgD({kappa}) and IgD({lambda}) sera but not to IgG, IgM, or IgA myeloma sera. Results obtained by introducing five amino acids randomly into MID962-1200 using transposons suggested that {alpha}-helix structures are important for the IgD binding. Ultracentrifugation experiments and gel electrophoresis revealed that native MID962-1200 is a tetramer. It is interesting that tetrameric MID962-1200 attracts IgD more than 20-fold more efficiently than the monomeric form.

The goals of the present paper were to examine the B cell stimulatory capacity of MID1-2139-deficient M. catarrhalis and to investigate the interactions between soluble and membrane-bound IgD and recombinant, truncated MID962-1200. Mutated M. catarrhalis strains devoid of MID showed a 75% decreased activation as compared with wild-type counterparts. In parallel, the MID-deficient strains did not bind to IgD-expressing B cells. In addition, MID962-1200 specifically bound IgD-expressing Chinese hamster ovary (CHO) transfectants. MID962-1200 activated peripheral blood lymphocytes (PBL) up to 15-fold as compared with untreated controls. It is interesting that MID962-1200 fused to green fluorescent protein (GFP) was found to bind to human B cells and stimulate PBL to the same degree as MID962-1200. Finally, the finding that purified B cells were strongly activated by MID962-1200 when coincubated with T cell cytokines further strengthens the evidence that the protein functions as a T cell-independent antigen.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Reagents
Retinal pigment epithelial (RPE)-conjugated mouse anti-human CD19 monoclonal antibody (mAb), fluorescein isothiocyanate (FITC)-conjugated rabbit anti-human IgD, and swine anti-rabbit polyclonal antibody (pAb) were from Dakopatts (Glostrup, Denmark). A human IgD standard serum was from Dade-Behring (Marburg, Germany). For blocking experiments, a mixture of neutralizing mouse anti-human CD154 [CD40 ligand (CD40L)] mAb (n=8) was obtained from BioLegend (San Diego, CA) and used according to the manufacturer’s instructions. Horseradish peroxidase (HRP)-conjugated goat anti-human IgD pAb were purchased from BioSource International (Camarillo, CA). The anti-MID902-1200 antiserum was prepared as described previously [21 ]. Briefly, rabbits were immunized intramuscularly with 200 µg purified recombinant MID902-1200 fragment emulsified in complete Freund’s adjuvants (Difco, Becton Dickinson, Heidelberg, Germany) and boosted on days 18 and 36 with the same dose of protein in incomplete Freund’s adjuvants. Blood was drawn 3 weeks later. The anti-MID902-1200 antiserum reacted with recombinant MID962-1200 and MID1000-1200. The Ig fraction of rabbit anti-human IgD–functional antibody fragment (Fab) was prepared as described before [9 , 21 ]. Human recombinant interleukin (IL)-2 was from Roche (Mannheim, Germany), and human recombinant IL-4 and IL-10 were obtained from Peprotech (London, UK). Phytohemagglutinin (PHA) and phorbol myristate actetate (PMA) were purchased from Sigma Chemical Co. (St. Louis, MO).

Bacteria and culture conditions
M. catarrhalis strains RH4, BBH18, and Bc5 were clinical isolates as described previously [17 ]. All bacterial strains were grown on blood agar base solid medium or brain heart infusion (BHI) liquid medium. MID-deficient M. catarrhalis were constructed using a standard protocol. Briefly, a kanamycin-resistance gene cassette from pUC4K was amplified by polymerase chain reaction (PCR) using specific primers introducing the restriction enzyme site for EcoRV. The resulting PCR product was digested and ligated into the EcoRV site of the mid gene [14 ]. M. catarrhalis strains RH4 and BBH18 were transformed by electroporation using a Genepulser apparatus (Bio-Rad, Sundbyberg, Sweden) and the settings 2.5 kV, 25 µF, and 200 Ohm. After transformation, bacteria were first cultured in BHI liquid medium without kanamycin for 6 h and thereafter, grown on BHI solid medium supplemented with kanamycin. Resulting mutants were deficient for MID, as revealed by Western blot using pAb directed against full-length MID and the truncated MID fragments A–I [18 ]. Furthermore, the two mutants were devoid of IgD binding, as revealed by Western blots and flow cytometry.

For formaldehyde killing, bacteria were grown overnight in nutrient broth (Oxoid, Basingstoke Hampshire, UK), harvested, and washed in phosphate-buffered saline (PBS), pH 7.2, by centrifugation. Thereafter, they were resuspended immediately in 0.5% formaldehyde for 3 h at room temperature, followed by heat treatment at 80°C for 3 min. After being washed in PBS, the bacteria were suspended in RPMI-1640 medium (Gibco, Paisley, UK) and stored in aliquotes at –20°C.

DNA cloning and protein expression
The truncated MID constructs MID962-1200 and MID1000-1200 were amplified by PCR using specific primers introducing the restriction enzyme sites BamHI and HindIII [21 ]. The primers were: 5'-GGATCCTGACCAAACCAAAGGCTTAAC-3' (forward primer MID962-1200), 5'-GCCAAGCTTGGTTTGGGCTTGGGCGACC-3' (forward primer MID1000-1200), and 5'-CAAGGATCCAACCGATGCCACCAAC-3' (reverse primer for both constructs). The open-reading frame of the mid gene from M. catarrhalis (pET26-MID) was used as template [14 ]. The resulting PCR products were cloned into pET26b(+; Novagen, Darmstadt, Germany). To avoid presumptive toxicity, the resulting plasmids were first transformed into the host Escherichia coli DH5{alpha}. Thereafter, the plasmids encoding for the MID fragments were transformed into the expressing host BL21(DE3; Novagen). To produce recombinant proteins, bacteria were grown to mid-log phase [optical density (OD)600 0.5–1.0], followed by 3.5 h of induction with 1 mM isopropyl-1-thio-ß-D-galactoside, resulting in overexpression of the proteins.

