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Relative Neutralizing Activity in Polyspecific IgM, IgA, and IgG Preparations against Group A Streptococcal Superantigens Anna Norrby-Teglund,1 Nahla Ihendyane,1 Rita Kansal,3,4 Hesham Basma,3,4 Malak Kotb,3,4 Jan Andersson,1 and Lennart Hammarstro¨m2
Divisions of 1Infectious Diseases and 2Clinical Immunology, Karolinska Institute, Huddinge University Hospital, Huddinge, Sweden; 3Veterans Affairs Medical Center, Research Service, and 4Departments of Surgery, Microbiology, and Immunology, University of Tennessee, Memphis
In this study we compared the ability of different immunoglobulin (Ig) preparations containing IgG, IgM, and/or IgA to neutralize the activity of streptococcal pyrogenic exotoxin A (SpeA) or culture supernatant from a clinical group A streptococcal isolate. All Ig preparations markedly inhibited the mitogenic and cytokine-inducing activity of SpeA and culture supernatant at concentrations of 0.05–0.5 mg/mL, and at 0.5 mg/mL, most caused 95–100% inhibition of both stimuli. A significantly higher (P < .05 ) inhibition of SpeA was achieved by Pentaglobin (IgG, IgM, and IgA) and IgAbulin (IgA and IgG), as compared with pure IgG preparations. IgM- and IgA-enriched preparations had significantly higher inhibitory activity against SpeA than against culture supernatant, whereas the reverse was true for the IgG preparations (P < .05 ). The data show that IgM and IgA are potent inhibitors of specific streptococcal superantigens. These findings may have implications for the optimization of immunotherapy in invasive streptococcal infections.
Polyspecific human immunoglobulin given intravenously (IVIG) has been reported to be efficacious in a variety of human diseases, such as autoimmune diseases, immunodeficiencies, Kawasaki disease, and sepsis [1–3]. Furthermore, a clinical beneficiary effect of IVIG as adjunctive therapy in toxic shock was suggested by several case reports [4–9] and was further supported by a comparative observational cohort study [10] that underscored the benefit of this therapy in reducing morbidity and mortality among patients with streptococcal toxic shock syndrome. Several modes of action have been described for IVIG, including inhibition of complement activation, Fc interaction, anti-idiotypic neutralization, toxin-neutralizing antibodies, and modulation of cytokine responses [1–3, 11, 12]. The various mode of actions of IVIG are not mutually exclusive, and it is likely that they work together to modulate the host response. Streptococcal toxic shock syndrome is one of the most severe manifestation caused by group A streptococci; it is usually associated with high mortality rates of 30%–80% [13–16]. The systemic effects seen in severe invasive group A streptococcal infections have been suggested to be mainly attributed to the
Received 13 January 2000; revised 13 April 2000; electronically published 10 November 2000. Financial support: Swedish Society for Medicine, Swedish Medical Research Council, US Veterans Administration (to M.K.), and National Institutes of Health grant AI40198 (to M.K.). Reprints or correspondence: Dr. Anna Norrby-Teglund, Division of Infectious Diseases I63, Huddinge University Hospital, SE-141 86 Huddinge, Sweden (
[email protected]). Clinical Infectious Diseases 2000; 31:1175–82 q 2000 by the Infectious Diseases Society of America. All rights reserved. 1058-4838/2000/3105-0012$03.00
action of streptococcal superantigens [17–22]. Group A streptococci produce several different superantigens, including the streptococcal pyrogenic exotoxins (Spe) A, B, C, F, G, H, and J [22, 23], streptococcal superantigens [24], and the streptococcal mitogenic exotoxin Z and Z-2 [23, 25]. As superantigens, they activate large numbers of T cells and antigen presenting cells, resulting in excessive production of inflammatory cytokines [18–20, 22]. In vitro experiments have demonstrated a marked inhibition of the mitogenic and cytokine-inducing capacity of streptococcal and staphylococcal superantigens by IVIG [26–29]. Furthermore, IVIG has been shown to contain opsonic antibodies against the surface-bound M protein of the group A streptococcal bacteria [30]. The majority of studies of Ig therapy of toxic shock caused by gram-positive bacteria have focused on IgG; however, several recent studies have suggested that IgM-enriched IVIG