Intestinal immune responses in humans. Oral cholera vaccination induces strong intestinal antibody responses and interferon-gamma production and evokes local immunological memory.

We have examined secretory antibody and cell-mediated immune responses to oral cholera vaccine in the human gastrointestinal mucosa. Freshly isolated peripheral blood lymphocytes and intestinal lymphocytes obtained by enzymatic dispersion of duodenal biopsies were assayed for numbers of total and vaccine specific immunoglobulin-secreting cells by enzyme-linked immunospot assay (ELISPOT) techniques; the frequency of cells secreting interferon-gamma (IFN-gamma) was also examined by a new modification of the ELISPOT technique. After booster immunizations with oral cholera vaccine, large numbers of cholera toxin-specific antibody-secreting cells (ASC) appeared in the small intestine. The responses were dominated by IgA ASC. A single immunization, performed 5 mo after the initial vaccinations, gave rise to an ASC response similar to that seen after the first booster immunization, with respect to both magnitude and isotype distribution. Each of the immunizations also evoked an ASC response in blood which was of lower magnitude than that seen in the small intestine, and comprised similar proportions of IgA and IgG ASC. A booster immunization also resulted in increased frequencies of IFN-gamma-secreting cells, but this increase was confined to the duodenal mucosa. This study establishes the feasibility of studying, at the single-cell level, intestinal immune reactivity in humans. Furthermore, it indicates that the small intestinal mucosa is an enriched source of IFN-gamma. It also demonstrates marked differences between intestinal and peripheral blood immune responses after enteric immunization, and confirms the notion that the mucosal immune system in humans displays immunological memory.

Effects of the two partial D2 agonists (+)- and (-)-3-PPP on prolactin release in EEDQ treated male rats.

The effects of pretreatment with the irreversible inactivator of brain D2 receptors N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ) on the suppression of prolactin (PRL) release induced by the two partial D2 agonists (+)- and (-)-3-(3-hydroxyphenyl)-N-n-propyl-piperidine [(+)-3-PPP, (-)-3-PPP] were investigated in gamma-butyrolactone (GBL) treated male rats. Pretreatment with a high dose of EEDQ (20 mg/kg, 24 h) did not influence the PRL response to (+)-3-PPP. In contrast, the effect of (-)-3-PPP was dose-dependently antagonized by EEDQ administration; thus, while pretreatment with EEDQ 2 mg/kg (24 h) failed to influence the efficacy of (-)-3-PPP, a complete antagonism of the PRL suppressive effect of the (-) enantiomer was obtained by administration of EEDQ 16 or 20 mg/kg (24 h). Moreover, in EEDQ (20 mg/kg, 24 h) treated rats (-)-3-PPP effectively antagonized the PRL inhibiting effect of the (+) enantiomer. Also, in EEDQ (20 mg/kg, 24 h) treated animals not receiving GBL (-)-3-PPP induced a dose-dependent increase in plasma levels of PRL. The data indicate that higher doses of EEDQ are required in order to reduce the responsiveness of lactotroph D2 receptors than that of various populations of brain D2 receptors. Also, the data lend support for the assumption that an altered receptor responsiveness may dramatically modify the response to a partial agonist with low intrinsic efficacy without affecting the response to a partial agonist with higher intrinsic efficacy.

Phenotypic characterization of circulating antibody-secreting cells after mucosal and systemic immunizations in humans.

We have developed a simple approach for immunophenotyping of functional lymphoid cell subpopulations. Using this approach we have partly characterized circulating antigen-specific ASC after systemic versus mucosal immunization with respect to maturational and activation stages, and to their anatomic commitment(s). Comparative analyses of mucosally versus systemically activated circulating ASC reveal differences with respect not only to utilization of homing receptor molecules but also to certain activation markers, and especially cell surface MHC class II molecules. We have now extended these studies to several other markers including early and late B-cell maturation markers, and we are currently examining whether differential expression of HLA-DQ molecules on the majority of mucosally activated blood ASC may explain the relative inability of these cells to present soluble antigens to class II-restricted T-cells.

Induction of specific immunity at mucosal surfaces: prospects for vaccine development.

We have demonstrated the feasibility of studying antigen-specific immune responses in a variety of mucosal tissues in humans after vaccination and during infection. In this respect, we have documented the usefulness of both oral cholera and ETEC vaccines for assessing in functional terms specific subpopulations of B- and T-cell immunocytes during an immune response initiated and/or expressed in human mucosal tissues. Circulating specific IgA antibody-secreting cells in blood appear to reflect recent or ongoing antigen exposure of mucosal surfaces. This implies that the detection of such cells in blood, the most accessible lymphoid compartment in humans, represents the simplest way to assess the immunogenicity of mucosal vaccines and to supplement the diagnostic and monitoring of active mucosal infections. Our studies indicate that while the concept of an integrated mucosal immune network is clearly operational in humans (at least in regards to induction of secretory antibody responses), its generalization appears somewhat simplistic as illustrated by the compartmentalization of immune responses initiated in certain mucosal organs such as the small intestine and the tonsils. Finally, the potential of the cholera toxin B subunit as a carrier for delivery of chemically or genetically linked foreign epitopes for induction of disseminated mucosal immune responses raises hope for the development of broadly applicable vaccines to control mucosal infections.