For construction of the fusion protein MID962-1200–GFP, the MID962-1200 cassette was amplified using the specific primers 5'-AAGGCATGCTGACCAAACCAAAGGCTTAACCACGCC-3' and 5'-GGTCGCCCAAGCCCAAACCCCGTCGACCAC-3', introducing the SphI and SalI restriction enzyme sites. The resulting product was thereafter cloned into plasmid GFP (pGFP; Clontech, Palo Alto, CA). To be able to purify the recombinant fusion protein, a histidine tag was introduced in the C-terminal of GFP using the oligonucleotide 5'-CGGCATGGACGAGCTGTACAAGCTTCACCACCACCACCACCACTGATCAACGAATTCCC-3'. The histidine tag was also introduced in pGFP. The resulting plasmids were transformed into DH5{alpha}. To produce recombinant proteins, bacteria were grown overnight.

To purify the recombinant proteins, bacteria were sonicated, and the proteins were applied to columns containing a nickel resin (Novagen), according to the manufacturer’s instructions for native conditions. The concentrations of eluted proteins were determined using the bicinchoninic (BCA) protein assay kit (Pierce, Rockford, IL). The resulting proteins were examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and further confirmed by Western and dot blots [21 ].

IgD-expressing cell line
To manufacture the membrane-bound human IgD-expressing CHO cell line (CHO IgD), the IgD coding sequences were amplified by PCR from the vector pAZ6812 (kindly provided by Dr. Sherie L. Morrison, Department of Microbiology and Molecular Genetics, University of California, Los Angeles) using the specific primers 5'-GAGCGCTAGCCAGTGTGATGGATATCCACCATGTACTTGGGAC-3' and 5'-GAGGGATCAGGGGGGTCATGGCCAGATAGCTGACTTCTAGGCTCCGGC-3'. The membrane anchor was amplified from B cell complementary DNA (cDNA) using the primers 5'-GCCGGAGCCTAGAAGTCAGCTATCTGGCCATGACCCCCCTGATCCCTC-3' and 5'-CGCGGATCCCTACTTCACCTTGATGAAAGTGACAATGCCGCTGTAG-3'. These two products were spliced together using overlap extension PCR. After digestion with the restriction enzymes NheI and BamHI, the spliced product was cloned into the pcDNA5/Flp recognition target (FRT) vector (Invitrogen, Carlsbad, CA). The CHO Flp-In cell line (Invitrogen) was transfected with cDNA encoding the human {kappa}-light chain (KLC), resulting in a cell line designated CHO-KLC, which was supertransfected with the pcDNA5/FRT vector harboring cDNA encoding for IgD heavy chain, including the transmembrane part. All transfections were done using Lipofectin reagent (Invitrogen).

Gel electrophoresis
SDS-PAGE (12%) was run as described [14 ]. Briefly, samples of purified MID962-1200, MID1000-1200, and MID962-1200–GFP were mixed with SDS sample buffer at 100°C and applied on a SDS-PAGE. Gels were stained with Coomassie brillant blue R-250 (Bio-Rad).

Enzyme-linked immunosorbent assay (ELISA)
The interactions between IgD and the MID-derived proteins were analyzed by ELISA. Microtiter plates (F96 Maxisorp, Nunc-Immuno Module, Roskilde, Denmark) were coated with 50 µl 100 nM recombinant MID962-1200, MID1000-1200, or MID962-1200–GFP in 0.1 M Tris-HCl (pH 9.0) at 4°C overnight. The plates were washed four times with PBS containing 0.05% Tween 20 (PBS-Tween) and blocked for 2 h at room temperature with PBS-Tween supplied with 1.5% ovalbumin (blocking buffer). After four washings, the plates were incubated for 1 h at room temperature with recombinant human IgD standard serum diluted in threefold steps in blocking buffer. Thereafter, HRP-conjugated goat anti-human IgD pAb was added and incubated at room temperature for 45 min. After four additional washings, the plates were developed and measured at 450 nm.

Conjugation of MID962-1200, MID1000-1200, and MID962-1200–GFP to cyanogen bromide (CNBr)-Sepharose
The MID962-1200, MID962-1200, and MID962-1200–GFP were conjugated to CNBr-Sepharose according to the manufacturer’s instructions. Briefly, 1.0 mg each recombinant protein was diluted in 1 ml coupling buffer (0.1 M NaHCO3 containing 0.5 M NaCl, pH 8.3). CNBr-activated Sepharose 4B (0.5 g, Amersham Pharmacia Biotech, Uppsala, Sweden) was pre-swelled and washed in 1 mM HCl. The recombinant proteins and CNBr-Sepharose were mixed and rotated overnight at 4°C. Excess ligand was quantitated by the BCA protein assay kit and thereafter discarded. The remaining active groups were blocked with 0.1 M Tris-HCl, pH 8.0, for 2 h. Finally, the conjugated proteins were 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 final product was diluted in 4 ml 0.1 M NH4CO3 (pH 8.0). The estimated concentration of the proteins bound to CNBr-Sepharose was 180 µg/ml for MID962-1200, 250 µg/ml for MID1000-1200, and 200 µg/ml for MID962-1200–GFP.

