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The Journal of Immunology, 2003, 170: 1854-1861.
Copyright © 2003 by The American Association of Immunologists

Immunoglobulin Superantigen Protein L Induces IL-4 and IL-13 Secretion from Human Fc{varepsilon}RI+ Cells Through Interaction with the {kappa} Light Chains of IgE 1

Arturo Genovese*, Guglielmo Borgia{dagger}, Lars Björck{ddagger}, Angelica Petraroli*, Amato de Paulis*, Marcello Piazza{dagger} and Gianni Marone2,*

Divisions of * Clinical Immunology and Allergy and {dagger} Infectious Disease, School of Medicine, University of Naples Federico II, Naples, Italy; and {ddagger} Department of Cell and Molecular Biology, University of Lund, Lund, Sweden


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Peptostreptococcus magnus protein L is a multidomain bacterial surface protein that correlates with virulence. It consists of up to five homologous Ig-binding domains (B1–B5) that interact with the variable domain of Ig {kappa} L chains. Intact protein L stimulates the synthesis and the release of IL-4 and IL-13 from human basophils in vitro. A protein L fragment covering the Ig-binding domains B1–B4 also induced IL-4 and IL-13 release from basophils. There was an excellent correlation (rs = 0.82; p < 0.001) between the maximal percent IL-4 release induced by protein L and that induced by anti-IgE and between intact protein L and the B1–B4 fragment (rs = 0.90; p < 0.01). Removal of IgE bound to basophils markedly reduced the IL-4 release induced by anti-IgE, protein L, and B1–B4. Preincubation of basophils with protein L or anti-IgE caused complete cross-desensitization to subsequent challenge with the heterologous stimulus. IgE purified from myeloma patients PS and PP ({lambda} chains) blocked anti-IgE-induced IL-4 release, but not the releasing activity of protein L. In contrast, IgE purified from myeloma patient ADZ ({kappa} chains) blocked both anti-IgE- and protein L-induced secretion. Cyclosporin A, but not cyclosporin H, inhibited protein L-induced release of IL-4 and IL-13 from basophils. Thus, protein L acts as a bacterial Ig superantigen to induce the synthesis and release of IL-4 and IL-13 from basophils by interacting with {kappa} L chains of the IgE isotype.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Human basophils and mast cells are the only cells expressing the tetrameric high affinity receptor Fc{varepsilon}RI and synthesizing histamine (1). Fc{varepsilon}RI+ cells exert a central role in the pathophysiology of allergic disorders through the elaboration and release of a myriad of proinflammatory and immunoregulatory molecules (2, 3) and the expression of a wide spectrum of surface receptors for cytokines (4, 5, 6, 7, 8, 9) and chemokines (10, 11, 12, 13).

Mast cells, widely distributed in all vascularized tissue (1, 2, 3), and circulating basophils ensure early contact with bacterial and viral pathogens. The strategic location of Fc{varepsilon}RI+ cells at the host-environment interface and their ability to release proinflammatory mediators, cytokines, and chemokines suggest that these cells are implicated in bacterial (14, 15, 16, 17, 18, 19, 20) and viral infections (21, 22, 23, 24, 25). Results obtained with in vivo experimental models demonstrate that mast cells are central to the host’s immune response to various bacterial agents (15, 16, 17). Moreover, there is in vitro evidence that several bacterial products can activate human Fc{varepsilon}RI+ cells to release preformed and de novo synthesized mediators (14, 26). This observation is generating great interest because of its therapeutic implications. At least three mechanisms of activation of Fc{varepsilon}RI+ cells have been identified: 1) the natural pentapeptide pepstatin A and the formyl-containing tripeptide (FMLP) bind with high affinity to N-formyl peptide receptor (FPR),3 a seven transmembrane receptor, present on human basophils but not on mast cells (26, 27); 2) several bacterial proteins bind with high affinity to different domains of Fc{varepsilon}RI-bound IgE, thereby acting as Ig superantigens (28, 29, 30); and 3) Toll-like receptors, a family of pattern recognition receptors crucial for cellular responses to a variety of microbial agents (19), have been identified on mouse bone marrow-derived mast cells (19, 20) and on human umbilical cord blood-derived mast cells (31).

Taken together, these studies demonstrate that several bacterial peptides and proteins can be chemotactic (32) and/or induce the release of proinflammatory mediators from human Fc{varepsilon}RI+ (26, 28, 29, 30). However, thus far none of these proteins has been shown to induce the synthesis and the release of immunoregulatory cytokines from human basophils or mast cells.

