Development and distribution of B lineage cells in the domestic cat: analysis with monoclonal antibodies to cat mu-, gamma-, kappa-, and lambda-chains and heterologous anti-alpha antibodies

To trace the development and distribution of B lineage cells in the domestic cat (Felis catus), we have produced monoclonal antibodies against mu-, gamma-, kappa-, and lambda-chains of feline immunoglobulins (Ig). Goat antibodies against human mu-, alpha-, and lambda-chains, which are reactive with shared determinants on their feline counterparts, were used in conjunction with the panel of mouse monoclonal antibodies. Cytoplasmic mu+ pre-B cells and surface IgM+ B lymphocytes were observed in 42 day fetal liver in which pre-B cells were more abundant than IgM+ B cells. Pre-B cells also were found in bone marrow in young cats, and continued to be generated in the marrow throughout life. In the spleen, adult levels of B cells were attained by 12 wk of age, at which time the frequencies of surface IgM+, IgG+, and lambda+ cells were 49, 3, and 40%, respectively. The distributions of Ig isotypes also were determined among plasma cells as a function of age and tissue localization. IgM plasma cells were predominant in the bone marrow of 1-wk-old cats, whereas IgG plasma cells were the prevalent isotype in adult bone marrow. In the mesenteric lymph nodes of adult animals, the frequency distributions of IgM, IgG, and IgA plasma cells were similar to the frequency distributions of IgM, IgG, and IgA isotypes among bone marrow plasma cells. IgA+ plasma cells predominated in the intestinal lamina propria, in which IgG+ and IgM+ plasma cells were relatively infrequent. In the tissues of both young and adult animals, the ratio of lambda:kappa expression was approximately 3:1. We conclude that the pattern of B cell development in the cat resembles that found in other mammals, except that the kappa to lambda ratio is reversed.     

Extraintestinal pathogenic isolates of Escherichia coli do not possess active IgA1, IgA2, sIgA or IgG proteases.

Infections outside of the intestinal tract due to pathogenic strains of Escherichia coli result in significant morbidity, mortality and increased healthcare costs. The ability of these strains to cause both mucosal and systemic infections, as well as recurrent infections due to the same (homologous) strain suggests the hypothesis that strains of E. coli that cause infection outside of the intestinal tract possess proteases that are capable of cleaving IgA1, IgA2, sIgA or IgG. To test this hypothesis the ability of eight E. coli strains, isolated from sites outside of the urinary tract and 14 homologous and 11 heterologous strains of E. coli that were isolated from women with recurrent UTI, to cleave IgA1, IgA2, sIgA or IgG was evaluated. Our experimental design allowed for detection of cell-associated and secreted immunoglobulin proteases in both log and stationary phase. Surprisingly, none of these 33 human clinical isolates when grown in iron depleted Luria-Bertani medium or human urine were able to degrade the immunoglobulins assessed. Despite previous studies suggesting otherwise, the findings from this study support the concept that strains of E. coli that cause infection outside of the intestinal tract do not possess proteases that cleave the human immunoglobulins IgA1, IgA2, sIgA or IgG.     

Generalized systemic and mucosal immunity in mice after mucosal stimulation with cholera toxin

