Immunoglobulins in nasal secretions of healthy humans: structural integrity of secretory immunoglobulin A1 (IgA1) and occurrence of neutralizing antibodies to IgA1 proteases of nasal bacteria

Abstract

Certain bacteria, including overt pathogens as well as commensals, produce immunoglobulin A1 (IgA1) proteases. By cleaving IgA1, including secretory IgA1, in the hinge region, these enzymes may interfere with the barrier functions of mucosal IgA antibodies, as indicated by experiments in vitro. Previous studies have suggested that cleavage of IgA1 in nasal secretions may be associated with the development and perpetuation of atopic disease. To clarify the potential effect of IgA1 protease-producing bacteria in the nasal cavity, we have analyzed immunoglobulin isotypes in nasal secretions of 11 healthy humans, with a focus on IgA, and at the same time have characterized and quantified IgA1 protease-producing bacteria in the nasal flora of the subjects. Samples in the form of nasal wash were collected by using a washing liquid that contained lithium as an internal reference. Dilution factors and, subsequently, concentrations in undiluted secretions could thereby be calculated. IgA, mainly in the secretory form, was found by enzyme-linked immunosorbent assay to be the dominant isotype in all subjects, and the vast majority of IgA (median, 91%) was of the A1 subclass, corroborating results of previous analyses at the level of immunoglobulin-producing cells. Levels of serum-type immunoglobulins were low, except for four subjects in whom levels of IgG corresponded to 20 to 66% of total IgA. Cumulative levels of IgA, IgG, and IgM in undiluted secretions ranged from 260 to 2,494 (median, 777) microg ml(-1). IgA1 protease-producing bacteria (Haemophilus influenzae, Streptococcus pneumoniae, or Streptococcus mitis biovar 1) were isolated from the nasal cavities of seven subjects at 2.1 x 10(3) to 7.2 x 10(6) CFU per ml of undiluted secretion, corresponding to 0.2 to 99.6% of the flora. Nevertheless, alpha-chain fragments characteristic of IgA1 protease activity were not detected in secretions from any subject by immunoblotting. Neutralizing antibodies to IgA1 proteases of autologous isolates were detected in secretions from five of the seven subjects but not in those from two subjects harboring IgA1 protease-producing S. mitis biovar 1. alpha-chain fragments different from Fc(alpha) and Fd(alpha) were detected in some samples, possibly reflecting nonspecific proteolytic activity of microbial or host origin. These results add to previous evidence for a role of secretory immunity in the defense of the nasal mucosa but do not help identify conditions under which bacterial IgA1 proteases may interfere with this defense.

FIG. 1

Size exclusion chromatography profiles of IgA in a sample of nasal wash from subject B (sample B_3,1 [Tables 2 and 3]). (A) OD profiles obtained by analysis of eluent fractions in ELISA for total IgA (▴) and S-IgA only (●). Distinct peaks of S-IgA and monomeric serum IgA were identified immunochemically and by reference to column calibration standards. Elution volumes for standards of 11S S-IgA and monomeric (7S) serum IgA are indicated. (B) Plot of IgA concentrations determined by titration of eluent fractions, using, within each peak, the relevant ELISA. By planimetry, S-IgA was found to account for 86% of total IgA in the sample.

FIG. 2

Selected immunoblots from three adults (subjects E, F, and G) and a child (subject I), illustrating the patterns of anti-α-chain antibody-reactive bands obtained from samples of nasal secretions. Samples obtained by different methods from individual adults were collected during one day. The dilutions of individual samples in reducing sample buffer prior to analysis are indicated. Note that all samples display a prominent band corresponding to intact α chain (M_r, ∼58K). For interpretation of other bands, see the text. Lane 9 represents sample I_w,3 from the child, from which H. influenzae was cultured (Table 3). Numbers on the left indicate molecular weight standards in kilodaltons.

FIG. 3

Immunoblots providing evidence for the lack of detectable IgA1 protease-induced IgA1 fragments in nasal secretions from subject B (A) and subject C (B), harboring IgA1 protease-producing S. mitis biovar 1 and H. influenzae, respectively. Blots of synchronous samples obtained by the three methods (lanes 1, 2, 3) are shown together with blots of aliquots of the method 3 sample incubated (incub.) with various amounts of homologous IgA1 protease (lanes 5 to 9) or with buffer (lanes 4). Note that faint bands displayed by secretion samples do not correspond to IgA1 protease-induced α-chain fragments. Numbers on the left indicate molecular weight markers in kilodaltons.

FIG. 4

Inhibition titers against the IgA1 protease of an autologous H. influenzae isolate relative to total immunoglobulin concentration in sequential nasal wash samples from a 9-year-old child. H. influenzae was isolated from one sample (hatched bar) only.