Diverse Ig-binding bacterial cell wall proteins bind specifically to different regions of human Ig. Protein A of Staphylococcus aureus binds to the Fc{gamma} region of human IgG subclasses 1, 2, and 4 (33) and also interacts with the VH3 domain of human Ig (34, 35). Protein L is an Ig L chain-binding cell-wall protein isolated from the anaerobic bacterium Peptostreptococcus magnus (36). P. magnus is part of the indigenous flora of the skin, the oral cavity, and the gastrointestinal and genitourinary tracts. These two bacteria are also the causative agents of a variety of infections (37, 38).

Protein L consists of up to five repeated Ig-binding domains (B1–B5) (39) and appears to be a virulence determinant (38, 40). Each homologous domain binds with high affinity to {kappa} L chains (39) without disrupting the function of the Ag-binding site (41, 42, 43). Specifically, protein L binds to the variable domain of the VKI, VKIII, and VKIV subgroups but does not bind to Ig H chains, {lambda} L chains, the CL domains of {kappa} L chains, or the variable domain of the VKII subgroup (44). Thus, protein L binds human Ig regardless of the H chain class, is mitogenic for B cells (45), and is an Ig superantigen (46).

We have previously demonstrated that protein L induces the release of proinflammatory mediators from human basophils and mast cells, presumably by interacting with the IgE bound to Fc{varepsilon}RI (29, 30). Several groups of investigators have demonstrated that immunologically challenged human basophils synthesize and release two important cytokines, IL-4 (21, 47, 48, 49) and IL-13 (50, 51, 52). This study has been undertaken to evaluate whether protein L and a fragment of protein L denoted B1–B4, which comprises four of the five Ig-binding protein L repeats (39), induce cytokine (IL-4 and IL-13) synthesis and secretion from human basophils.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents

The following were purchased: 60% HClO4 (Baker Chemical, Deventer, The Netherlands); FMLP (Calbiochem-Behring, La Jolla, CA); Eagle’s MEM and gentamicin (Flow Laboratories, Irvine, Scotland); FCS, HBSS, Trizol, and murine Moloney leukemia virus transcriptase (Life Technologies, Grand Island, NY); RPMI 1640 containing 25 mM HEPES buffer (BioWhittaker, Walkersville, MD); dextran 70, Percoll, Sephadex G-200, and Sepharose CL-4B-CMBr (Pharmacia Fine Chemicals, Uppsala, Sweden); DEAE 23-cellulose SS SERVACEL (Serva Finbiochemica, Heidelberg, Germany); and human serum albumin (HSA), L-glutamine, and PIPES (Sigma-Aldrich, St. Louis, MO). Anti-IgE, produced by rabbit immunization with the Fc fragment of a human IgE myeloma (patient PS) and then adsorbed with the IgE Fab as previously described (49), was generously provided by K. and T. Ishizaka (La Jolla Institute for Allergy and Immunology, La Jolla, CA). Cyclosporin A (CSA) and cyclosporin H (CSH) were obtained from D. Romer and E. Rissi (Novartis, Basel, Switzerland).

Protein L

Purified intact protein L from strain 312 of P. magnus or a recombinantly expressed fragment covering the Ig-binding domains B1–B4 were obtained as previously described (36, 39).

Buffers

The PIPES buffer used in these experiments consisted of 25 mM PIPES (pH 7.4), 110 mM NaCl, and 5 mM KCl, the mixture referred to as P. PCG contains, in addition to P, 5 mM CaCl2, and 1 g/L D-glucose (11). PACGM contains, in addition to P, HSA 3%, 1 mM CaCl2, 1 g/L dextrose, and 0.25 g/L MgCl2 · 6H2O (pH 7.4). PGMD contains 0.25 g/L MgCl2 · 6H2O, 10 mg/L DNase, and 1 g/L gelatin in addition to P (pH 7.4). PBS contains 8 g/L NaCl, 1.15 g/L Na2HPO4, 200 mg/L KCl, and 200 mg/L KH2PO4 (pH 7.4). Experiments for cytokine release were performed in IMDM with L-glutamine and 25 mM HEPES (Invitrogen, Paisley, U.K.).

