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. Author manuscript; available in PMC: 2008 Jan 23.
Published in final edited form as: Cell Host Microbe. 2007 Mar 15;1(1):51–61. doi: 10.1016/j.chom.2007.01.002

The Broad Antibacterial Activity of the Natural Antibody Repertoire is due to Polyreactive Antibodies

Zhao-Hua Zhou 1, Yahong Zhang 2, Ya-Fang Hu 1, Larry M Wahl 2, John O Cisar 3, Abner Louis Notkins 1,*
PMCID: PMC2212603  NIHMSID: NIHMS26550  PMID: 18005681

Abstract

Polyreactive antibodies bind to a variety of structurally unrelated antigens. The function of these antibodies, however, has remained an enigma and because of their low binding-affinity their biological relevance has been questioned. Using a panel of monoclonal polyreactive antibodies, we showed that these antibodies can bind to both Gram-negative and Gram-positive bacteria and acting through the classical complement pathway can inhibit bacterial growth by lysis, generate anaphylatoxin C5a, enhance phagocytosis and neutralize the functional activity of endotoxin. Polyreactive antibody-enriched, but not polyreactive antibody-reduced, IgM prepared from normal human serum displays anti-bacterial activity similar to that of monoclonal polyreactive IgM. We conclude that polyreactive antibodies are a major contributor to the broad anti-bacterial activity of the natural antibody repertoire.

Keywords: Bacteria, Endotoxin, Innate Immunity, Polyreactive Antibodies, Natural Antibodies

INTRODUCTION

What is now known as natural antibodies was first described nearly 100 years ago (Tauber and Podolsky, 1997). Innumerable studies since have shown that serum containing these antibodies has bactericidal activity and, in fact, to distinguish antigen-induced antibodies from natural antibodies, serum is routinely diluted 10 - 50 fold before testing to reduce the background activity of the natural antibodies (Notkins, 2004). Natural antibodies, however, have remained an enigma to immunologists because they are found in serum in the apparent absence of antigenic stimulation and are present in newborns and germ-free animals. Absorption of serum with a specific protein or bacterium can result in the loss of antibody activity not only to the antigen used for absorption, but also to other unrelated proteins or bacteria (Gordon and Carter, 1932; Michael et al., 1962 ). Adding further to the enigma of natural antibodies is the observation that many natural antibodies react with a variety of normal host proteins suggesting that some of these antibodies are autoantibodies, which would seem to contradict the well-accepted view that the host is immunologically tolerant to most self antigens. The fact that normal serum contains millions of different Ig molecules, all in small quantities, has made it difficult to characterize these antibodies.

With the advent of hybidoma technology in the mid-1970s it became possible to prepare large quantities of individual Ig molecules. Analysis of these monoclonal Ig molecules, prepared from normal individuals, revealed that many were polyreactive; that is, they could bind to a variety of structurally unrelated self (e.g., proteins, lipids, carbohydrates, DNA) and non-self (e.g., bacteria, viruses) antigens (Haspel et al., 1983; Dighiero et al., 1983; Casali and Notkins, 1989; Prabhakar et al., 1984; Burastero et al., 1988 ). These polyreactive antibodies bind to antigens with low affinity (Kd = 10−3 to 10−7 mol l−1) as compared to monoreactive antibodies (Kd = 10−7 to 10−11 mol l−1) and each polyreactive antibody has a distinct antigen-binding pattern that can vary for different antigens by as much as 1000-fold (Haspel et al., 1983; Dighiero et al., 1983; Casali and Notkins, 1989; Prabhakar et al., 1984; Burastero et al., 1988 ). Many of the polyreactive antibodies have a germ-line or near germ-line sequence and are primarily IgM, but some are also IgG and IgA. Contrary to the classic “lock and key” rigid-structure hypothesis of antigen-antibody interaction, the antigen binding pocket of polyreactive antibodies, perhaps because of their germ-line configuration, are believed to be more flexible and therefore can accommodate different antigenic configurations (Gordon and Carter, 1932). The B cells that make these antibodies can be identified by the binding of fluorescein-labelled antigens to the polyreactive B cell receptors on their surface (Zhou and Notkins, 2004; Wang et al., 2001). Some of those polyreactive antigen-binding B (PAB) cells have a B-1 positive, whereas others have a B-1 negative, phenotype (Zhou and Notkins, 2004). In the newborn up to 50% of peripheral blood B-lymphocytes can make polyreactive antibodies. In the adult the number is reduced to between 15% and 20% (Chen et al., 1998). Polyreactive antibodies therefore are a major component of the natural antibody repertoire.