Cholera toxin (CT) has been found to be an extremely potent immunogen for mucosal IgA responses when administered via the intestine. This study has examined both mucosal and systemic immune responses after feeding CT and compared these responses with those obtained after feeding keyhole limpet hemocyanin (KLH), another protein that is strongly immunogenic in mice. Feeding CT to mice resulted not only in IgA antibody in intestinal secretions but also resulted in substantial plasma IgG and IgA antibody levels. Feeding KLH in much larger quantity resulted in little or no antibody response in intestinal secretions or plasma. Lymphoid cells from various tissues of mice fed CT were cultured in vitro for 10 days and the supernatant was tested for antibody to CT. Spontaneous antibody synthesis (no antigen added to cultures) was present in cultures of each cell type, but IgG anti-CT was found mainly in cultures of spleen and mesenteric lymph node cells and IgA anti-CT mainly in cultures of Peyer's patch and lamina propria cells. Peyer's patch cells cultured with CT as antigen synthesized both IgG and IgA anti-CT, suggesting that the antibody response to both isotypes originated in this site. Helper T cell activity for both IgA and IgG anti-CT was detected in spleens, mesenteric lymph nodes, and Peyer's patches. Lastly, when KLH and CT were fed to mice at the same time, an intestinal IgA anti-KLH and plasma IgG anti-KLH response was stimulated, a response pattern similar to that occurring to CT after CT was fed alone. We conclude that mucosal stimulation by CT generates both a systemic IgG and mucosal IgA response to this antigen, and that CT can cause a similar pattern of response to an unrelated protein antigen when both are administered into the intestine at the same time. The data favor the idea that both the IgG and IgA responses originate in GALT and then disseminate to other tissues. We propose that CT accomplishes these effects by altering the regulatory environment within GALT.     

Dynamics of the accumulation of the basic immunoglobulin classes in the intestinal contents in food toxinfections]

The work presents the data on the dynamics of accululation of the main classes of immunoglobulins (A, G and M) in coprofiltrates obtained from patients with alimentary toxicoinfections (bacteriologically confirmed salmonellosis and diseases of unknown etiology). The levels of immunoglobulins of all classes (mainly IgA and IgG) were shown to be elevated in the process of the disease. The dynamics of the increase in the level of IgA (both general and secretory) was supposed to indicate the formation of local immunity in the intestinal wall. The presence of serum IgA and the characteristic IgG dynamics seemed to be indicative of destructive processes occurring in the intestinal wall. Thus, the dynamics of accumulation of immunoglobulins in coprofiltrates obtained from patients with alimentary toxicoinfections reflects the main local pathological and immunological processes.     

Antibodies in the small intestine: mucosal synthesis and deposition of anti-glycosyl IgA, IgM, and IgG in the enterocyte brush border

Synthesis and deposition of immunoglobulins in the brush border was studied in organ-cultured pig small intestinal mucosal explants. Surprisingly, comparable amounts of IgM and IgA were synthesized during a 6-h pulse, and also newly made IgG was detected in media and explants, including the microvillar fraction. For IgA and IgM, this subcellular distribution is consistent with basolateral-to-apical transcytosis, mediated by the polymeric immunoglobulin receptor. IgG is a ligand for the Fc receptor FcRn, and beta2-microglobulin, the light chain of FcRn, coclustered in immunogold double labeling with IgG in subapical endosomes and in the basolateral membrane of enterocytes. In addition, beta2-microglobulin was copurified with IgG on protein G-Sepharose. Apical endocytosis of IgG, as judged by internalization of fluorescent protein G, was not detectable except in a few isolated cells. This suggests that IgG in the adult small intestine is transported across the enterocyte mainly in the basolateral to apical direction. Significant fractions of all immunoglobulins bound to lactoseagarose, indicating that "anti-glycosyl" antibodies, raised against commensal gut bacteria, are synthesized locally in the small intestine. By partial deposition in the brush border, these antibodies therefore may have a protective function by preventing lectin-like pathogens from gaining access to the brush border surface.     

Immunohistochemical localization of immunoglobulins A, G and M in the mouse female genital tract

The localization of immunoglobulins A, G and M (IgA, IgG, IgM) in the mouse genital tract was studied by immunoperoxidase techniques at oestrus, on the day of mating and at the time of implantation. In the horns and body of the uterus, IgA and IgG were located in plasma cells in the endometrium surrounding uterine glands and in the gland lumina. The numbers of these plasma cells increased markedly between Day 1 and Days 4 and 5 of pregnancy and the ratio of plasma cells containing IgA and IgG was about 3 or 4 to 1 at all stages. Area measurements indicated that the increased number of plasmacytes was not due to an increase in the amount of endometrial, myometrial or glandular tissues. Plasma cells were not detected in the cervix and vaginal fornix at oestrus and Day 1, but a few were present on Day 5. In the oviduct, plasma cells containing IgA and IgG were present only in the preampulla and both immunoglobulins were present in the extracellular space of the lamina propria only in this region. No IgM was detected in any part of the reproductive tract at any of the times studied. Uteri on Day 1 of pregnancy contained bacteria of several kinds, some of which were aggregated and coated with IgA. This suggests that the uterine lumen at this time may contain specific anti-bacterial IgA antibodies. Our observations indicate that the horns and body of the uterus and the preampulla of the oviduct are major sites of a local immune system in the female mouse genital tract.     