Purification of human monoclonal IgE

IgE myeloma proteins were purified from the sera of three myeloma patients (PS, ADZ, and PP) by repeated gel filtration on Sephadex G-200 followed by elution through a Sepharose CL-4B column. Analysis by SDS-PAGE of purified human monoclonal IgE proteins demonstrated a single protein with a molecular mass of 180–200 kDa. Immunoelectrophoresis of PP and PS sera with anti-{lambda} chain antiserum showed a bowing of the anodal extremity of the arc, corresponding with that of IgE. Immunoelectrophoresis of ADZ serum showed a bowing of the anodal extremity of the arc, corresponding with that of IgE, with anti-{kappa} chain antiserum (53). Analysis by radioimmunoassay showed no IgG, IgM, or IgA contamination (28, 30).

Purification of human polyclonal IgG

Human IgG was prepared by precipitation of normal human serum with 50% satured ammonium sulfate followed by chromatography on a DEAE-cellulose column equilibrated with 0.01 M PBS (pH 7.4) (28).

Purification of peripheral blood basophils

Basophils were purified from peripheral blood cells of healthy volunteers, aged 18–46 years (mean age, 38.2 ± 6.7 years), undergoing hemapheresis. Buffy coat cell packs provided by the Immunohematology Service at the University of Naples Federico II were reconstituted in PBS containing 0.5 g/L HSA and 3.42 g/L sodium citrate and were loaded onto a countercurrent elutriator (model J2-21; Beckman Coulter, Fullerton, CA). Several fractions were collected, and fractions containing basophils in large numbers (>20 x 106 basophils) and of improved purity (>15%) were enriched by discontinuous Percoll gradients (21). Basophils were further purified to near homogeneity (>98%) by depleting B cells, monocytes, NK, dendritic cells, erythrocytes, platelets, neutrophils, eosinophils, and T cells, using a cocktail of hapten-conjugated CD3, CD7, CD14, CD15, CD16, CD36, CD45RA, and anti-HLA-DR Abs and MACS MicroBeads coupled to an anti-hapten mAb (54). The magnetically labeled cells were depleted by retaining them on a MACS column in the magnetic field of the MidiMACS (Miltenyi Biotec, Bergisch Gladbach, Germany). Yields ranged from 3 to 10 x 106 basophils with purity usually 98%, assessed by basophil staining with Alcian Blue and counting in a Spiers-Levy eosinophil counter (54).

Histamine and cytokine release assay

Basophils (~6 x 104 basophils/tube) resuspended in PCG (histamine release) or IMDM (cytokine release) and 0.1 ml of the cell suspension were placed in 12 x 75-mm polyentylene tubes and warmed to 37°C; 0.1 ml of each preformed stimulus for release was added, and incubation was continued at 37°C for 45 min (histamine release), 4 h (IL-4 secretion), or 18 h (IL-13 secretion) (21, 49). At the end of this step, the reaction was stopped by centrifugation (1000 x g, 22°C, 2 min), and the cell-free supernatants were stored at -20°C for subsequent assay of histamine and cytokine content. The cell-free supernatants were assayed for histamine with an automated fluorometric technique (55). Total histamine content was assessed by lysis induced by incubation of cells with 2% perchloric acid before centrifugation. To calculate histamine release as a percentage of total cellular histamine, the spontaneous release of histamine from basophils (0–8% of the total cellular histamine) was substracted from both the numerator and denominator (49). All values are based on the means of duplicate or triplicate determinations. Replicates differed from each other in histamine content by <10%.

IL-4, IL-13, and IFN-{gamma} ELISA

The harvested supernatants were assayed for IL-4, IL-13, or IFN-{gamma} with the IL-4, IL-13, or IFN-{gamma} Quantikine high sensitivity kit (R&D Systems, Minneapolis, MN). The standard curve for these kits was run in the medium used for the release experiments (21, 49).