Natural antibodies have long been thought to be a first line of defense against bacterial and viral infections (Casali and Notkins, 1989), but the biological function of polyreactive antibodies has never been determined. In fact, because of their low affinity their biological relevance has been questioned. The present experiments were initiated to determine whether polyreactive antibodies have antibacterial activity.

RESULTS

Properties of polyreactive antibody 2E4

Mouse hybridomas were prepared, cloned and screened for polyreactivity. Polyreactive antibody 2E4 was chosen for detailed study. Of the antigens screened the strongest binding was to ssDNA and β-gal with weaker binding to insulin, Tg and LPS (Fig. 1A). The dissociation constants (Kd) for ssDNA, β-gal and insulin were 4.0×10−8, 7.0×10−7 and 1.0×10−5, respectively. As with other polyreative antibodies (Haspel et al., 1983), 2E4 bound to a variety of organs and cell types as determined by indirect immunofluorescence (not shown). Gene sequencing revealed that 2E4 is an IgM antibody with a germline sequence 99.7% identical to J558.8 (Supplementary Table 1).

Figure 1.

Figure 1

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Properties of polyreactivity antibody 2E4. (A) Dose-dependent binding of 2E4 to different antigens as measured by ELISA and the calculated dissociation constants (Kd) . (B) Polyreactive antibody 2E4 binds to a variety of Gram-positive and Gram-negative bacteria. (C) Non-polyreactive MAb2507 only binds to its cognate antigen, E. coli O157: H7. (D) Polyreactive antibody 2E4 (right panel), but not non-polyreactive MAb2507 (left panel), fixes complement as measured by FACS analysis with anti-C3.

Abbreviation: So, S. oralis; Sm, S. mitis; Sg, S. gordonii; An, A. naeslundii; Ec, E. coli.

Polyreactive antibody 2E4 binds to a variety of different bacteria

The binding of 2E4 to a variety of Gram-positive and Gram-negative bacteria, including different serotypes of three antigenically distinct genera, was determined by FACS analysis. Representative data (Fig. 1B) show that 2E4 binds strongly to some bacteria (Streptococcus oralis J22, Streptococcus oralis 10557, Streptococcus mitis 15914 and E. coli BL21), moderately to other bacteria (Streptococcus oralis C104, Streptococcus oralis 34, Actinomyces naeslundii T14V and E. coli K12) and weakly or not at all to still other bacteria (Streptococcus gordonii 38, Streptococcus gordonii DL1, Actinomyces naeslundii 12104 and E. coli O157: H7). In contrast, monoclonal antibody MAb2507 reacted only to its cognate antigen E. coli O157: H7 (Fig. 1C).

Polyreactive antibody 2E4 fixes complement and inhibits E. coli growth

Bacteria (E. coli BL21) that had been incubated with polyreactive antibody 2E4 or the non-binding monoreactive antibody, MAb2507, were incubated with complement. The fixation of complement was determined by FACS analysis with an antibody to the third component of complement (C3). As seen in Fig. 1D, complement bound strongly to the polyreactive antibody-treated bacteria, but not to the non-binding monoreactive antibody-treated bacteria.

The effect of complement on the growth of bacteria treated with polyreactive antibody 2E4 was determined by measuring the incorporation of 3H-thymidine. At a 1:10 dilution of complement there was little if any incorporation of 3H-thymidine into bacteria as compared to bacteria that had been treated with PBS or with MAb2507 (Fig. 2A). At higher dilutions of complement the growth inhibitory effect decreased and with heat-inactivated complement (Fig. 2B) there was no inhibition of growth.

Figure 2.

Figure 2

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Polyreactive 2E4 is bactericidal in the presence of complement. Growth of 2E4-treated E. coli BL21 as measured by the incorporation of 3H-TdR in the presence of different concentrations of (A) untreated and (B) heat-inactivated complement. (C) Bacteria colony formation on agar plates in the presence of complement with or without 2E4. (D) Lysis of 2E4-treated bacteria as measured by the release of 3H-TdR in the presence of different concentrations of complement. (E) Growth of 2E4-treated bacteria as determined by the incorporation of 3H-TdR in the presence of different concentrations of C4+/+ or C4−/− serum or (F) heat-inactivated C4+/+ or C4−/− serum. (G) Lysis of 2E4-treated E. coli BL21 as measured by the release of H3-TdR in the presence of different concentrations of C4+/+ or C4−/− serum. Bars represent s.d.

The inhibitory effect of 2E4 and complement also was demonstrated by plating the 2E4-treated and un-treated bacteria on agar plates and evaluating the number and size of the bacterial colonies. Bacteria treated with 2E4 and complement showed little if any colony formation as compared to bacteria treated with PBS and complement (Fig. 2C).