Lymphotoxin Alpha-Deficient Mice Clear Persistent Rotavirus Infection after Local Generation of Mucosal IgA

Rotavirus is a major cause of pediatric diarrheal illness worldwide. To explore the role of organized intestinal lymphoid tissues in infection by and immunity to rotavirus, lymphotoxin alpha-deficient (LTα^−/−) mice that lack Peyer''s patches and mesenteric lymph nodes were orally infected with murine rotavirus. Systemic rotavirus was cleared within 10 days in both LTα^−/− and wild-type mice, and both strains developed early and sustained serum antirotavirus antibody responses. However, unlike wild-type mice, which resolved the intestinal infection within 10 days, LTα^−/− mice shed fecal virus for approximately 50 days after inoculation. The resolution of fecal virus shedding occurred concurrently with induction of intestinal rotavirus-specific IgA in both mouse strains. Induction of intestinal rotavirus-specific IgA in LTα^−/− mice correlated with the (late) appearance of IgA-producing plasma cells in the small intestine. This, together with the absence of rotavirus-specific serum IgA, implies that secretory rotavirus-specific IgA was produced locally. These findings indicate that serum IgG responses are insufficient and imply that local intestinal IgA responses are important for the clearance of rotavirus from intestinal tissues. Furthermore, they show that while LTα-dependent lymphoid tissues are important for the generation of IgA-producing B cells in the intestine, they are not absolutely required in the setting of rotavirus infection. Moreover, the induction of local IgA-producing B cell responses can occur late after infection and in an LTα-independent manner.     

Evolution of mouse intestinal and blood immunoglobulins after oral route vaccination]

The authors have studied the mice immunoglobulins level after vaccination by oral route with a killed-pathogenic strain of Salmonella typhimurium and an avirulent mutant of the same bacteria. The obtained results show an increase of the intestinal IgA and IgG1 levels and a slighter one of sera IgG between the 10 th and 30 th day following immunization. No correlation was observed concerning the IgM, IgG and IgA levels and the mice protection against a challenge of pathogenic bacteria.     

An immunohistochemical study of cells with surface and cytoplasmic immunoglobulins in situ in Peyer's patches and lamina propria of rat small intestine

The distribution of cells with surface and cytoplasmic immunoglobulins was studied in Peyer's patches (PP) and intestine of rats, using both frozen and paraffin sections, with a two-step peroxidase technique. Anti IgM, IgG, IgA and IgE sera were used. Surface staining was found within PP with all antisera used. Although the villi contained predominantly IgA plasma cells (PC), IgG PC and a few IgM and IgE PC were also found. Within PP, however, no IgA PC were found but IgM and IgG PC were present in all stages of development, mainly in the dome. PC of all types, but mostly IgA cells, were present in and around high endothelial venules (HEV). The results suggest that IgM and IgG PC precursors can develop to PC within PP whereas IgA precursors do not. PC appear to home to the gut preferentially via HEV.     

VP4-specific intestinal antibody response to rotavirus in a murine model of heterotypic infection.