Isolation of cellular mRNA and RT-PCR for IL-4 and IL-13

In the RT-PCR experiments, 1.0 x 106 of purified basophils were suspended in IMDM supplemented with 5% FCS and 1% antibiotic-antimycotic solution. Cells were incubated with medium alone or with protein L (30–3 x 103 ng/ml) for 2 or 4 h to evaluate the mRNA expression of IL-4 and IL-13, respectively. At the end of incubation, RNA was isolated by the TRIzol technique (Invitrogen) according to the manufacturer’s instructions. Diethylpyrocarbonate-treated water without SDS was used for the final resuspension step; RNA was stored at -80°C. Reverse transcription was performed with 5 mM MgCl2, oligo(t)16 primer, and murine leukemia virus reverse transcriptase according to the manufacturer’s instructions (Applied Biosystems, Foster City, CA). PCR was performed with AmpliTaqGold DNA polymerase (1–2.5 U/reaction) at the annealing temperature of 60°C with target-specific primers for IL-4 (5'-ATGGGTCTCACCTCCCAACTGCT-3' and 3'-GTTTTCCAACGTACTCTGGTTGGC-5' at 0.2–1 µm/primer) and IL-13 (5'-GGAAGCTTCTCCTCAATCCTCTCCTGTT-3' and 3'-GCGGATCCGTTGAACCGTCCCTCGCGAAA-5' at 0.2–1 µm/primer). Numbers of cycles were 40 for IL-4 and 35 for IL-13. Normalization of RNA was achieved by RT-PCR for the constitutive marker gene {beta}-actin (30 cycles). All PCR products, together with a DNA ladder as a size standard, were separated on 2.5% agarose gel, stained with ethidium bromide, and photographed. Semiquantitative measurements of relative cytokine mRNA expression were obtained by digital scanning and densitometric analysis (Scion Image, Frederick, MD) (56).

Statistical analysis

The results are expressed as the mean ± SEM. Differences were considered significant when p < 0.05 (57).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Protein L induces release of IL-4 from human basophils

Initial experiments were performed to determine whether protein L induced the release of IL-4 from peripheral blood basophils purified (>98%) from healthy individuals. The cells were incubated (37°C, 4 h) with various concentrations of protein L and anti-IgE. Fig. 1 shows the results of five experiments in which we examined the effects of protein L (30–3 x 103 ng/ml) and anti-IgE (30–3 x 103 ng/ml) on the levels of extracellular IL-4 and IFN-{gamma}. These experiments demonstrated that protein L and anti-IgE stimulated the release of IL-4 from basophils. In contrast, IFN-{gamma} was not detected in any of the basophil preparations stimulated with protein L or anti-IgE. In 16 experiments, basophils (enriched to 95–98% purity) were challenged with different protein L concentrations (102–3 x 103 ng/ml). Protein L concentration dependently induced both IL-4 and histamine release from basophils and, as shown in Fig. 2, there was a correlation between histamine release and IL-4 secretion (rs = 0.82; p < 0.01).



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  		FIGURE 1. Effects of various concentrations of protein L and anti-IgE on IL-4 and IFN-{gamma}. Secretion from human basophils obtained from normal donors. Purified basophils (>98%) were incubated with protein L or anti-IgE for 4 h at 37°C. Each point represents the mean ± SEM obtained from five experiments. Error bars are not shown when graphically too small.

 


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  		FIGURE 2. Correlation between the maximum percentage of histamine secretion and IL-4 release caused by protein L from human basophils (n = 16) obtained from normal donors. Each point represents the mean of duplicate determinations from separate experiments.

 
Kinetics of protein L-induced IL-4 and IL-13 release and histamine secretion from human basophils

Fig. 3 compares the kinetics of histamine, IL-4, and IL-13 release from basophils challenged with an optimal concentration of protein L (103 ng/ml). Histamine release peaked at 30 min, IL-4 release at 3–4 h, and IL-13 release at 18–24 h. These data demonstrate that protein L induces the secretion of IL-4 and IL-13, which are important cytokines for the polarization of Th2 cells (58, 59, 60, 61), from basophils.



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  		FIGURE 3. Kinetics of IL-4, IL-13, and histamine release from human basophils induced by protein L (103 ng/ml). Each point represents the mean of duplicate determinations. Similar results were obtained in three additional experiments.

 
Effect of protein L on IL-4 and IL-13 mRNA

The latency of the effects of protein L on IL-4 and IL-13 release from basophils suggested that this effect may be mediated by induction of IL-4 and IL-13 gene expression. To test this hypothesis, we measured IL-4 mRNA in basophils exposed to protein L (30 and 3 x 102 ng/ml) in three separate experiments. Fig. 4 depicts the {beta}-actin- and IL-4-specific RT-PCR amplification products from a representative experiment in which basophils were cultured for 2 h with medium alone or with protein L (30 and 3 x 102 ng/ml). Adequate normalization of RNA for each sample was confirmed by equal RT-PCR amplification products for the constitutive marker gene of {beta}-actin. Exposure to two concentrations of protein L increased IL-4 mRNA vs unstimulated cells. These findings suggest that protein L induces IL-4 production through activation of IL-4 gene expression.