To determine if the inhibition of growth was due to bacterial lysis, the release of 3H-TdR from radiolabeled E. coli BL21 was measured. Fig. 2D shows that considerably more 3H-TdR was released from the cells treated with 2E4 than those treated with MAb2507 or PBS and that the effect was complement dependent.

Bactericidal activity of 2E4 is mediated through the classical complement pathway

The classical complement pathway, in contrast to the alternative complement pathway, is antigen-antibody specific and requires C4 (Carroll, 2004). To see if polyreactive 2E4 exerted its growth inhibitory effect through the classical complement pathway, C4−/− serum was compared to C4+/+ serum as a source of complement. Fig. 2E shows that the growth of 2E4-treated bacteria, as measured by the uptake of 3H-TdR, was markedly inhibited by C4+/+ serum, but not by C4−/− serum or by heat-inactivated C4+/+ serum (Fig. 2F). Release of 3H-TdR from radialabelled 2E4-treated bacteria by C4+/+ serum, but not by C4−/− serum, showed that the inhibition of growth was due to bacterial lysis mediated through the classical complement pathway (Fig. 2G).

Polyreactive antibody 2E4 binds to and lyses Pseudomonas aeruginosa

To see if polyreactive antibody 2E4 would inhibit the growth of other bacteria, Pseudomonas aeruginosa serotype 10 (PSA-10), was chosen for study. Figure 3A shows that 2E4 binds to PSA-10 as demonstrated by FACS analysis and fixes complement (not shown). In the presence of complement, polyreactive antibody 2E4, but not non-polyreactive MAb8512, produced significant lysis of PSA-10 (Fig. 3B).

Figure 3.

Figure 3

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Lysis of Pseudomonas aeruginosa (PSA-10) by MAb2E4 and three additional polyreactive antibodies that inhibit the growth of E. coli BL21 in the presence of complement. (A) MAb 2E4 (right panel), but not MAb8512 (left panel), binds to PSA-10. (B) Enhanced lysis of 2E4-treated PSA-10 as compared to MAb8512-treated PSA-10 in the presence of two different concentrations of complement as measured by the release of 3H-adenine. (C) Polyreactive MAbs ZH-6, ZH-14 and ZH-20, but not non-polyreactive MAb2507, bind to E. coli BL21. (D) Inhibition of growth of antibody-treated E. coli BL21 as measured by incorporation of 3H-TdR in the presence of complement (left) or heat-inactivated complement (right). (E) Lysis of antibody-treated E. coli BL21 as measured by the release of 3H-TdR in the presence of complement.

Other monoclonal polyreactive antibodies with antibacterial activity

Three additional monoclonal polyreactive antibodies were prepared and tested (Supplementary Table 1). Monoclonal antibody ZH-6 and ZH-20 were IgMs, belonged to the J558 family and were 100% and 99.3% identical to J558.2 and J558.13, respectively. Their light chains belonged to VK-21 and VK-12 family, respectively. In contrast, ZH-14 was an IgG3, belonged to the VGAM3.8 (VH9) family and was 96.8% identical to VH9.1. The light chain belonged to the VK-21 family. These antibodies then were tested for their binding to a variety of antigens and bacteria. In contrast to 2E4 (Fig. 1B), all three antibodies showed an intermediate level of binding to E. coli BL21(Fig. 3C), but in the presence of complement the three antibodies inhibited the growth of E. coli (Fig. 3D) which was mediated through bacterial lysis (Fig. 3E).

Generation of anaphylatoxin C5a in the absence of bacterial lysis

Polyreactive antibody 2E4 can bind to a variety of Gram-positive bacteria (Fig. 1B) and also fix complement (Fig. 4A). However, in contrast to Gram-negative bacteria, Gram-positive bacteria are not lysed (Fig. 4B) nor is their growth inhibited (data not shown). Despite the failure to inhibit growth, the interaction of polyreactive antibodies with Gram-positive bacteria, in the presence of complement, generates solid phase C5 convertase and, in turn, the anaphylatoxin C5a, an important chemotaxis factor (Fig. 4C).

Figure 4.