Cell preparations
Human PBL were isolated from healthy donors by Lymphoprep (Nycomed, Oslo, Norway) density-gradient centrifugation as described [22 ]. CD19+ B lymphocytes were isolated using anti-CD19-conjugated magnetic beads 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% human leukocyte antigen-DR+, as assessed by flow cytometry. A series of experiments was also performed by using negatively selected B cells (B cell isolation kit, Miltenyi Biotec) with an additional step using anti-CD3-conjugated magnetic beads (Miltenyi Biotec). Cells were cultured in RPMI-1640 medium (Life Technologies, Paisley, Scotland) supplemented with 10% fetal calf serum, 2 mM glutamine, and 10 µg/ml gentamicin (complete medium). A total of 2.5 x 105 cells was cultured in 96-well round-bottom plates (Nunc-Immuno Module) in triplicates in a final volume of 200 µl complete medium supplemented with various reagents. In CD40L blocking experiments, PBL were preincubated with 50 µg/ml anti-CD40L mAb for 1 h at 37°C. In some experiments, MID962-1200 was immobilized on flat-bottom plates, which gave the same results as MID962-1200 conjugated to CNBr-Sepharose. To analyze whether the folding of MID962-1200 affected the proliferation, MID962-1200 was incubated at various temperatures (20, 37, 60, and 100°C). After 15 min, MID962-1200 was coated immediately on flat-bottom plates in 0.1 M Tris-HCl (pH 9.0) at 4°C overnight. The plates were washed thoroughly before addition of PBL. Proliferation was measured by [methyl-3H]-thymidine incorporation (5 µCi/well, Amersham Pharmacia Biotech) using an 18-h pulse period.

In some experiments, a T cell cytokine-rich supernatant was used. To produce T cell cytokines, T lymphocytes, which were obtained from the "flow-through" during positive B cell selection (as described above), were incubated with 1 µg/ml PHA and 50 ng/ml PMA for 24 h. After centrifugation, the supernatant was dialyzed thoroughly (dialysis filter: molecular weight cut-off, 6–8000 kDa; Spectrum Laboratories, Rancho Dominguez, CA) for 24 h at 4°C to remove the mitogens. For the cytokine protein arrays, 1 x 106 negatively selected B cells were cultured with or without MID962-1200 in 12-well flat-bottom plates (Nunc-Immuno Module) in a final volume of 1 ml complete medium. The cell-free supernatant was harvested after 96 h, and the cytokine protein arrays were performed according to the manufacturer’s instructions (RayBio Human Antibody Array VI, RayBiotech, Inc., Norcross, GA).

Fluorescence labeling of M. catarrhalis
Bacteria (OD600 0.4) were washed in PBS, pH 7.4, and then resuspended in 1 ml PBS containing 1% bovine serum albumin (BSA). FITC (Sigma Chemical Co.) was added at a final concentration of 0.2 mg/ml, and the suspension was incubated at room temperature. After 20 min under rotation, Moraxella was washed three times in PBS-BSA and resuspended in PBS before addition to the cells. The bacterial viability was not affected by the labeling, as judged by counting colony-forming units on agar plates.

Flow cytometry analyses
For flow cytometry analyses, bacteria (108) were washed twice with PBS containing 1% BSA and incubated with a human IgD standard serum or rabbit anti-MID902-1200 pAb in a final volume of 100 µl PBS, 1% BSA, on ice for 30 min. After washing, the bacteria were incubated with FITC-conjugated anti-IgD pAb or FITC-conjugated swine anti-rabbit pAb for 30 min on ice. After two additional washes, the bacteria were analyzed by flow cytometry (EPICS, XL-MCL flow cytometer, Coulter, Hialeah, FL). In another set of experiments, the FITC-labeled M. catarrhalis strain BBH18 or its derived MID mutant was incubated with 5 x 105 PBL in 100 µl PBS containing 1% BSA for 30 min on ice. After washings, a RPE-conjugated anti-CD19 mAb was added for 30 min on ice followed by additional washings and analysis by flow cytometry.

To determine the ability of MID962-1200–GFP fusion protein to bind to human B cells, 10 µg MID962-1200–GFP was incubated with 5 x 105 PBL in 100 µl PBS-BSA. After 30 min on ice and washings, anti-CD19-RPE mAb was added for another 30 min on ice followed by additional washings and flow cytometry analysis. In blocking experiments, PBL were preincubated with 30 µg anti-human IgD-Fab pAb for 1 h on ice before incubation with the MID962-1200–GFP fusion protein. After two additional washes, the cells were analyzed by FACSCalibur flow cytometer (Becton Dickinson, Franklin Lakes, NJ). FITC-conjugated rabbit anti-human IgD and GFP were included as controls.

To investigate the presence of membrane-anchored IgD on the surface of CHO-IgD, 3 x 105 cells were incubated with an anti-human IgD FITC mAb for 1 h on ice. Thereafter, the cells were washed in PBS-BSA and analyzed in the FACSCalibur flow cytometer (Becton Dickinson, Franklin Lakes, NJ). The binding of MID962-1200 to CHO-IgD was investigated by incubating 3 x 105 cells with 10 µg MID962-1200 for 1 h on ice. After washing in PBS-BSA, anti-MID962-1200 pAb were added. Cells were washed and incubated with FITC-conjugated goat anti-rabbit pAB. Cells were analyzed by flow cytometry. CHO-KLC was used as a negative control.

