The inter-heavy chain disulfide bonds of IgG4 are in equilibrium with intra-chain disulfide bonds.

Unlike other immunoglobulin G (IgG) subclasses, IgG4 antibodies in plasma have been reported to be functionally monovalent. In a previous paper, we showed that the apparent monovalency of circulating IgG4 antibodies is caused by asymmetry of plasma IgG4-a large fraction has two antigen-binding sites resulting in bispecificity. We postulated that the generation of bispecific antibodies was caused by a post-secretion mechanism, involving the exchange of IgG4 half-molecules (i.e. one heavy and one light chain). This hypothesis was based on the observed instability of the inter-heavy chain disulfide bonds of IgG4. To investigate this instability, we constructed IgG4 mutants and analyzed the covalent interaction between the heavy chains by sodium dodecyl sulfate-poly acrylamide gel electrophoresis (SDS-PAGE) under non-reducing conditions. The mutation to serine of one of the hinge cysteines involved in the inter-heavy chain bond formation, Cys226, resulted in a more stable rather than a more labile inter-heavy chain linkage. Moreover, we confirmed that mutating the IgG4 hinge sequence Cys-Pro-Ser-Cys to the IgG1 hinge sequence Cys-Pro-Pro-Cys also markedly stabilizes the covalent interaction between the heavy-chains. These two observations suggested an explanation for the observed instability of the inter-heavy chain disulfide bonds: the formation of an alternative, intra-chain cystine. Obviously, this intra-chain cystine cannot be formed in the mutant where Cys226 is replaced by Ser, and cannot easily be formed in the mutant with the IgG1 hinge sequence (Cys-Pro-Pro-Cys) due to the restricted torsional freedom of prolines. We, therefore, postulate that the lack of a covalent heavy-chain interaction in a subpopulation of IgG4 reflects an equilibrium between inter- and intra-chain cystines. Based upon the published structure of the IgG4-related hinge-deleted IgG1 myeloma protein Mcg, we propose a model for the two forms of IgG4 and for the half-molecule exchange reaction, which might result in the formation of bispecific IgG4 antibodies.

Stimulation of bacterial adherence by neutrophil defensins varies among bacterial species but not among host cell types.

Adherence of Haemophilus influenzae to bronchial epithelial cells is enhanced by neutrophil defensins, which are released from activated neutrophils during inflammation [Gorter et al. (1998) J. Infect. Dis. 178, 1067-1078]. In this study, we showed that the adherence of H. influenzae to various epithelial, fibroblast-like and endothelial cell types was significantly enhanced by defensins (20 microg ml(-1)). Defensins stimulated also the adherence of Moraxella catarrhalis, Neisseria meningitidis and nonencapsulated Streptococcus pneumoniae to the NCI-H292 cell line. In contrast, defensins did not affect the adherence of Pseudomonas aeruginosa, encapsulated S. pneumoniae, Escherichia coli and Staphylococcus epidermidis. These results suggest that the defensin-enhanced adherence might support the adherence and possibly persistence of the selected bacterial species using the respiratory tract as port of entry.

Stimulation of the adherence of Haemophilus influenzae to human lung epithelial cells by antimicrobial neutrophil defensins.

Patients with chronic obstructive pulmonary disease (COPD) frequently have recurrent lower respiratory tract infections with nonencapsulated Haemophilus influenzae. The infected mucosa of these patients is infiltrated with neutrophils, which upon activation may release antimicrobial peptides, including defensins. It was shown that defensins isolated from neutrophils or from sputum samples of COPD patients did not kill H. influenzae from these patients, but they did stimulate its adherence to human bronchial epithelial cells in a time- and dose-dependent manner. Maximal stimulation was observed after 3 h in the presence of > or = 10 micrograms/mL defensins, resulting in 65 +/- 36 cfu/cell (61-fold increase). The enhanced adherence was not solely due to charge effects and was specifically blocked by alpha 1-proteinase inhibitor. Because adherence is the first step in the onset of respiratory tract infections, our findings indicate that neutrophil defensins likely contribute to the pathogenesis of H. influenzae infection in the lower respiratory tract.