Figure 4

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Polyreactive antibodies in the presence of complement do not lyse Gram-positive bacteria (i.e., Streptococcus mitis, Streptococcus oralis) but generate the anaphylatoxin, C5a. (A) Polyreactive antibody 2E4 fixes complement as measured by FACS analysis with anti-C3 antibody (red line) as compared to non-binding MAb8512 (green shadow). (B) Neither polyreactive 2E4-treated nor non-polyreactive MAb8512-treated Gram-positive bacteria are lysed in the prescence of complement (1:10 dilution) as determined by 3H-TdR release. (C) Monoclonal antibody treated Gram-positive bacteria were incubated with guinea pig complement (1:10 dilution) followed by incubation with human C5. MAb 2E4-treated, but not MAb8512-treated, Gram-positive bacteria generated anaphylatoxin C5a as measured by ELISA.

Polyreactive MAb 2E4 enhances phagocytosis

The fixation of complement to antigen-antibody complexes can enhance phagocytosis. Bacteria E. coli BL21 were labeled with FITC, treated with polyreactive 2E4 or non-binding MAb2507, and then incubated with complement. Phagocytosis of the bacteria by mouse macrophages was evaluated by confocal microscopy and FACS analysis. As seen in Fig 5A, bacteria treated with polyreactive 2E4 and complement were readily phagocytosed as compared to bacteria treated with non-binding MAb2507 and complement or with 2E4 alone or complement alone (Fig. 5B). Maximum fluorescence (phagocytosis) was detected at 150 minutes.

Figure 5.

Figure 5

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Polyreactive antibodies enhance phagocytosis and inhibit the activity of LPS. (A) Immunofluorescence staining shows bacteria E. coli BL21 (green) being taken up by macrophages (red) with blue nuclei. Left panel: bacteria treated with MAb2507 and complement. Right panel: bacteria treated with MAb2E4 and complement. (B) Phagocytosis of 2E4-treated bacteria in the prescence of complement as determined by FACS analysis and measured by mean fluoresce intensity (MFI). (C) Human monocyte MMP-1 is increased by incubation with LPS (Salmonella abortus-equi, SAE) in the presence of PBS or non-binding MAb8512, but is decreased by polyreactive 2E4. (D) MMP-1 is not produced by monocytes incubated with PBS or with different polyreactive (i.e., ZH-14, ZH-6 and ZH-20) or non-polyreactive MAbs (i.e., 8512 and 2507) in the absence of LPS. (E-G) Monocyte MMP-1 production is increased by incubation with LPS from (E) SAE, (f) E. coli O157: H7 or (G) Pseudomonas aeruginosa (PSA) in the presence of PBS or non-binding MAb8512, but is decreased by polyreactive MAbs ZH-14, ZH-6 and ZH-20. Non-polyreactive MAb2507 decreased only the production of MMP-1 induced by LPS from E. coli O157: H7, its cognate antigen (F). (H) Polyreactive-enriched, but not polyreactive-reduced, human IgM inhibits LPS-induced production of MMP-1.

Polyreactive antibodies neutralize the functional activity of endotoxin (LPS)

Exposure of human monocytes to LPS resulted in the production of matrix metalloproteinase 1 (MMP-1) (Zhang et al., 1997). Incubation of LPS from Salmonella abortus-equi (SAE) with polyreactive antibody MAb2E4, but not irrelevant MAb8512, reduced the production of MMP-1 (Fig. 5C). Similar results were obtained with three other polyreactive antibodies (i.e., ZH-14, ZH-6 and ZH-20) using LPS from three different bacteria (i.e., SAE, E. coli O157: H7 and PSA) (Figs. 5E, F and G). MAb2507 reduced only the production of MMP-1 by LPS from Ec-O157: H7, its cognate antigen (Fig. 5F), but not the production of MMP-1 by LPS from SAE or PSA (Fig. 5E and G). None of the monoclonal antibodies bound directly to monocytes as evaluated by immunostaining and FACS analysis (not shown). These findings show that polyreactive antibodies can neutralize the functional activity of LPS in the absence of complement.

Polyreactive antibodies from serum are bactericidal

Polyreactive antibodies from normal serum were enriched by serial passage of affinity purified human IgM through three different antigen-affinity columns (i.e., ssDNA, β-gal, thyroglobulin). The polyreactive-enriched, as compared to the polyreactive-reduced, IgM bound to a variety of different antigens (Fig. 6A), including bacteria (Fig. 6B), and in the presence of complement lysed radiolabelled E. coli BL21 (Fig. 6C). In the absence of complement polyreactive-enriched, but not polyreactive-reduced, human IgM decreased the production of LPS-induced MMP-1 (Fig. 5H). These findings show that polyreactive antibodies from human sera have antibacterial properties similar to monoclonal polyreactive antibodies.

Figure 6.