Immunohistochemistry
For fluorescence microscopy, 2 x 106 PBL were incubated with 0.5 µg MID962-1200–GFP or GFP together with RPE-conjugated anti-CD19 mAb in PBS containing 1% BSA for 30 min on ice. Thereafter, PBL were then washed twice in PBS. Samples were examined using a Nikon Eclipse E800 fluorescence microscope (Nikon, Osaka, Japan), and images were analyzed using Image-Pro Plus 4.0 software (Media Cybernetics, Silver Spring, MD).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
M. catarrhalis-dependent activation of human PBL is caused by the outer membrane protein MID
M. catarrhalis stimulates B cells to proliferate, and MID, purified from Moraxella bacteria, has been demonstrated to induce B cell proliferation [11 , 13 ]. To further examine the importance of MID in M. catarrhalis-dependent activation, two Moraxella clinical isolates (BBH18 and RH4) were chosen for inactivation of MID. A kanamycin-resistance gene cassette was introduced into the chromosome, and the resulting mutants were screened for the absence of MID expression and further confirmed by PCR. Flow cytometry analysis of the two MID-deficient mutant M. catarrhalis strains, using an anti-MID902-1200 pAb, proved the absence of MID at the cell surface. In Figure 1A , the flow cytometry analysis of the M. catarrhalis BBH18 strain is exemplified. The wild-type BBH18 strongly bound soluble IgD, whereas the corresponding MID-deficient mutant was negative for IgD binding (Fig. 1B) .


Figure 1
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  		Figure 1. MID-deficient M. catarrhalis does not bind soluble or membrane-bound IgD. Flow cytometry profiles of M. catarrhalis BBH18 and the MID-deficient mutant BBH18{Delta}mid showing MID expression (A) or IgD binding (B). PBL were incubated with the FITC-conjugated BBH18 wild-type (C), the MID-deficient mutant BBH18{Delta}mid (D), or anti-human IgD pAb (E). (A and B) The bacteria were grown on solid medium overnight. After incubation with anti-MID902-1200 pAb or human IgD standard serum, followed by a FITC-conjugated anti-rabbit pAb or anti-human IgD pAb and several washings, bacteria were analyzed by flow cytometry. (C–E) PBL were isolated from heparinized human blood using Lymphoprep one-step gradients, incubated with a RPE-conjugated anti-CD19 mAb, washed, and further incubated with FITC-conjugated M. catarrhalis or FITC-conjugated anti-human IgD pAb. After final washings, PBL were analyzed by flow cytometry.

 
To demonstrate the specificity of the MID-dependent binding to human B cells, the M. catarrhalis wild-type and the MID-deficient M. catarrhalis mutant were FITC-conjugated followed by incubation with PBL. B lymphocytes were visualized by RPE-conjugated anti-CD19 mAb. The high MID-expressing wild-type M. catarrhalis BBH18 bound to CD19+ B cells (Fig. 1C) , whereas the MID-deficient mutant did not (Fig. 1D) . Furthermore, neither the MID-expressing Moraxella strain nor the mutant bound to the T cells (the CD19– cell population). A FITC-conjugated anti-human IgD pAb was used as positive control (Fig. 1E) . Similar results were obtained with the M. catarrhalis RH4 strain and the corresponding RH4{Delta}mid mutant (data not shown).

We also incubated human PBL with the M. catarrhalis BBH18 and RH4 wild-type bacteria and their MID-deficient counterparts. Cells were pulsed with [3H]-thymidine, and the uptake was analyzed by scintillation counting. It is interesting that PBL incubated with BBH18{Delta}mid and RH4{Delta}mid showed a 75% reduced proliferation as compared with cells stimulated with the wild-type MID-expressing bacteria (Fig. 2 ). Thus, the IgD-binding protein MID was the major Moraxella protein responsible for binding to and consequently activating human B cells.


Figure 2
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  		Figure 2. MID-expressing M. catarrhalis strongly activates PBL as compared with MID-deficient M. catarrhalis. PBL were incubated with formaldehyde-killed M. catarrhalis BBH18 or RH4 wild-type (wt) strains or their MID mutant counterparts. After 3 days, lymphocyte proliferation was analyzed by determination of [3H]-thymidine uptake. Control indicates the background proliferation without any added bacteria. Three separate experiments with different donors were done. Error bars indicate SD. cpm, Counts per minute.

 
MID962-1200 fused to GFP retains its IgD- and B cell-binding capacity
The smallest fragment with essentially preserved, soluble IgD binding, as compared with the full-length molecule MID, comprises 238 amino acid residues (MID962-1200; Fig. 3A ). MID962-1200 was produced recombinantly in E. coli and purified by affinity chromatography. In addition, a non-IgD-binding fragment (MID1000-1200) was manufactured to be included as a negative control (Fig. 3A and 3B) . To further characterize the interaction between MID962-1200 and B cells, a fusion protein of MID962-1200 and GFP was constructed. MID962-1200 as well as the MID962-1200–GFP fusion protein bound soluble, recombinant IgD in a dose-dependent manner, as revealed by an IgD ELISA, whereas MID1000-1200 did not bind IgD (Fig. 3C) . Thus, the ability of MID962-1200–GFP to bind soluble IgD was not affected by the GFP fusion partner.