We have adapted a murine model of heterotypic rotavirus infection for the purpose of evaluating the intestinal antibody response to an infection that mimics human vaccination. Neonatal mice were infected with the rhesus rotavirus (RRV). The enzyme-linked immunospot assay was used in order to avoid common artifacts in the quantitation of intestinal immune responses inherent in measurements of luminal or serum immunoglobulins and to obtain easily quantifiable data in a flexible and convenient format. Functionally active lymphocytes were harvested from the spleen, small intestinal lamina propria, Peyer's patches, and mesenteric lymph nodes and processed into single-cell suspensions. Antibody-secreting cells (ASC) were quantitated from 5 to 50 days after infection for total, RRV-specific, baculovirus-expressed VP4-specific, and single-shell RRV-specific ASC secreting either immunoglobulin G (IgG), IgM, or IgA. The response to VP4 constituted less than 1.5% of the total virus-specific response, which was located almost exclusively in the gut and was 90% IgA. Intestinal ASC were directed overwhelmingly toward proteins incorporated in the single-shell particle, predominantly VP2 and VP6. We conclude that the antibody response to VP4, thought to be the site of the important neutralization sites conserved among several rotavirus serotypes, is an extremely small portion of the overall antibody response in the intestinal tract.     

Lymphocyte recirculation and the gut: the cellular basis of humoral immunity in the intestine

During the last 15 years evidence has accumulated which shows that the B immunoblasts generated in the gut associated lymphoid tissue (GALT) in response to antigenic stimuli from the intestinal tract are discharged into the intestinal lymph. The lymph stream carries them to the blood but most of them soon extravasate in the gut (and some other mucosae) and develop into plasma cells which synthesise secretory immunoglobulins. In sheep, there is some evidence that these cells may be derived initially from a sub-set of small lymphocytes which circulate preferentially through the GALT, but the situation in rodents is less clear. However, in most species it is becoming clear that although some of the antibodies produced by the sub-mucosal plasma cells are secreted directly into the lumen of the gut many find their way, via the lymph, into the blood and, if in the form of polymeric IgA, they are rapidly and actively transported by the hepatocytes into the bile and thus gain direct access to the duodenum in concert with the entry of freshly ingested food and bacteria.     

Organ and isotype distribution of plasma cells producing specific antibody after oral immunization: evidence for a generalized secretory immune system.

Mice were induced to produce IgA antibodies against ferritin after oral immunization. Such antibodies were detected by immunofluorescence in plasma cells in the intestinal mucosa as well as in secretory sites located elsewhere, such as the lactating mammary gland, salivary gland, and respiratory tract. The observation suggested that cells immunized locally via the gut could home to distant secretory sites. To confirm this hypothesis, lymphocyte transfer studies were done with mesenteric node (MN) versus peripheral node (PN) cells from orally immunized donors into nonimmunized recipients. IgA anti-ferritin cells from MN homed to exocrine targets, whereas IgM and IgG anti-ferritin cells homed to PN. The findings overall support the concept of a generalized and interrelated secretory immune system.     

Differential effects of vasoactive intestinal peptide, substance P, and somatostatin on immunoglobulin synthesis and proliferations by lymphocytes from Peyer's patches, mesenteric lymph nodes, and spleen

We examined the effect of vasoactive intestinal peptide, substance P, and somatostatin on concanavalin A (1 microgram/ml)-induced lymphocyte proliferation and immunoglobulin (IgA, IgM, and IgG) synthesis by cells from spleens, Peyer's patches, and mesenteric lymph nodes. These neuropeptides (10(-7) to 10(-12) M) modulated immune responses in a dose-dependent manner. For a comparative study, neuropeptides were used at 10(-8) M concentration. Both vasoactive intestinal peptide and somatostatin significantly decreased DNA synthesis (30 to 50%), whereas substance P increased synthesis (40%) in lymphocytes from all organs tested. IgA synthesis was significantly altered by all of the neuropeptides tested, whereas IgM synthesis was less affected and IgG synthesis was virtually unchanged. Somatostatin inhibited IgA (20 to 50%) and IgM (10 to 30%) synthesis in lymphocytes from all three organs. Substance P increased IgA synthesis in mesenteric lymph nodes (50%), spleens (70%), and Peyer's patches (300%). It also increased IgM synthesis in Peyer's patches (20%) and spleens (30%), but was without effect on IgM synthesis in mesenteric lymph nodes. Vasoactive intestinal peptide increased the IgA response in mesenteric lymph nodes (20%) and spleens (30%), but inhibited IgA synthesis in lymphocytes from Peyer's patches (60%). Interestingly, in Peyer's patches, IgM synthesis was increased by vasoactive intestinal peptide (80%), whereas it was unchanged in mesenteric lymph nodes and spleen. Thus, not only did these neuropeptides have different effects on the production of different immunoglobulin isotypes, but their effect was also organ-specific. Because neuropeptides which are abundant in the intestine can modulate IgA and other immunoglobulin synthesis in vitro, they may play a significant regulatory role in mucosal immune responses in vivo.     