Figure 6

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Bactericidal activity of polyreactive antibody-enriched human IgM. (A) Binding of polyreactive antibody-enriched, as compared to polyreactive antibody-reduced, IgM to different antigens and to (B) E. coli BL21. (C) Polyreactive-enriched, but not polyreactive-reduced, IgM lyses E. coli BL21 in the presence of complement as measured by the release of 3H-TdR. Equal concentrations (50 μg/ml) of polyreactive-enriched and polyreactive-reduced IgM were used in all experiments.

DISCUSSION

Natural antibodies, by definition, are produced in the apparent absence of antigenic stimulation (Tauber, 2000 ). The most likely explanation for their occurrence is that they are the result of the millions of normally occurring VDJ rearrangements and exist in germ-line or near germ-line configuration. Because of their diversity, the percentage of antibodies in normal serum that are polyreactive has been difficult to determine. However, since up to one-half of the B cells in the cord blood of newborns and one-fifth of the B cells in the peripheral circulation of adult are capable of making polyreactive antibodies it is evident that these antibodies are highly represented (Chen et al., 1998; Wardemann et al., 2003).

The high representation of polyreactive antibodies raises questions about the nature and function of the non-polyreactive antibodies in the natural antibody repertoire. Assuming that they too exist in germ-line or near germ-line configuration, it is unclear whether they have sufficient specificity and affinity to be biologically relevant in natural defense against invading pathogens without further antigenic stimulation and somatic mutation. If, in fact, the genes encoding these non-polyreactive antibodies are not in germ-line configuration and instead show many somatic mutations they rightfully should not be considered part of the natural antibody repertoire. Perhaps more important, even if some of these germ line-encoded non-polyreactive antibodies do have a potential biological function, their concentration in serum as an individual molecular species may be too low to have immediate biological value when confronted with invading pathogens.

In contrast to non-polyreactive antibodies, the great number and diversity of polyreactive antibodies, with the ability of many of them to recognize the same bacteria and multiple antigens on those bacteria, make them a major component of the natural antibody repertoire and potentially an important first line of defense. This is not meant to negate the possibility that there may be a population of germ-line-encoded non-polyreactive natural antibodies directed against evolutionally- conserved epitopes found on pathogens such as phosphocholine (Briles et al., 1981; Claflin and Berry, 1988 ). In fact, such antibodies have been found by hybridoma technology, but in many cases it was not clear whether these antibodies were: monoreactive, oligoreactive or polyreactive (Notkins, 2004 ); truly encoded by germ-line sequences; or presented in sufficient concentration in the serum to have biological value.

Polyreactive antibodies are an ancient part of the immune system and have been found in jawed vertebrates going back as far as the shark (Marchalonis et al., 2001; Marchalonis et al., 2002). The ability of polyreactive antibodies to bind to a variety of different antigens suggests that these antibodies represent the humoral component of the innate immune system. In fact, upon entering the host, pathogens will be exposed almost immediately to polyreactive antibodies since these antibodies are constantly present in serum. This exposure, in the presence of complement, could result in lysis of bacteria which in turn could lead to the release of ligands (e.g., CpG-DNA) that bind to Toll-like receptors. In this way the innate immune system could be activated (Krieg, 2002). Of particular interest is the fact that even though the interaction of polyreactive antibodies with Gram-positive bacteria does not result in lysis, it does activate the complement cascade. Cleavage products such as C5a then bind to a whole range of cell surface receptors on myeloid and lymphoid cells and thereby contribute to the host's inflammatory response (Guo and Ward, 2005; Huber-Lang et al., 2006). Thus the initial encounter of polyreactive antibodies with pathogens could serve as a first line of defense and give the adaptive immune system time to be activated.

A variety of studies involving the passive transfer of normal serum to animals that subsequently were challenged with bacteria or viruses showed that normal serum enhanced protection (Ochsenbein et al., 1999; Baumgarth et al., 2000; Quan et al., 1997; Kroese et al., 1996; Boes et al., 1998). To what extent this protection was due to the polyreactive antibodies in the transferred serum, however, has never been determined. The demonstration here that both monoclonal polyreactive antibodies and enriched, but not reduced, polyreactive antibodies prepared from normal serum can lyse bacteria, generate C5a, enhance phagocytosis and neutralize endotoxin argues that polyreactive antibodies contribute in an important way to the antibacterial activity of normal serum. The effectiveness of polyreactive antibodies depends on the nature of the antigens on the particular pathogen and the titer and binding capacity of the circulating polyreactive antibodies.