Figure 3
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  		Figure 3. Schematic map of the recombinant proteins used in this study. MID962-1200 and MID962-1200–GFP binds soluble human IgD in a dose-dependent manner, whereas MID1000-1200 does not. Localization of the two truncated protein fragments MID962-1200 and MID1000-1200 within full-length MID1-2139 (A) [17, 21]. Coomassie-stained gel, demonstrating the purity of the recombinant proteins (B). A molecular weight standard is indicated on the left. Soluble IgD bound to MID962-1200 and MID962-1200–GFP but not to MID1000-1200 as analyzed by ELISA (C). Recombinant proteins were expressed in E. coli and purified using His-tags, followed by analysis in SDS-PAGE. (C) An immunoplate was coated with MID962-1200, MID962-1200–GFP, or MID1000-1200, followed by incubation with human IgD, diluted in threefold steps and added in duplicates. After incubation and washings, bound IgD was analyzed by HRP-conjugated goat anti-human IgD pAb. The experiment was repeated twice, and error bars show SD.

 
To analyze whether the MID962-1200–GFP fusion protein bound to the B cell receptor (BCR) IgD, human PBL were isolated. PBL were incubated with MID962-1200–GFP and anti-human CD19 RPE, followed by flow cytometry analyses and fluorescence microscopy. As can be seen in Figure 4B , MID962-1200–GFP efficiently bound to the CD19+ B cells. The interaction was blocked completely when the PBL were preincubated with anti-human IgD-Fab (Fig. 4C) . Furthermore, the specificity for the IgD receptor was verified by staining B cells with anti-CD19 mAb and FITC-conjugated anti-IgD pAb in the presence of MID962-1200–GFP (Fig. 4E) . This combination of antibodies, in addition to MID962-1200–GFP, did not further increase the fluorescence (mean fluorescence intensity), excluding other surface molecules involved in MID962-1200–GFP binding. The binding of MID962-1200–GFP to B cells was also confirmed by fluorescence microscopy. MID962-1200–GFP (green fluorescence) bound to CD19+ cells (red fluorescence), whereas CD19– cells did not bind MID962-1200–GFP (Fig. 4H and 4I) . In contrast, any green fluorescence could not be detected when PBL were incubated with GFP alone (Fig. 4L) . Taken together, MID962-1200–GFP specifically bound to IgD-expressing B cells, as revealed by flow cytometry and immunohistochemistry.


Figure 4
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  		Figure 4. The fusion protein MID962-1200–GFP specifically bound to CD19+ IgD+ B cells. Flow cytometry profiles demonstrate PBL incubated with RPE-conjugated anti-CD19 mAb (A–F) together with MID962-1200–GFP (B, C, and E) or FITC-conjugated anti-human IgD (D and E). (C) PBL were preincubated with anti-IgD-Fab pAb before addition of MID962-1200–GFP, and GFP was used as a negative control (F). (E) Anti-IgD-FITC mAb and MID962-1200–GFP were added simultaneously. Panels with immunohistochemistry show bright fields (G and J) and filters for the emission wavelengths of RPE (575 nm; H and K) or GFP (508 nm; I and L). PBL without added mAb or recombinant proteins were used to exclude autofluorescence (not shown). (G–I) PBL were incubated with RPE-conjugated anti-CD19 mAb and MID962-1200–GFP. Moreover, cells were also incubated with RPE-conjugated anti-CD19 mAb and GFP without the IgD-binding protein MID962-1200 (J–L). PBL were purified from heparinized human blood using Ficoll gradients, incubated with RPE-conjugated anti-CD19 mAb, washed, and incubated further with MID962-1200–GFP or GFP as indicated. After final washings, PBL were analyzed by flow cytometry or fluorescence microscopy. Representative results from two separate experiments are shown.

 
MID962-1200 binds recombinantly expressed membrane-anchored IgD
To further prove that MID962-1200 binds to the IgD BCR, IgD-expressing CHO cells were manufactured. A cell line was established by transfecting cDNA encoding KLC. Thereafter, the CHO-KLC cell line was supertransfected with cDNA coding for the IgD heavy chain containing the cell membrane-bound anchor. The resulting CHO-IgD transfectants were confirmed by flow cytometry using a mAb directed against human IgD (Fig. 5A ). The negative control, i.e., the CHO-KLC cell line, did not express IgD. To analyze binding of MID962-1200 to the IgD BCR, IgD-expressing CHO cells were incubated with MID962-1200, followed by rabbit anti-MID902-1200 pAb and FITC-conjugated goat anti-rabbit pAb. Flow cytometry profiles revealed that MID962-1200 bound to the CHO-IgD transfectants (Fig. 5B) . In contrast, MID962-1200 did not bind to CHO-KLC, indicating that the binding was specific for IgD. The apparent binding of MID962-1200 to CHO-KLC in Figure 5B was background binding of the anti-MID902-1200 pAb to the CHO-KLC cells. Thus, our results further strengthen the specificity of MID962-1200 for membrane-anchored IgD.


Figure 5
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  		Figure 5. MID962-1200 specifically binds to membrane-anchored, recombinant IgD. (A) CHO cells expressing KLC (CHO-KLC) or KLC in addition to the IgD heavy chain (CHO-IgD) were analyzed by flow cytometry using FITC-conjugated anti-human IgD. (B) Flow cytometry profiles of the two CHO cell lines preincubated with MID962-1200 followed by detection with rabbit anti-MID902-1200 and a FITC-conjugated goat anti-rabbit pAb.