The role of immune pig colostrum,serum and immunoglobulins IgG,IgM, and IgA in local intestinal immunity against enterotoxic strain ofEscherichia coli 055 in germfree piglets

The protective effect of pig immune colostrum, serum and immunoglobulins IgG, IgM and IgA against the enterotoxic strain ofEscherichia coli O55, was studied in newborn germfree piglets. This strain produced accumulation of fluid and dilatation of intestine when injected into the ligated ileal segment of germfree piglets, which is considered to be the typical effect of enterotoxins. Erosion of the intestinal epithelium and penetration of bacteria into the submucosa were also observed. Immune serum, colostrum and all the immunoglobulin classes used produced a local protective effect, IgA being most effective. The mechanism of protection conferred by these immunoglobulins is discussed with respect to the possible pathogenic action of enterotoxicEscherichia coli O55 in the intestinal tract of immunologically virgin germfree piglets.     

Selective chemotaxis of subsets of B lymphocytes from gut-associated lymphoid tissue and its implications for the recruitment of mucosal plasma cells

As they differentiate, precursor cells from the gut-associated lymphoid tissue are known to travel via the lymphatic system to the blood and then preferentially to home to various mucosal and exocrine sites such as the lamina propria of the gut and the lactating mammary gland, where they give rise to IgA-secreting plasma cells. The present study, directed at the mechanism by which the circulating precursors of mucosal IgA plasma cells selectively lodge in characteristic locations, explored the hypothesis that such homing is due to a locally produced chemotactic factor and that milk might be a source of such a factor. Subsets of lymphocytes bearing particular surface markers and purified by panning from lymph nodes of mice were examined in a micropore chemotaxis assay to search for the presence of chemotactic activity in mouse milk. The globulin fraction of whey was shown to contain a nondialyzable factor that is chemotactic for IgA (and also IgG)-positive lymphocytes when these are obtained from mesenteric lymph nodes as a source of mucosal-associated lymphoid tissue. Lymphocytes from peripheral lymph nodes, nonmucosal associated, were unaffected as were surface IgM-positive lymphocytes and T lymphocytes obtained from mesenteric nodes. Chemotactic activity for IgA lymphocytes was undetectable in mouse serum. The data are consistent with the idea that precursors of mucosal IgA plasma cells home to mucosal and exocrine sites in response to a specific chemotactic factor elaborated by local differentiated epithelial cells.     

An immunofluorescent study of lung, hilar lymph nodes and spleen in a series of autopsied cases

Direct immunofluorescence reactions were performed with conjugated antihuman IgA, IgG, and IgM sera on bronchi, lung, lymph nodes and spleen, sampled at 12-72 h after death in a series of 20 cases submitted to autopsy. The IgA cells were preponderent in the lung where they concentrate preferentially in the bronchial tree, whereas the IgG and IgM cells were found in a relative higher proportion in the pulmonary parenchyma. The most intense lung immunofluorescence reactions were identified in pneumonias; in relation of these types they acquire distinct aspects: a bronchio-peribronchial disposition in bronchopneumonia and an interstitial-perivascular one in plasma cell pneumonitis. The lymph nodes and spleen of the same cases presented mostly IgG cells, but also, in a smaller amount, IgA and IgM plasma cells.     