In addition to their role in defense, a variety of other functions have been attributed to natural antibodies including clearance of degraded proteins (e.g., red blood cells, bacteria), the enhancement of immunogenicity by trapping of antigens in lymphoid organs and immunoregulatory functions (Ochsenbein et al., 1999; Sapir and Shoenfeld, 2005; Miller and Rodriguez, 1995; Trebst and Stangel, 2006; Robey et al., 2002 ). In none of these cases, however, has the role of polyreactive antibodies been evaluated. It is of particular interest that the intravenous administration of natural Ig (IVIG) is being used to treat a variety of disorders (Sapir and Shoenfeld, 2005; Siragam, et al. 2006). There is no consensus as to how natural Ig works, but again the role of polyreactive antibodies needs to be evaluated.

Although polyreactive antibodies can bind to normal mammalian tissues, under ordinary circumstances, they do not appear to be deleterious to the host, presumably because a variety of regulatory proteins (e.g., factor H, DAF, CR-1) inhibit the lytic action of complement on eukaryotic cells (Zipfel, 2001; Kinoshita et al., 1985; Wagner et al., 2006). Moreover, it is well established that B cells capable of making high affinity self-reactive antibodies are eliminated from the host early in life by negative selection, but because of their low affinity, there may be little need for the host to eliminate the B cells that make polyreactive antibodies. In fact, because of their low affinity and germ-line configuration, polyreactive antibodies do not appear to be true autoantibodies and certainly do not fit into the same category as antigen-specific, somatically-mutated, high affinity pathogenic autoantibodies (Brard et al., 1999; Roark et al., 2002; Rahman, 2004; Wellmann et al., 2005). It is possible, however, that the B cells that make polyreactive antibodies might be the precursor population from which at least some high affinity autoantibodies are derived (Ichiyoshi et al., 1995). There also is some evidence that the polyreactive B cells population may be the result of positive selection. Transgenic mouse models provide support for the positive selection hypothesis (Julien et al., 2002; Hayakawa et al., 2003; Wong et al., 2002; Hardy and Hayakawa, 2005; Gaudin et al., 2004; Casola et al., 2004) but which self antigens are involved in positive selection of polyreactive B cells is still not clear.

In conclusion, the demonstration that passive transfer of normal sera containing natural antibodies can enhance resistance to bacteria and viruses (Ochsenbein et al., 1999; Boes et al., 1998; Reid et al., 1997) and the demonstration here of the anti-bacteria, anti-toxin (i.e., LPS) and phagocytosis enhancing effects of polyreactive antibodies raise the intriguing, although still distant, possibility that passive administration of a cocktail of monoclonal polyreactive antibodies might prove useful in the prevention and treatment of some infections. If this turns out to be the case, developing methods to activate in vivo the B cell subset that makes polyreactive antibodies might be of value in broadly enhancing natural resistance during times of bacterial and viral epidemics.

MATERIALS AND METHODS

Mice, cell lines and bacterial strains

Three to 12 week-old BALB/c mice were bred at the NIH animal facility (Bethesda, MD, USA). All experiments were carried out in compliance with institutional guidelines and approved by the NIDCR ACUC (Bethesda, MD). Gram-negative E. coli bacteria were cultured in Luria-Bertani (LB) broth, Pseudomonas aeruginosa Migula serotypic 10 (PSA-10) in Nutrient broth (BD234000) and different Grame-positive bacteria (i.e., S. gordonii, S. oralis, S. mitis and A. naeslundii) in Todd Hewitt Broth (Krackeler Scientific, Inc., Albany, NY) until an OD600 of 0.3 was reached. Mouse macrophage cell line Raw264.7 was cultured in RPMI1640 with 10% FBS and replaced with serum-free medium before use.

Preparations of hybridomas

Splenocytes were isolated from three to 12-month-old Balb/C mice and fused with Sp2/0-Ag14 myeloma cells (Hartman et al., 1989). Positive clones were subcloned by limiting dilution at least two times. Large amounts of antibody were prepared by culturing hybridoma cells in serum-free CD Hybridoma Medium (Invitrogen, Carlsbad CA). Supernants were concentrated and quantitated by Coomassie Plus Protein Assay Kit (Pierce Biotechnology, Inc, Rockford, IL). In the current study four new monoclonal polyreactive antibodies (2E4, ZH-6, ZH-16, ZH-20) were prepared and characterized (Supplementary Table 1). Two monoclonal monoreactive mouse IgM antibodies (2507 and 8512) were obtained from ATCC (ATCC No: CRL-2507, HB-8512) and used as controls. 2507 is specific for E. coli O157: H7 O-antigen and 8512 is against bacterial cell wall peptidoglycan.

