Takashi Tanikawa

Induction of preferential chemotaxis of unstimulated B-lymphocytes by 2-arachidonoylglycerol in immunized mice

2‐Arachidonoylglycerol (2‐AG) is an endogenous ligand for cannabinoid receptors. There are two types of cannabinoid receptors, CB1 and CB2. We investigated the chemotactic activity of 2‐AG using mouse lymphocytes because cells in the immune system are known to express CB2. Spleen cell migration toward 2‐AG was observed, which was completely inhibited by SR144528, a CB2‐specific antagonist 2‐AG has been reported to induce a preferential B cell chemotaxis. We examined whether there is any difference in responsiveness during the activation of B cells. When spleen cells from immunized mice were tested, naive B cells but not germinal center B cells (GL7‐positive) were increased in the fraction attracted by 2‐AG. Furthermore, when Peyers patch lymphocytes were tested after oral administration of cholera toxin, the number of IgA– B cells was increased in the fraction attracted by 2‐AG. These results suggested that 2‐AG preferentially attracts unstimulated naive B cells rather than activated and/or class‐switched B cells. This property may influence the structure of B cell compartments in secondary lymphoid tissues.

Production of Secretory Immunoglobulin A against Shiga Toxin-Binding Subunits in Mice by Mucosal Immunization

ABSTRACT The toxicity of Shiga toxins (Stx) depends on the binding of their B subunits to carbohydrate ligands on host cells. The production of antibodies against B subunits, especially immunoglobulin A (IgA) secreted on the mucosal surface, should contribute to host defense. One of the major problems in attempts to produce IgA against Stx was the poor immunogenicity of B subunits. We were able to produce serum IgA as well as IgG against Stx1B in mice of the H-2d haplotype by means of intranasal immunization with recombinant B subunits of Stx (Stx1B) together with cholera toxin as a mucosal adjuvant. Secretory IgA (S-IgA) was detected in nasal washes but not in feces. We prepared chemically cross-linked Stx1B for use as an immunogen, and the formation of stable oligomers was revealed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and mass spectrometry. When the cross-linked Stx1B was used together with cholera toxin for the intranasal immunization of BALB/c mice, strong enhancement of the immune response was observed. The S-IgA titers in nasal washes were 16- to more than 64-fold higher than those in mice immunized with native Stx1B plus cholera toxin. Furthermore, fecal IgA was detectable when the cross-linked Stx1B was used. The use of cholera toxin was necessary for the induction of high titers of S-IgA in the nasal washes. However, the effect of cross-linking was dependent on the major histocompatibility complex haplotype; that is, no enhancement of IgA production was observed in C57BL/6 mice. The present results provide a practical means of producing IgA against Stx1B in BALB/c mice.

Characterization of Monoclonal Immunoglobulin A and G Against Shiga Toxin Binding Subunits Produced by Intranasal Immunization

Immunoglobulin A (IgA) is considered to play a major role in protection of the mucosal surface. However, its immunological and biological properties have not been extensively studied because the production of IgA class monoclonal antibodies (mAbs) is difficult. We compared the properties of IgA and IgG mAbs against Shiga toxin B subunits (Stx1B). These mAbs were secreted from hybridomas that had been produced from mice after intranasal immunization with recombinant Stx1B and cholera toxin. The dose response curves for the binding of the IgA (clone G2G7) and IgG (clone D11C6) mAbs to immobilized Stx1B were similar, as revealed on ELISA. The majority of the IgA mAb formed dimers while the IgG mAb was monomeric, as judged by immunoblot analysis. The IgG mAb completely inhibited the binding of Stx1B to Burkitt’s lymphoma cell line Ramos, while the inhibition by the IgA mAb was only partial. The IgG mAb was able to neutralize the cytotoxicity of Stx1 holotoxin towards Vero cells, whereas the IgA mAb was not. The binding affinity of each binding site was compared by means of surface plasmon resonance analysis involving a capture method, with which the binding of soluble Stx1B to immobilized mAb was detected. The association rate was similar but the dissociation rate was twofold faster in the case of the IgA mAb, resulting in twofold higher affinity of the IgG mAb. These results suggest that one can obtain high affinity IgA mAb but toxin neutralization is another challenge as to therapeutic antibodies of the IgA class.

Establishment of Recombinant Hybrid-IgG ⁄ IgA Immunoglobulin Specific for Shiga Toxin

Shiga toxin 1 produced by enterohaemorrhagic Escherichia coli is an AB5 toxin that is involved in the life‐threatening haemolytic‐uraemic syndrome. The B subunits (Stx1B) are cell‐binding subunits. We previously established mouse hybridoma cell line producing IgA and IgG monoclonal antibodies (mAbs) against Stx1B. Here, we cloned cDNAs encoding each of the heavy, light and joining (J) chains from the hybridoma cell lines by means of the 5′ rapid amplification of cDNA ends (RACE) PCR method. Upon assignment of the variable regions of the heavy and light chains to known germline sequences, we found substantial somatic hypermutation in the complementarity‐determining regions in both the IgA and IgG mAbs. We also established a hybrid‐IgG/IgA heavy chain having variable regions of the IgG mAb by means of recombinant PCR methods. Upon transient expression of the hybrid‐IgG/IgA heavy, IgG‐associated light and J chains in COS‐1 cells, the translated dimeric hybrid‐IgG/IgA bound to immobilized Stx1B, as revealed on ELISA. The production of dimeric hybrid‐IgG/IgA was revealed on immunoblot analysis. The dimeric hybrid‐IgG/IgA inhibited the binding of digoxigenin‐conjugated Stx1B to natural ligands (CD77) displayed on Burkitt’s lymphoma cell line Ramos. These results indicate that the replacement of variable regions resulted in the production of more useful recombinant dimeric IgA against Stx1B.