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  		FIGURE 4. RT-PCR analysis of expression of IL-4 mRNA in human basophils stimulated by protein L. Total cellular RNA was extracted from purified basophils (>98%) after incubation with protein L (30 and 3 x 102 ng/ml) at 37°C for 2 h. mRNA for IL-4 was amplified by RT-PCR, subjected to electrophoresis, and visualized by ethidium bromide. Lower panel, Densitometric analysis of IL-4 RT-PCR product expressed as the ratio of signal intensity of the bands normalized for that of {beta}-actin.

 
In another set of three experiments, we evaluated the effect of protein L on IL-13 mRNA in basophils. In these experiments, basophils were cultured with medium or protein L for 4 h. The incubation time was longer for this group of experiments because of the delayed kinetic of IL-13 synthesis (see Fig. 3). Fig. 5 shows a representative result of three experiments in which two concentrations of protein L (30 and 3 x 102 ng/ml) increased IL-13 mRNA compared with unstimulated cells.



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  		FIGURE 5. RT-PCR analysis of expression of IL-13 mRNA in human basophils stimulated with protein L. Total cellular RNA was extracted from purified basophils (>98%) after incubation with protein L (30 and 3 x 102 ng/ml) at 37°C for 4 h. IL-13 mRNA was amplified by RT-PCR, subjected to electrophoresis, and visualized by ethidium bromide. Lower panel, Densitometric analysis of IL-4 RT-PCR product expressed as the ratio of signal intensity of the bands normalized for that of {beta}-actin.

 
Correlations between the maximum protein L-, B1–B4-, and anti-IgE-induced IL-4 release from human basophils

In 13 experiments, basophils of a purity of 76–96% were challenged with different concentrations of protein L (102-3 x 103 ng/ml), B1–B4 (102-3 x 103 ng/ml), and anti-IgE (102-3 x 103 ng/ml). A significant correlation was found between the maximum IL-4 release caused by anti-IgE and that induced by protein L (rs = 0.89; p < 0.01) and between IL-4 release induced by B1–B4 and protein L (rs = 0.90; p < 0.01). These results support the hypothesis that both intact protein L and the B1–B4 fragment induce IL-4 release from basophils by interacting with IgE.

Effect of IgE stripping on protein L- and B1–B4-induced IL-4 release from human basophils

Brief exposure to a low pH removes most of the IgE bound to basophils, thus markedly reducing the activating properties of IgE-mediated stimuli (21, 36). Fig. 6 illustrates the results of three experiments showing that brief exposure to lactic acid completely blocks the effects of protein L and of anti-IgE on IL-4 release from basophils. In contrast, the response to mAb cross-linking the {alpha}-chain of Fc{varepsilon}RI (anti-Fc{varepsilon}RI{alpha}) (30, 62) was not affected by this treatment. Similarly, brief exposure to lactic acid markedly reduced IL-4 release induced by B1–B4 (data not shown).



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  		FIGURE 6. Effect of lactic acid on IL-4 release from normal human basophils induced by protein L, anti-IgE, and anti-Fc{varepsilon}RI. Purified basophils were treated with buffer or lactic acid (0.01 M (pH 3.9), 5 min, 22°C) and were washed twice (4°C). Cells were then resuspended in IMDM buffer and challenged (4 h at 37°C) with protein L (103 ng/ml), anti-IgE (103 ng/ml), or anti-Fc{varepsilon}RI{alpha} (103 ng/ml). Each bar represents the mean ± SEM of IL-4 release obtained from three experiments. *, p < 0.01 when compared with the corresponding group preincubated with buffer.

 
Cross-desensitization between protein L and anti-IgE

We further examined the relationship between anti-IgE and protein L using cross-desensitization between these two stimuli. Basophils were preincubated with anti-IgE (3 x 103 ng/ml) or protein L (3 x 103 ng/ml) in PIPES buffer containing 4 mM EDTA for 30 min at 37°C. At the end of incubation, cells were washed (two times) and resuspended in IMDM buffer. The results obtained in one of three experiments are illustrated in Fig. 7. Cells preincubated with PIPES buffer and then challenged with anti-IgE (103 ng/ml), protein L (103 ng/ml), or anti-Fc{varepsilon}RI{alpha} (103 ng/ml) released IL-4. In contrast, the release of IL-4 induced by protein L or anti-IgE was markedly reduced when challenged with either anti-IgE or protein L. Basophils desensitized with protein L or anti-IgE were still responsive to anti-Fc{varepsilon}RI{alpha}, which activates a specific epitope on the {alpha}-chain of Fc{varepsilon}RI at the basophil membrane (62). Thus, it appears that the releasing property of protein L is mediated mainly by interaction with IgE present at the basophil surface.