High frequency of spontaneous interferon-gamma-producing cells in human tonsils: role of local accessory cells and soluble factors.

The frequency of mononuclear cells (MNC) spontaneously secreting interferon-gamma (IFN-gamma) has been examined in freshly isolated cell suspensions from human palatine tonsils. Two-site reverse enzyme-linked immunospot (ELISPOT) analyses, involving short term (20 h) incubation of MNC in the absence of any added exogenous stimulus, revealed that tonsillar MNC suspensions contain exceptionally large numbers of cells secreting IFN-gamma. No significant differences were observed when comparing the frequency of IFN-gamma-producing cells between cell suspensions obtained from hyperplastic and tonsillitis specimens. Cell-sorting experiments disclosed that spontaneous tonsillar IFN-gamma production was essentially contributed by CD4+ T cells, and required the presence of accessory cells and/or soluble factors to be detected. Thus, depletion of plastic adherent cells or monocytes from the tonsillar MNC suspensions resulted in reduced numbers of detectable IFN-gamma-secreting cells. Addition of very small numbers of autologous monocytes restored spontaneous IFN-gamma production in tonsillar MNC cultures depleted of monocytes. Neutralization of endogenous IL-1 beta and IL-2, as well as blocking of the IL-2 receptor, also decreased IFN-gamma production from unfractionated tonsillar cells. Addition of exogenous IL-1 beta restored IFN-gamma production in cultures of tonsillar MNC depleted of plastic adherent cells. Furthermore, IL-1 beta synergized with IL-2 by tonsillar MNC depleted of plastic adherent cells. Furthermore, IL-1 beta synergized with IL-2 by increasing intracellular as well as cell-free levels of IFN-gamma in cultures of unfractionated tonsillar MNC. This study further establishes that the tonsils are highly active immunological organs containing large numbers of T cells spontaneously producing IFN-gamma whose detection is contingent upon the presence of functional accessory cells. It also demonstrates that concomitant production of IL-1 beta and IL-2 occurs in tonsils and is necessary to maintain ongoing synthesis and extracellular accumulation of IFN-gamma in these organs.

Antibody-producing cells in peripheral blood and salivary glands after oral cholera vaccination of humans.

We examined whether immunization with a newly developed oral cholera vaccine would elicit gut-derived antibody-producing cells in the blood and in distant mucosal tissues, such as the minor salivary glands, in 30 adult Swedish volunteers. The results of this study demonstrated that this vaccine indeed induced production of specific antibody-producing cells against the cholera toxin B subunit in both peripheral blood and salivary glands. The response in blood, which after primary and booster immunizations comprised both immunoglobulin A (IgA) and IgG antibody-forming cells, was highly transient and preceded the response in salivary glands; the latter response was restricted to the IgA isotype. The results provide further evidence of the existence of a common mucosal immune system in humans. Furthermore, these findings support previous observations that in animals, the cholera toxin B subunit may be a useful carrier protein for preparing enteric vaccines against pathogens encountered at intestinal and extraintestinal mucosal sites.

Intestinal and circulating antibody-forming cells in IgA-deficient individuals after oral cholera vaccination.

In search for a possible explanation for the different susceptibility to mucosal infections in IgA-deficient (IgAd) individuals, the frequency of total immunoglobulin-secreting cells (ISC) and vaccine-specific antibody-secreting cells (ASC) in intestinal mucosa and peripheral blood was determined by the enzyme-linked immunospot (ELISPOT) assay before and after peroral vaccination with a B subunit-whole cell cholera vaccine. Two groups of IgAd individuals, frequently infected and non-infected respectively, and normal controls were studied. Before cholera vaccination there were significantly higher frequencies of total IgM and IgG ISC in the gut, but not in the blood, in the IgAd individuals than in the controls. However, there were no significant differences between healthy and infection-prone IgAd individuals in this respect. In response to oral cholera vaccination, intestinal cholera toxin (CT)-specific IgG and IgM ASC were significantly more abundant among the IgAd individuals with a history of frequent infections than among the healthy IgAd individuals and controls. A similar difference in IgG and IgM ASC, although not significant, was also noted in blood. In IgAd individuals with frequent infections the vaccine induced variable anti-CT IgM ASC responses in the gut, ranging from no increase to a few strikingly high responses. In the controls, the CT-specific responses were dominated by IgA ASC. The data show that oral cholera vaccination evoked strong CT-specific IgG ASC responses, and in some cases also strong IgM ASC responses in the intestinal mucosa of IgAd patients with a history of frequent infections. The healthy IgAd individuals unexpectedly responded with lower numbers of CT-specific IgG ASC and did not show any increase of CT-specific IgM ASC in the intestinal mucosa. Thus, inability to mount a mucosal immune response to an oral antigen cannot in itself explain recurrent infections among many IgAd individuals.