RT-PCR and sequencing of variable region of immunoglobulins

Total RNA from hybridomas was isolated with TRIZOL (Invitrogen, Carlsbad, CA) according to the manufacturer's protocol and the constant and variable regions of IgM and IgG amplified by PCR using Mouse Ig-Primer Sets from Novagen (EMD Biosciences, Inc. La Jolla, CA). PCR fragments were cloned in pCR2.1 vector (Invitrogen) and sequenced.

ELISA and affinity assays

Approximately 10 μg/ml of antigens (beta-galactosidase, thyroglobulin, insulin, ssDNA, lipopolysacride, etc.) (Sigma, St. Louis MO) in coating buffer (15 mM Na2CO3, 35 mM NaHCO3) were adsorbed onto Maxisorp® microplates (Nalge Nunc, Copenhagen, Denmark). Nonspecific binding sites were blocked by incubation with 1% BSA in phosphate-buffered-saline (PBS). Different concentrations of purified monoclonal antibody were loaded in wells. The second antibody (biotin-labeled rat anti-mouse IgM or biotin-labeled polyclonal goat anti-mouse Igs at 2μg/ml) (BD Pharmigen, San Diego, CA) then was added. The reaction mixture was developed with Avidin-HRP (1:3000 dilution) and ABTS substrate (Zymed Laboratories, South San Francisco, CA) and absorbance read at 415nm by an Elisa microplate reader (Molecular Devices Corporation, Sunnyvale, CA). Affinity assays were performed as described (Nakamura et al., 1988) by competitive ELISA and the dissociation constants (Kd) calculated according to Friguet et al (Friguet et al., 1985).

Affinity isolation of human polyreactive IgM

Affinity columns of ssDNA, beta-gal, and thyroglobulin were prepared with HiTrap™ NHS-activated HP Columns (GE Healthcare, Piscataway, NJ ) according to the manufacture's instruction. Purified human IgM (>96% purification) isolated from normal human serum was obtained from Research Diagnostics, Inc., Flanders, NJ. Human IgM was sequentially passed through and eluted from DNA, beta-gal and thyroglobulin columns. The pass-through fractions were designed polyreactive IgM-reduced and the eluted fractions polyreactive IgM-enriched. IgM was eluted by washing with 2M NaCl and desalted with HiTrap Desalting columns (GE Healthcare). The polyreactive-enriched and polyreactive-reduced IgM fractions were concentrated by Macrosep centrifugation (Pall Life Sciences, Ann Arbor, MI) and the protein concentration determined by Coomassie Plus Protein Assay Kit (Pierce Biotechnology, Inc., Rockford, IL).

Immunofluorescence staining and flow cytometry assays

To measure the binding of antibody, 2 ×107 bacteria or 3.0 to 4.0 ×105 mammalian cells were preincubated with 1% BSA and 2 mm EDTA at 4°C for 30 min to minimize the effect of nonspecific binding sites. Mammalian cells also were preincubated with 20 μg/ml Mouse Fc Block™ (BD PharMingen). The cells then were incubated with mouse monoclonal polyreactive or monoreactive antibodies (50 μg/ml) followed by FITC-labeled goat anti-mouse IgM (Sigma-Aldrich Inc, St Louis, MO) and fixed with 4% paraformaldehyde. Unless indicated otherwise, to measure the binding of complement, antibody-treated bacteria were incubated with 1:15 dilution of guinea pig complement serum (Cedarlane Laboratories Limited, Ontario, Canada) that had been pre-adsorbed by four rounds of incubation with the bacteria to be tested to eliminate bacteria-binding immunoglobulin that might be present in the guinea pig serum. This was followed by incubation with FITC-labeled goat anti-guinea pig C3 polyclonal antibody (VitroStat Inc., Portland, ME) and fixation with 4% paraformaldehyde. Fluorescence intensity was analyzed by FACSCalibur using CELL Quest software (BD, San Jose, CA, USA).

Bacterial growth

2 ×107 bacteria were treated with 50 μg/ml polyreactive or non-polyreactive antibodies or PBS, followed by incubation with different dilutions of bacterial adsorbed guinea pig complement serum (to eliminate bacteria-binding Ig) at 37°C for 1h. Incorporation of [3H]TdR or [3H]adenine (20 μCi/ml) (GE Healthcare) was used to measure bacterial growth. At different time points, 100 μl of medium containing bacteria were removed, placed in triplicate on 0.45μm filters of a 96-well filtration plate (Millipore, Bedford, MA ), washed 8 times and the amount of radioactivity remaining on the filters determined ( Microbeta 1450 Trilux liquid scintillation counter, Wallac, Gaithersburg, MD). Growth also was determined by seeding 1:1000 dilution of bacterial cultures onto LB-agar plates and colonies were counted after overnight incubation at 37°C. All experiments were repeated at least three times.

