Properties and function of polyreactive antibodies and polyreactive antigen-binding B cells

Abstract

The advent of hybridoma technology has made it possible to study in-depth individual antibody molecules. These studies have revealed a number of surprises that have and are continuing to change our view of the immune system. None of these was more surprising than the demonstration that many antibody molecules are polyreactive - that is they can bind to a variety of different and structurally unrelated self- and non-self-foreign antigens. These findings make it clear that self-reactivity is a common and not necessarily forbidden or pathogenic feature of the immune system and that the well-known broad antibacterial activity of natural antibodies is largely due to polyreactive antibodies. In this brief review we will discuss these insights and their impact on basic and clinical immunology.

Fig. 1

Binding of a murine monoclonal polyreactive IgM antibody (PAb2E4) to different normal tissues.

Fig. 2. Binding of antigens by monoclonal polyreactive (PAb2E4) and moclonal monoreactive (MAb GAL-40) antibodies

(A) Polyreactive antibody PAb2E4 binds strongly to β-galactase (β-gal) and single-stranded DNA (ss-DNA) and moderately to insulin, thyroglobulin (Tg) and LPS, whereas (B) while monoreactive antibody MAbGal-40 only recognizes its cognate antigen, β-gal [23].

Fig. 3. Binding of FITC-labeled antigens to polyreactive (PAB-18) and monoreactive hybridomas (anti-hFc) cells

Polyreactive hybridoma clone PAB-18 binds FITC-labeled β-galactase (β-gal), thyroglobulin (Tg), ovalbumin (OVA) and human IgG Fc (hFc). In contrast, the monoreactive hybridoma clone anti-hFc only binds its cognate antigen, hFc [15].

Fig. 4

Separation of PAB⁺ from PAB⁻ cells by antigen-coated magnetic beads.

Fig. 5. Antigen-binding of PAB⁺ and PAB⁻ cells from peritoneal cavity (PerC) and spleen (SPL)

PAB⁺ and PAB⁻ cells from PerC and SPL of BALB/c mice were positively selected as described in Figure 4. The binding of Ags (β-gal, Tg, actin and insulin) was determined by FACS analysis. The percentage of positively stained cells is indicated [16].

Fig. 6. Polyreactive antibody binds to various bacteria

(A) Polyreactive antibody PAb2E4 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, (B) monoclonal antibody MAb2507 binds only to its cognate antigen E. coli O157: H7. Antibody binding measured by fluorescence intensity with anti-IgM [23].

Fig. 7. Polyreactive antibody PAb2E4 inhibits bacterial growth through lysis of bacteria

The effect of complement on the growth of bacteria treated with polyreactive antibody PAb2E4 was determined by measuring the incorporation of ³H-thymidine. (A)There was little if any incorporation of ³H-thymidine into bacteria treated with PAb2E4 as compared to bacteria that had been treated with PBS or with MAb2507. (B) In the presence of heat-inactivated complement there was no inhibition of growth. (C) Lysis of PAb2E4-treated bacteria as measured by the release of ³H-TdR in the presence of complement. Considerably more ³H-TdR was released from the cells treated with PAb2E4 than those treated with MAb2507 or PBS [23].

Fig. 8. Polyreactive antibody PAb2E4 enhances phagocytosis

FITC-labeled bacteria (green) were treated with monoclonal antibodies and complement and then added to murine macrophages cultured in serum-free medium. The nuclei of the macrophages were prestained (blue) with Hoechst 33342. The cytoplasm of the macrophages was stained with ethidium bromide (red). (A) Substantial phagocytosis by macrophages of bacteria treated with PAb2E4 and complement. (B) Minimal phagocytosis of bacteria treated with MAb2507 and complement. (C) Phagocytosis of 2E4-treated bacteria in the presence of complement as determined by FACS analysis and expressed as mean fluorescence intensity (MFI) [23].

Fig. 9. Polyreactive-enriched IgM antibodies lyse bacteria

Purified human IgM was sequentially passed through and eluted from ssDNA, β-gal and thyroglobulin affinity columns. The pass-through fractions were designated “polyreactive-depleted IgM” and the eluted fractions “polyreactive-enriched IgM”. Lysis of IgM-treated bacteria was measured by the release of ³H-TdR in the presence of complement.