 
MID962-1200 and MID962-1200–GFP activate human PBL, whereas MID1000-1200 is inert
To analyze the capacity of MID962-1200 and the GFP fusion protein to stimulate human lymphocytes, PBL were isolated and incubated for various times with MID962-1200 or MID962-1200–GFP at increasing concentrations. In these experiments, MID962-1200 or MID962-1200–GFP was conjugated to Sepharose beads to achieve an efficient cross-linking of the IgD BCRs. During the last 18 h of incubation, PBL were pulsed with [3H]-thymidine. As can be seen in Figure 6A , MID962-1200 at 0.5 µg/ml was optimal to induce a vigorous proliferation. It is interesting that similar results were obtained with the MID962-1200–GFP fusion protein (Fig. 6B) . However, as the GFP protein is almost twice as large as MID962-1200, more molecules were required to achieve the same level of proliferative response. Analysis of the kinetics revealed that the strongest proliferation was observed at 96 h (Fig. 6C) . A similar kinetics was seen with MID962-1200–GFP (not shown). In contrast, the recombinant non-IgD-binding MID1000-1200 fragment, which was included as a negative control, did not induce any proliferation. Thus, MID962-1200 and the GFP fusion protein induced lymphocyte proliferation.


Figure 6
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  		Figure 6. MID962-1200 and MID962-1200–GFP stimulate untouched PBL to vigorous proliferation, and blocking with anti-CD40L mAb inhibits the activation. MID962-1200 (A) or MID962-1200–GFP (B) activates human PBL in a dose-dependent manner (range 0.1–5 µg/ml). (C) Kinetics of PBL proliferation. (D) PBL preincubated with anti-CD40L mAb (50 µg/ml). PBL were isolated and incubated with formaldehyde-killed M. catarrhalis or the indicated recombinant proteins conjugated to CNBr-Sepharose for 96 h (A, B, and D) or 48–120 h (C). For the last 18 h, the PBL were pulsed with [3H]-thymidine. Results are presented as means of three separate experiments with different donors. (D) The control with M. catarrhalis Bc5 in each experiment was defined as 100 arbitrary units. Error bars indicate SD.

 
To determine the importance of the CD40/CD40L interaction between T cells and MID962-1200-induced B cells, PBL were preincubated with a mixture of neutralizing anti-CD40L mAb. As can be seen in Figure 6D , blocking of the CD40L completely inhibited the proliferation. This result proved that the physical B and T cell interaction was crucial for MID962-1200-induced PBL activation.

We recently demonstrated that MID962-1200 is a tetramer under native conditions and that the tetramer bound IgD 23-fold more efficiently as compared with the monomeric form [21 ]. To analyze whether the folding of MID962-1200 affected its ability to stimulate human lymphocytes, MID962-1200 was incubated at increasing temperatures to create monomers. Thereafter, cell-culture plates were coated with MID962-1200 followed by incubation with PBL. It is interesting that the proliferation of the PBL decreased dramatically when MID962-1200 had been incubated at 60 and 100°C (Fig. 7 ), i.e., when MID962-1200 was denaturated and existed as a monomer [21 ]. This implies that a stable tetramer formation of MID962-1200 is required for efficient activation.


Figure 7
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  		Figure 7. The native folding of tetrameric MID962-1200 is crucial for efficient activation of PBL. MID962-1200 was treated at room temperature, 37, 60, or 100°C, and thereafter, flat-bottom cell-culture plates were coated with the resulting tetramers (native MID962-1200) or monomers. After incubation with PBL for 96 h, the [3H]-thymidine uptake was determined. Results are presented as means of three separate experiments with different donors. The highest value in each experiment was defined as 100 arbitrary units. Error bars indicate SD.

 
MID962-1200 stimulates B cells in a T cell-independent manner requiring T cell cytokines
To investigate the importance of T cell cytokines for the MID962-1200-dependent activation of human B lymphocytes, purified B cells were incubated with MID962-1200 in the absence or presence of a cell-free supernatant obtained from T lymphocytes, which had been stimulated with PMA and PHA for 24 h. Only a minor stimulation of purified B cells was seen in the presence of 0.5 or 1.0 µg/ml MID962-1200 without cytokines (Fig. 8A ). In contrast, when the T cell cytokine-rich supernatant was supplemented together with MID962-1200, a strong proliferation was observed, demonstrating the need for a costimulatory signal. When B lymphocytes were incubated with T cell cytokines only (in the absence of MID962-1200), no proliferation was found. This control ensured that the T cell supernatant did not contain trace amounts of the mitogens PHA and PMA. Furthermore, the non-IgD-binding recombinant fragment MID1000-1200, which in addition to MID962-1200 was also produced in E. coli, did not activate B cells in the presence of the T cell cytokine-rich supernatant, excluding that trace amounts of E. coli lipopolysaccharide (LPS) induced the B lymphocytes (Fig. 8A) .


Figure 8
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  		Figure 8. Purified B cells are activated in the presence of MID962-1200 and T cell cytokines. B cells were incubated with MID962-1200 at 0.5 or 1 µg/ml, with or without a supernatant obtained from stimulated T lymphocytes (A). The cytokine-rich supernatant was manufactured by stimulating purified T lymphocytes with PHA and PMA, followed by extensive dialysis. MID1000-1200 was included as a negative control. MID962-1200, at 0.5 µg/ml, induced a strong B cell proliferation when incubated with the T helper cell type 2 (Th2) cytokine IL-4 (10 ng/ml), whereas a lower activation was observed in the presence of IL-2 (10 units/ml); IL-10 was used at 50 ng/ml (B). Human B lymphocytes were purified by positive selection and incubated with the recombinant proteins for 96 h as indicated. Proliferation was analyzed by pulsing cells with [3H]-thymidine overnight. Three different donors were analyzed at three separate occasions. The highest value in each experiment was defined as 100 arbitrary units. Data are presented as means, and error bars show SD.