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  		FIGURE 7. Effect of desensitization to one stimulus in response to a second stimulus. Human basophils were desensitized to protein L or to anti-IgE by incubation with the stimuli in Ca2+-free PIPES buffer containing 4 mM EDTA for 30 min at 37°C. Cells were washed twice (4°C), resuspended in IMDM buffer, and challenged (4 h at 37°C) with protein L (103 ng/ml), anti-IgE (103 ng/ml), or anti-Fc{varepsilon}RI{alpha} (103 ng/ml). Each bar represents the mean of duplicate determinations of a typical experiment. Similar results were obtained in two additional experiments.

 
Interactions between protein L or anti-IgE with different IgE myeloma proteins

We used a different approach to establish that the histamine releasing activity of protein L was directed against antigenic determinants on human IgE. The binding and physicochemical properties of protein L have been characterized (36, 39, 40, 41, 42). Protein L binds specifically to the L chains of Ig, and the affinity constant for IgG, IgA, and IgM is ~1010 M-1 (41). In addition, the multiple binding sites for {kappa} L chains (39, 41) make protein L similar to divalent anti-IgE Abs (21). As mentioned above, protein L does not show affinity for {lambda} L chains (41).

To evaluate the mechanism of activation of human basophils by protein L, protein L or anti-IgE were preincubated with three IgE myelomas (obtained from patients PS, ADZ, and PP, respectively) or with human polyclonal IgG. IgE myelomas from patients PS and PP display {lambda} L chains, whereas IgE myeloma from patient ADZ possesses {kappa} chains (29, 30). Human polyclonal IgG displays ~60% {kappa} chains. In a first set of three experiments, one of which is illustrated in Fig. 8, we found that IgE purified from patients ADZ, PS, and PP (0.1–1 µg/ml) concentration dependently inhibited the releasing activity of anti-IgE. Human polyclonal IgG (1–3 µg/ml) did not modify the IL-4 releasing activity of anti-IgE. In contrast, IgE purified from patients PS and PP ({lambda} chains) did not modify the releasing activity of protein L, whereas IgE from patient ADZ ({kappa} chains) and human polyclonal IgG blocked the IL-4 releasing activity of protein L (Fig. 9).



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  		FIGURE 8. Effect of preincubation of anti-IgE with human monoclonal IgEs (ADZ, PS, or PP) and human polyclonal IgG on IL-4 release from basophils. Anti-IgE (3 x 102 ng/ml) was preincubated for 15 min at 37°C with human monoclonal IgE ADZ (0.1–1 µg/ml), PS (0.1–1 µg/ml), PP (0.1–1 µg/ml), or IgG (1–3 µg/ml). Basophils were then added, and incubation continued for another 4 h at 37°C. Each bar shows the mean of duplicate determinations. Similar results were obtained in two additional experiments.

 


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  		FIGURE 9. Effects of preincubation of protein L with human monoclonal IgEs (ADZ, PS, or PP) and human polyclonal IgG on IL-4 release from basophils. Protein L (3 x 102 ng/ml) was preincubated for 15 min at 37°C with human monoclonal IgE ADZ (0.1–1 µg/ml), PS (0.1–1 µg/ml), PP (0.1–1 µg/ml), or polyclonal IgG (1–3 µg/ml). Basophils were then added, and incubation continued for another 4 h at 37°C. Each bar shows the mean ± SEM of IL-4 release obtained from three experiments. *, p < 0.01 when compared with the group not preincubated with human Ig.

 
In four additional experiments, we found that the IL-4 releasing activity of a wide spectrum of concentrations of protein L (30–3 x 103 ng/ml) was unaffected by preincubation with IgE from patients PS and PP. In contrast, the inhibitory effect of IgE from patient ADZ ({kappa}) and of human polyclonal IgG decreased with increasing concentrations of protein L. The results of these experiments, one of which is illustrated in Fig. 10, indicate that human monoclonal IgE ADZ ({kappa}) and polyclonal IgG competitively inhibit the interaction between protein L and IgE at the surface of basophils.