preparations are superior in the treatment of neonatal and gram-negative septicemia, as compared with IVIG preparations containing only IgG [31–34]. Furthermore, the effect of IgA, the major Ig in mucosal secretion, on superantigen responses has never been investigated. The goal of this study was to investigate the comparative neutralization effects of polyspecific IgM, IgA, and IgG on streptococcal superantigens. Material and Methods Reagents and toxins. The Escherichia coli BL21 containing pET15b-speA [35] was kindly provided by Dr. C. Collin and Dr. B. Kline, University of Miami, Florida. Recombinant streptococcal pyrogenic exotoxin A (rSpeA) was expressed and purified as a His fusion protein according to the recommendation of the manufacturer (Novagen, Madison, WI). The N-terminal His tag was re-
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moved by digestion of the fusion protein with 1 U of thrombin per milligram of rSpeA for 16 h at room temperature with the thrombin cleavage capture kit (Novagen). The digested rSpeA was treated with polymyxin B agarose (Boehringer Mannheim, Indianapolis, IN) for 6 h at room temperature to remove any contaminating endotoxin. Thrombin-digested rSpeA contains 4 additional amino acids at the amino terminus compared with native SpeA [35], but the superantigenic activity was comparable to that of native SpeA with regards to mitogenic- and cytokine-inducing activity [36, 37] as well as Vb specificity [38]. A concentrated and dialyzed culture supernatant prepared from an overnight culture of a group A streptococcal strain isolated from a patient with streptococcal toxic shock syndrome was used as a crude preparation of superantigens produced by this isolate, as previously described [22, 23]. The isolate was serotype T3/B3264, and the culture supernatant was shown to contain SpeB and SpeF, but no detectable SpeA, as determined by Western blot analysis. The optimal concentration of SpeA and culture supernatant were 10 ng/mL respectively, 1:100 dilution, as determined by proliferation experiments with peripheral blood mononuclear cells (PBMC) from healthy donors. Phytohemagglutinin-L (Sigma, St. Louis, MO) was used at a concentration of 0.5 mg/mL in the proliferation assays. Ig preparations. Four IVIG preparations, Gammagard S/D (Baxter, Lessines, Belgium), IVIG-CP (IVIG-CP [chromatography purified]; Biotest Pharma, Dreieich, Germany), Pentaglobin (Biotest Pharma), and IgAbulin (Immuno AG, Vienna, Austria), were included in the study. The Ig composition of each preparation is presented in table 1. An IgM (Hu299) preparation was prepared from Cohn fraction III of pooled human plasma and purified by octanoic acid precipitation and anion exchange chromatography to a purity of 82%, as determined by ELISA (table 1). IgG and IgM were removed from IgAbulin by several passages through protein G and anti-IgM columns to yield a 99.9% pure IgA preparation, as determined by ELISA or nephelometry (table 1). Two IgA preparations purified from human colostrum (Sigma and Dr. J. Mestecky, Department of Microbiology, Birmingham, AL) were also included. Measurement of anti-Spe antibodies by ELISA. Anti-SpeA, -SpeB, and -SpeF antibodies in Ig preparations were measured by ELISA, as detailed elsewhere [39]. Recombinant SpeA, purified SpeB (Toxin Technology, Sarasota, FL), or recombinant SpeF were used as coating antigens, and wells with no antigen were used as controls for nonspecific binding of the Ig preparations. Fetal bovine serum (FBS) diluted 1:100 was used as a negative control. Each Ig preparation (diluted 1:200 and 1:500) was analyzed for antibodies of IgA, IgM, and IgG isotypes by use of the following isotype-specific antibodies conjugated to horseradish peroxidase: mouse anti-human IgA1 and IgA2 monoclonal antibodies, mouse anti-human IgM monoclonal antibody (Southern Biotechnology, Birmingham, AL), and goat anti-human IgG polyclonal antibody (Cappel Research Reagents, Costa Mesa, CA). Color reaction was achieved by the addition of the substrate 2,20-azino-di-(3-ethylbenzthiiazoline-6-sulfonate) (ABTS Microwell peroxidase substrate systems, Kirkegaard & Perry Laboratories, Gaithersburg, MD), and the absorbance was measured at 410 nm. All samples were assayed in triplicate. The Spe-specific binding activities were nor-
Table 1.
CID 2000;31 (November)
Composition of IgM, IgA, and IgG preparations.