 
Experiments with the T cell supernatant revealed that cytokines were crucial for optimal B cell activation in the presence of MID962-1200. To further examine the specific need for T cell cytokine costimulatory activation, the cytokines IL-2, IL-4, and IL-10 were incubated separately with MID962-1200. It is interesting that when IL-4 or IL-2 was supplemented together with MID962-1200, an increased proliferation was observed as compared with cells incubated with the truncated MID962-1200 fragment only (Fig. 8B) . MID962-1200 at 0.5 µg/ml, together with IL-4, increased the proliferation up to ninefold, whereas MID962-1200, together with IL-2, resulted in a 4.5-fold increase. Addition of IL-10 slightly inhibited the proliferative response, and when IL-10 was added separately together with MID962-1200, no significant proliferation could be detected (Fig. 8B) . Furthermore, B cells did not proliferate when incubated with cytokines in the absence of MID962-1200. The data shown represent an experiment with positively selected B cells, but similar results were obtained with untouched (negatively selected) B cells. Taken together, MID962-1200-dependent B cell activation required the T cell cytokines IL-2 or IL-4 but no direct T cell contact.

MID962-1200 induces IL-6 production
We recently showed that full-length MID stimulates B cell to produce IL-6 [13 ]. To determine the cytokine production of B cells after stimulation with MID962-1200, B cells were isolated using negative selection with an additional anti-CD3 mAb purification step, reducing the T cell population to <0.1%. The final preparation contained >96% pure B cells. A human cytokine array containing antibodies directed against 60 different growth factors, chemokines, and ILs, including IL-10, IL-6, tumor necrosis factor {alpha}, transforming growth factor-ß, and IL-1, was used. A strongly increased IL-6 production was detected when B cells were activated with MID962-1200 as compared with unstimulated cells (Fig. 9 ). A slightly up-regulated IL-1ß synthesis was also observed with stimulated B cells. Finally, a low level of MCP-2 was found irrespective of activation. Taken together, amongst all cytokines and chemokines tested, IL-6 was the main IL produced by purified B cells incubated with MID962-1200.


Figure 9
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  		Figure 9. MID962-1200-induced B cells mainly produce IL-6. A minor up-regulation of IL-1ß was also seen. Purified B cells were incubated with (A) or without MID962-1200 (B) for 96 h. Thereafter, the cytokine contents in the B cell culture supernatants were determined using a human cytokine protein array. The protein microarray cut-off controls are indicated as positive controls. Complete medium was included as a negative control (data not shown). MCP-2, Macrophage chemoattractant protein-2.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The respiratory pathogen M. catarrhalis is a strong IgD-binding bacterium [14 , 17 ]. In the present paper, we show by using MID-deficient Moraxella mutants that MID was the only M. catarrhalis outer membrane protein interacting with IgD (Fig. 1) . Furthermore, the truncated protein MID962-1200 activated human B cells in the range 0.1–5 µg/ml when T cells (Fig. 6) or T cell cytokines (Fig. 8) were supplied. It is important that the non-IgD-binding fragment MID1000-1200, which in addition to MID962-1200 was produced in E. coli, did not induce any B cell proliferation. MID1000-1200 also failed to attract soluble IgD when analyzed in ELISA (Fig. 3) . MID962-1200 was shown to recognize membrane-anchored IgD on the surface of transfected CHO cells (Fig. 5) . Finally, MID962-1200 fused to GFP was found to bind and stimulate B cells in the same order as MID962-1200.

In addition to the IgD-binding M. catarrhalis, only a few examples of nonimmune Ig binding to Gram-negative bacteria are known. Binding of bovine IgM to Brucella abortus, equine IgG to Taylorella equigenitalis, and bovine IgG and IgM to Haemophilus somnus has been reported [23 24 25 ]. An Ig-Fc receptor has also been purified from H. somnus [26 ]. Furthermore, the respiratory pathogen H. influenzae strongly attracts human IgD, albeit, the particular Ig-binding protein has not yet been found [27 ].

In contrast to Gram-negative species, several examples exist of Gram-positive bacteria, which attract Igs in a nonimmune manner. Staphylococcus aureus protein A (SpA) is the most characterized protein and binds to the Fc part of IgG [28 , 29 ]. SpA also binds a fraction of Ig molecules of all classes as a result of the so-called "alternative" binding to VH3 [30 ]. In addition to SpA, a S. aureus gene encoding another Ig-binding protein has been found [31 ]. Protein G, isolated from groups C and G streptococci of human origin, has a distinct affinity for the same site on the human Fc fragment of IgG as SpA but also interacts with IgG Fab fragments [32 , 33 ]. Protein H, isolated from Streptococcus pyogenes, is able to compete for the same region of IgG Fc with SpA and protein G [34 ]. In addition, S. pyogenes produces an antigenically and functionally heterogenous group of M-like proteins with different Ig-binding specificities [35 ]. Protein Bac or the B antigen is an IgA-binding protein expressed by certain strains of group B streptococci [36 ]. Finally, protein L, a surface component of Peptostreptococcus magnus, has affinity for all classes of Ig through an interaction with determinants present in the variable region of KLC [37 ].