Bactericidal assay

E. coli were radio-labeled by culturing in LB broth with 20 μCi/ml [6-3H]Thymidine (GE Healthcare) and PSA-10 were radio-labeled by culturing in nutrient broth (BD234000) with 20 μCi/ml [8-3H]adenine (GE Healthcare). 3 ×107 radio-labeled bacteria, suspended in PBS-BSA, then were incubated with polyreactive or non-polyreactive monoclonal antibodies (50 μg/ml) to which was added different dilution of bacterial adsorbed guinea pig complement or bacterial adsorbed C4−/− guinea pig serum (to eliminate bacteria-binding Ig). After incubation at 37°C for 1 h the reaction mixtures were transferred to 96 well filtration plates (Millipore), washed and the cpm remaining on the filters determined with a Microbeta 1450 Trilux liquid scintillation counter. Percent lysis was calculated as [(cpm of bacteria treated with control sera) – (cpm of bacteria treated with polyreactive antibody)/ (cpm of bacteria treated with control sera)] × 100.

Measurement of C5a

2 ×107 Gram-positive bacteria were incubated with polyreactive or non-polyreactive monoclonal antibodies, followed by incubation with 1:10 dilution of guinea pig complement as described above. The bacteria then were washed twice and incubated with 10 μg/ml of complement C5 from human serum (Sigma) at 37°C for 1h. Generation of C5a was determined by human C5a ELISA kit (BD Bioscience, San Jose CA).

Opsonization of Bacteria

Bacteria were labeled with FITC using a FluoroTag™ FITC conjugation kit purchased from Sigma (St Louis, MO) as described by Weingart et al. (Weingart et al., 1999). The FITC-labeled bacteria then were incubated with monoclonal polyreactive or non-polyreactive antibodies or PBS for 30 min, washed and incubated with 1:15 dilution of bacterial adsorbed guinea pig complement for 30 min. The bacteria then were added to Raw264.7 macrophages cultured in 24-well plates with serum-free medium. At different time points, the cells were trypsinized, fixed with 4% paraformaldehyde and mean fluorescence intensity determined. Raw264.7 macrophages also were cultured on Lab-Tek Chambered Coverglass (Nunc, Inc. Naperville, IL). The nuclei of Raw264.7 macrophages were pre-stained (blue) by culturing in10 μg/ml of Hoechst 33342 (Bis-Benzimide, Sigma) for 90 min and FITC-labeled bacteria were added. At different time points, 50 μg/ml ethidium bromide was added for 5 min to stain the cytoplasm of the macrophages red. Oponization was determined by Laser scanning confocal microscopy with a Leica TCS-SP2 confocal scanning head mounted on a Leica DMRE-7 (SDK) upright microscope. (NA=1.40; Leica Microsystems Inc., Bannockburn, IL, USA).

Purification of Human Monocytes and Detection of MMP-1 by Western Blot

Human peripheral blood cells were obtained by leukapheresis from normal volunteers at the Department of Transfusion Medicine at the National Institutes of Health. Monocytes were prepared as described previously (Zhang et al., 1997) and the final preparation contained greater than 90% monocytes as determined by morphology, non-specific esterase staining, and flow cytometry. The purification procedure did not activate the monocytes as demonstrated by the fact less than 4% of the cells were IL-2R positive, a sensitive marker of monocyte activation. 5 × 106/ml purified monocytes then were stimulated with 100 ng/ml LPS or LPS premixed with 3.0 μg/ml antibodies (30 min at 4°C) for 36 to 48 hours as previously described (14). The supernatant fluids together with BSA (40 μg/ml) were added to cold ethanol and put at −70°C for 15 min. Proteins then were pelleted by microcentrifugation, lyophilized and then the resuspended proteins electrophoresed on a 10% Tris-glycine gradient polyacrylamide gel. The proteins then were transferred onto 0.45 μm nitrocellulose and the blots were incubated overnight with rabbit polyclonal antibodies against matrix metalloproteinase (MMP-1). Western blots were analyzed by the addition of Alexa Fluor 680 goat anti-rabbit IgG (Molecular Probes Inc., Eugene) and the fluorescence detected with the Odyssey infrared imaging system (LI-COR, Lincoln, NE).

Statistical analysis

Data are presented as mean ± s.d. and all paired comparisons were subjected to a two-tailed student t test. Significance was set at a P value of less than 0.05.

Supplementary Material

01

ACKNOWLEDGEMENTS

The authors have no conflicting financial interests. This research was supported by Intramural Research Program of the National Institute of Dental and Craniofacial Research, the National Institutes of Health.

Footnotes

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