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  		FIGURE 10. Effect of preincubation of increasing concentrations of protein L (30–3 x 103 ng/ml) with human monoclonal IgE, which displays {lambda} (PS and PP) or {kappa}. L chains (ADZ), or with human polyclonal IgG on IL-4 release from basophils. Protein L was preincubated for 15 min at 37°C with human monoclonal IgEs (1 µg/ml) or polyclonal IgG (3 µg/ml). Basophils were then added, and incubation continued for another 4 h at 37°C. Each point shows the mean of duplicate determinations. Similar results were obtained in three additional experiments.

 
Effects of CsA and CsH on protein L-induced IL-4 and IL-13 secretion from human basophils

CsA is a potent inhibitor of the IgE-dependent release of proinflammatory mediators and cytokines from human basophils and mast cells (21, 27). In contrast, CsH, a stereoisomer of CsA, does not inhibit IgE-dependent release of mediators from human Fc{varepsilon}RI+ cells, but it is a specific antagonist of FPR receptors activated by the bacterial tripeptide FMLP (27, 63, 64). In these experiments, we compared the effects of preincubation of low concentrations (8–800 nM) of CsA and CsH on the release of IL-4 and IL-13 from purified basophils activated by protein L. CsA concentration dependently inhibited protein L-induced release of IL-4 from basophils at concentrations as low as 8 nM to 90–95% at 800 nM, whereas CsH did not affect the release of IL-4 (Fig. 11). CsA and CsH exerted a similar effect on the protein L-induced release of IL-13 from basophils (Fig. 12). These results again suggest that protein L induced basophil activation through the engagement of the IgE-Fc{varepsilon}RI network and not through FPR.



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  		FIGURE 11. Effects of various concentrations of CsA and CsH on IL-4 release from human basophils induced by protein L. Cells were preincubated for 15 min at 37°C with the indicated concentrations of CsA or CsH and then were challenged (4 h at 37°C) with protein L (3 x 102 ng/ml). Each bar represents the mean of duplicate determinations of a typical experiment. Similar results were obtained in two additional experiments.

 


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  		FIGURE 12. Effects of various concentrations of CsA and CsH on IL-13 release from human basophils induced by protein L. Cells were preincubated for 15 min at 37°C with the indicated concentrations of CsA or CsH and then were challenged (18 h at 37°C) with protein L (3 x 102 ng/ml). Each bar represents the mean of duplicate determinations of a typical experiment. Similar results were obtained in two additional experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
This study shows that protein L from P. magnus and the fragment B1–B4 of protein L are potent inducers of the synthesis and release of IL-4 and IL-13 from human Fc{varepsilon}RI+ cells. The activity of protein L and B1–B4 appears to be, mediated by interaction with the {kappa} L chains of IgE, present on human basophils. This is the first demonstration that a bacterial protein triggers the release of two cytokines critical for Th2 polarization from human Fc{varepsilon}RI+ cells, thereby acting as a bacterial Ig superantigen.

Several observations indicate that activation of human inflammatory cells possessing high affinity receptors for IgE (Fc{varepsilon}RI) is mediated by interaction of the bacterial product with membrane-bound IgE: 1) protein L binds with high affinity (~1010 M-1) to all isotypes of human Ig (41), 2) there is a highly significant correlation between the maximal IL-4 release induced by anti-IgE and that induced by protein L, 3) there is complete cross-desensitization between protein L and anti-IgE, and 4) basophils from which IgE had been dissociated by brief exposure to lactic acid did not release cytokines in response to protein L. These findings also suggest that the single protein L molecule interacts with more than one cell surface-associated Ig molecule. Therefore, protein L can function as a natural cross-linking agent, which reproduces the releasing activity of rabbit IgG anti-IgE on human Fc{varepsilon}RI+ cells (21, 49).

This study provides some insight into the mechanism of interaction between protein L and IgE bound to the basophil membrane. Two IgE myeloma proteins (PS and PP), which possess {lambda} chains, did not prevent the release of histamine induced by protein L from basophils, whereas they suppressed the release induced by anti-IgE. In contrast, IgE myeloma purified from patient ADZ ({kappa} chains) and human polyclonal IgG completely blocked the releasing activity of protein L. This suggests that IgE possessing {kappa} chains and polyclonal IgG, ~60% of which possess {kappa} chains, act as competitive antagonists of membrane-bound IgE at the level of protein L binding sites. Combined, these results show that protein L interacts with the {kappa} L chains of IgE molecules at the basophil surface to induce the release of IL-4 and IL-13.