The outer-membrane glycolipids of M. catarrhalis lack the repeating O-antigen polysaccharides of LPS and are hence designated lipo-oligosaccharides (LOS) [38 ]. These occur commonly in nonenteric Gram-negative bacteria, such as those that colonize the mucosal surfaces of the upper respiratory tract [39 ]. The LOS of M. catarrhalis consists of lipid A plus a core polysaccharide and one oligosaccharide [40 ]. The lipid A component is similar to that of other Gram-negative bacteria [41 ] and cross-reacts with lipid A of the Enterobacteriaceae, but it lacks the 3-hydroxytetradecanoic acid, which is normally present in enteric bacteria [40 ]. We show that lymphocyte activation in the presence of MID-deficient M. catarrhalis mutants was decreased significantly compared with when cells were incubated with wild-type bacteria expressing high levels of MID (Fig. 2) . These experiments did not only demonstrate the importance of MID as the major B cell stimulus of moraxellas but also that M. catarrhalis LOS plays a minor role in human B lymphocyte activation.

When B cells are activated through IgD, an intracellular signal is evoked, resulting in cell division and an improved humoral response. MID962-1200 strongly activates human B cells in the presence and absence of T cells. This is in parallel to full-length MID1-2139 or MID1-2139 conjugated to Sepharose, which stimulates human peripheral B cells but not T cells [13 ]. IgM secretion was detected in B cell cultures stimulated with MID and IL-2, whereas secretion of IgG and IgA was induced in cultures with the combination of MID and IL-4, IL-10, and soluble CD40L. These findings suggest that Th2-derived cytokines are required for MID-dependent class-switch [13 ].

MID and other antigens, which can induce antibody responses without obvious ("physical") T cell help, are classified as T cell-independent (TI) antigens [13 , 42 ]. This phenomenon was initially evaluated by the ability of antigens to elicit antibody responses in T cell-deficient nude mice. In general, TI antigens have repeating determinants, which can be recognized by antibody receptors on B lymphocytes. TI antigens have been subdivided into two types based on their differential ability to induce antibody responses in neonates and in CBA/N mice, the latter having an X-linked immune deficiency [43 ]. Dextran-conjugated anti-IgD antibodies can at low concentrations induce a B cell signal and have as a representative TI-2 antigen been used in several experimental models [42 , 44 ]. In contrast to other TI-2 antigens, the dextran-conjugated anti-IgD antibodies do not result in antigen-specific antibodies but induce a polyclonal activation [45 ]. Activation by MID most likely also results in a pAb production, and by analogy, with dextran-conjugated anti-IgD antibodies, MID, as an activator through the IgD BCR, may also be regarded as a TI-2 antigen.

In spite of the fact that MID962-1200 activated the B cells without the presence of physical T cell help, addition of cytokines was required to achieve similar B cell proliferation as compared with cell cultures with PBL. The increased proliferation in cultures with PBL can be explained by costimulatory activation via the CD40-CD40L and cytokines. Indeed, blocking of the CD40L prior to stimulation with MID962-1200 resulted in a decreased proliferation (Fig. 6D) . It has also been shown that recombinant CD40L strongly enhances MID-dependent B cell proliferation [13 ]. As MID962-1200 was covalently attached to Sepharose beads or in some experiments, bound to the microtiter plate plastic surface, MID962-1200 was most likely not internalized, processed, or presented to T cells on major histocompatibility complex class II. The T cells might, however, be activated through the release of B cell cytokines, e.g., IL-6 (Fig. 9) . Finally, it cannot be excluded that monocytes in the PBL preparations were also activated by lymphocyte cytokines.

An interesting fact is that a high number of IgD-producing plasma cells have been observed in the lymphoid tissue from nasopharyngeal tonsils and lacrimal, parotid, and lactating mammary glands as compared with spleen lymph nodes and glandular tissue of the gastrointestinal tract [46 , 47 ]. A substantial, local IgD synthesis is found in nasopharynx and in the middle ear cavity [48 , 49 ]. In ~20% of middle ear effusions examined, a content of more than 600 mg/l IgD can be calculated. This finding indicates that IgD may have a crucial role in the humoral immunity of secretions from the upper respiratory tract. M. catarrhalis frequently colonizes the respiratory tract and also is an important pathogen in, for example, acute otitis media. It is an enigma why M. catarrhalis binds IgD, especially in light of a much higher bacterial turnover rate compared with the human host and thus, would mutate or turn off a nonbeneficial IgD-binding protein within a short period of time. Is it of value for the bacterium to bind IgD, and/or does the nonimmune IgD binding play a role in the innate (humoral) immune response? A possibility would be that M. catarrhalis, by inducing the BCR signaling pathway, entices the B cells to polyclonal Ig synthesis, which perhaps, would be potentially harmless for the bacterium itself and thus exclude production of specific monoclonal antimoraxella antibodies. It is tempting to speculate that a bacteria-IgD interaction on lymphocytes and in secretions of the mucous membranes may play an important role in the pathogenesis and host defense in upper respiratory tract infections. Thus, further studies about bacterial IgD-binding proteins are required to fully explain the M. catarrhalis/host interactions, and MID962-1200 will most likely be a basis of further studies.


   ACKNOWLEDGEMENTS
 
This work was supported by grants from the Alfred Österlund Foundation, the Anna and Edwin Berger Foundation, the Greta and Johan Kock Foundation, the Swedish Medical Research Council, the Swedish Society of Medicine, and the Cancer Foundation at the University Hospital in Malmö.

Received February 2, 2005; revised October 4, 2005; accepted October 5, 2005.


   REFERENCES
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 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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