Immunoglobulin preparation
IgG, %
IgM, %
IgA, %
Pentaglobin Hu299 (IgM) IgAbulin IgA (purified from IgAbulin) IgA colostrum 1 IgA colostrum 2 IVIG-CP Gammagard S/D
76 5 25 !0.1 NA NA 97 199
12 82 0 !0.1 NA NA 2.5 —
12 13 75 99.9 96 >98 !0.05 !0.01
NOTE. IVIG, polyspecific human Immunoglobulin given intravenously; NA, not available. The Ig composition is according to the manufacturers’ specification or as determined by nephelometry or ELISA. IgA colostrum 1 was obtained from Sigma; IgA colostrum 2 was obtained from Dr. J. Mestecky, Birmingham, Alabama.
malized to that in the wells with no antigen, according to the following equation: OD410 Sample:Spe OD410 Sample:No antigen
well well
–OD410 FBS:Spe well –OD410 FBS:No antigen
. well
Superantigen neutralizing assay. PBMC were isolated from healthy donors by Ficoll-Hypaque gradient centrifugation and were cultured in RPMI 1640 medium supplemented with 25 mM HEPES, 4 mM l-glutamine, 100 U penicillin/streptomycin per mL, and 5% heat-inactivated endotoxin-free FBS. PBMC (1 3 10 6 cells/ mL) were cultured with the different stimuli in the absence or presence of various concentrations of immunoglobulins (0.05–0.5 mg/mL). After 3 days, the cells were pulsed for 6 h with 1 mCi per well of [3H]-thymidine (specific activity, 6.7 Ci/mmol; DuPont, Wilmington, DE). All samples were assayed in triplicate, and the data are presented as the percentage inhibition of toxin mitogenicity, as related to cultures with no Ig added. Analyses of cytokine production at a single-cell level. PBMC (1 3 10 6 cells/mL) were stimulated with the various stimuli in the presence or absence of Ig preparations (0.5 mg/mL) and harvested after 24 and 72 h of culture. The cells were transferred to adhesion glass slides (Erie Scientific, Portsmouth, NH), fixed with freshly prepared 2% formaldehyde (Fisher Scientific, Fair Lawn, NJ) in PBS for 20 min, and stained for cytokines as previously described in detail [28]. The following cytokine-specific monoclonal antibodies were used at concentrations of 2 mg/mL: IL-1b (IL-1b; 2D8, 1437.96.5, murine IgG1; ImmunoContact, Lugano, Switzerland), TNF-b (TNF-b; LTX-21, murine IgG2b from Biosource, Camarillo, CA), and IFN-g (IFN-g; 1-D1K, 7-B6-1, murine IgG1; from MabTech, Stockholm, Sweden), and as secondary antibody, biotinylated goat anti-mouse IgG1 (Caltag Lab, San Francisco, CA) diluted 1:300 was used. Immunohistochemical staining was achieved by Vectastain-Elite (Vector Laboratories, Burlingame, CA) in combination with the substrate 3,3 diaminobenzidine (DAB; Vector Laboratories). IFN-g– and TNF-b–producing cells were characterized by a local dense juxtanuclear staining, whereas IL-1b results in a cytoplasmic staining, consistent with previous findings [40]. The data are presented as the percentage of cytokineproducing cells 5 SD. Statistical evaluation. Mann-Whitney U test or Kruskal Wallis analysis of variance were used to evaluate significant differences of the data. P ! .05 was considered significant.
CID 2000;31 (November)
IgM and IgA Inhibit Streptococcal Superantigens
Results Different IgM, IgA, and IgG preparations were tested for their ability to neutralize the mitogenic and cytokine-inducing activity of a defined superantigen SpeA, as well as a culture supernatant containing a mixture of superantigens and other extracellular virulence factors prepared from a clinical T3 strain. Western blot analysis of the culture supernatant revealed that it contained SpeB and SpeF, but no SpeA (data not shown). A total of 6 different Ig preparations with varying Ig isotype composition was studied, including 2 IgG preparations as controls (table 1). Proliferation experiments revealed that like IgG, IgM and IgA preparations could neutralize the mitogenic activity of both SpeA and group A streptococcal culture supernatant (figure 1). However, there were marked differences in the level of neutralizing ability against the stimuli among the different IgA, IgM, and IgG preparations, with the weakest inhibitory activity noted for the purified IgM preparation (Hu299). SpeA-induced proliferation was inhibited to a significantly higher extent by Pentaglobin and IgAbulin as compared to IgG. For example, at 0.1 mg/mL, Pentaglobin, IgAbulin, and IgG caused 95%, 94%, and 72% inhibition of SpeA response, respectively (P < .05). Differences in inhibitory capacity between preparations were only seen at relatively low concentrations (