The various proteins expressed at the bacterial cell wall differ greatly in their capacity to promote the release of proinflammatory mediators from human basophils and mast cells (26, 28, 29, 30). Protein L is a complete secretagogue, i.e., it releases proinflammatory mediators (histamine and 5(S)-hydroxy-6(R)-S-glutathionyl-7,9-trans-11,14-cis-eicosatetraenoic acid) (29, 30) and induces the de novo synthesis of immunoregulatory cytokines (IL-4 and IL-13) from basophils. Given the biologic importance of these cytokines (58, 59, 60, 61), this finding could have a biologic relevance as to the virulence of protein L-expressing peptostreptococci (38, 40). P. magnus is part of the indigenous flora of the skin, the gastrointestinal tract, and the genitourinary tract, but it is also sometimes a causative agent in various infections (37). Interestingly, in contrast to proteins A and G, the expression of protein L is correlated to bacterial virulence (38, 40). The possibility exists that the release of cytokines at sites of infections induced by protein L might contribute to the virulence of infections caused by protein L-expressing P. magnus strains. In vitro studies have demonstrated that certain bacterial products can induce cytokine release from rodent mast cells (31). In vivo studies have demonstrated that the release of TNF-{alpha} from mast cells is an important element in the role played by mast cells in innate immunity (15, 16, 17). Human basophils and mast cells respond to stimulation with different bacterial products by releasing both preformed (histamine and tryptase) and de novo synthesized eicosanoids (PGD2 and 5(S)-hydroxy-6(R)-S-glutathionyl-7,9-trans-11,14-cis-eicosatetraenoic acid) (26, 28, 29, 30). However, so far none of these bacterial proteins have been shown to induce the synthesis and release of cytokines from human Fc{varepsilon}RI+ cells. Thus, protein L appears to be the first bacterial protein found to induce in vitro synthesis and release of two important cytokines (IL-4 and IL-13) from human basophils.

Several bacterial products can activate human Fc{varepsilon}RI+ cells through specific mechanisms: a non-IgE-dependent and an IgE-dependent mechanism (65). The natural peptapeptide pepstatin A and FMLP activate a specific seven transmembrane receptor (FPR), independent of Fc{varepsilon}RI, on basophils to release proinflammatory mediators (26, 27). Basophil activation by FMLP is not inhibited by CsA, but it is specifically antagonized by CsH (27, 65). Different bacterial products can activate human Fc{varepsilon}RI+ cells through IgE-dependent mechanisms. For example, protein A activates human basophils by interacting with the F(ab')2 region of ~10% of human polyclonal IgE (28, 30). Protein L acts as a bacterial Ig superantigen by interacting with the {kappa} L chain of IgE on human basophils to induce the synthesis and release of IL-4 and IL-13. This is also supported by the fact that CsA inhibits protein L-induced cytokine release from basophils, whereas CsH does not.

In conclusion, our results demonstrate a novel mechanism by which the Ig superantigen protein L specifically activates human Fc{varepsilon}RI+ cells to release two important cytokines, which further supports the notion that protein L expression is correlated with peptostreptococcal virulence.


   Acknowledgments
 
We thank Giorgio Fratellanza for providing buffy coat cell packs and Aikaterini Detoraki for elaboration of the figures.


   Footnotes
 
1 This work was supported by grants from the Istituto Superiore Sanità (AIDS Projects 40B.64 and 40.B1); Ministero della Salute "Alzheimer Project"; Ministero dell’ Istruzione, dell’ Università e della Ricerca National Project "Helicobacter pylori infection: host-pathogen interactions"; Consiglio Nazionale delle Ricerche (Target Project Biotechnology Nos. 01.00191.PF31 and 01.00295.PF49; Rome, Italy); and the Swedish Research Council (Projects 7480 and 14379). G.M. is the recipient of the 2002 Esculapio Award (Accademia Tiberina, Rome, Italy). Back

2 Address correspondence and reprint requests to Dr. Gianni Marone, Division of Clinical Immunology and Allergy, University of Naples Federico II, Via S. Pansini 5, 80131 Napoli, Italy. E-mail address: marone{at}unina.it Back

3 Abbreviations used in this paper: FPR, N-formyl peptide receptor; HSA, human serum albumin; CsA, cyclosporin A; CsH, cyclosporin H. Back

Received for publication September 5, 2002. Accepted for publication December 11, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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