Peculiar secretory IgA system identified in chickens. II. Identification and distribution of free secretory component and immunoglobulins of IgA, IgM, and IgG in chicken external secretions.

A homologue of a free secretory component (SC) was identified in chicken intestinal secretion by criteria based on its antigenic relationship with intestinal secretory IgA (SIgA), molecular size, sugar content, and electrophoretic mobility, as well as its elution characteristic from ion-exchange chromatography. SC was obtained in a form free from IgA from the intestinal secretion by salting out and DEAE chromatography, followed by density ultracentrifuguation or Sephadex G-200 gel-filtration. However, the free SC revealed some antigenic deficiency when compared to bound SC of intestinal SIgA and showed a failure of binding to serum-type-polymeric IgA of biliary IgA in vitro. Several kinds of chicken external secretions were examined for detection of SC and immunoglobulin classes of IgG, IgA, and IgM. In spite of the wide distribution of immunoglobulins in the external secretions, SC antigen could be detected only in intestinal secretion. Most IgA in the secretions had a molecular structure of a tetramer of serum-type IgA, lacking in SC and having 17S to 18.5S and 600,000 to 700,000 daltons. On the other hand, IgA in the intestinal secretion showed close similarity to the mammalian SIgA, associated with SC and having 11.2S and 350,000 daltons. Presence of antibody activity in the intestinal IgA to avian reovirus was confirmed by plaque reduction tests.     

Cellular and humoral immunodeficiency in children with vaccine-associated paralytic poliomyelitis

Markers of humoral and cellular immunity in 16 patients with vaccine-associated paralytic poliomyelitis (VAPP) were evaluated. Signs of immunodeficiency (decrease of T- and B-lymphocytes counts, impaired synthesis of immunoglobulins, defects of phagocytosis, decrease of NK number) were revealed in all of the patients. Majority of them (81.3%) had defects in humoral immunity. Decrease of CD31, CD4+ and CD8+ was detected in 86.7, 35.7 and 91.7% of the patients respectively. Study of serum immunoglobulins performed in 15 patients showed decrease of IgG, IgM and IgA levels in 6 (40%), 1 (6.7%) and 6 (40%) of the patients respectively. Agammaglobulinemia was diagnosed in one patient in which only trace quantities of IgA and IgG were detected and IgM level was well below the normal. Congenital deficiency of IgA was diagnosed in 3 children. Majority of the children (11 from 12) had comorbidities (frequent respiratory infections, dermatitis, changes of intestinal microflora). Thus, immunocompromised condition of a child is a risk factor for VAPP after administration of alive oral poliovaccine.     

Immunoglobulins in the mouse uterus during the oestrous cycle

The distribution of IgA, IgG and IgM was studied by an immunoperoxidase technique on sections of mouse uteri at each stage of the oestrous cycle. Staining for IgG and IgA was highest at pro-oestrus, declined at oestrus and was very low during the other stages. At pro-oestrus IgG was found throughout the stroma, in the uterine lumen, and in 10% of glandular lumina; very few IgG-containing plasma cells were present. At pro-oestrus, IgA was found in the uterine lumen, and in most of the uterine glands, both in the lumen and in the epithelium; little IgA was present in the stroma. IgA-plasma cells were detected at each stage of the cycle and were particularly numerous at pro-oestrus and oestrus. These results suggest that IgA is secreted locally from plasma cells into the uterine gland through the glandular epithelium, but that IgG enters the stroma from the local capillaries. The obvious increase in IgG and IgA secretion at pro-oestrus, when plasma oestradiol levels are highest, supports the hypothesis that, during the oestrous cycle, the humoral immune response is regulated in the uterus by ovarian hormones.     

Hymenolepis citelli, H. diminuta and H. microstoma: immunoglobulin-containing cells in the lamina propria of the mouse gut during primary and secondary infections

The indirect immunofluorescent technique was used to determine the occurrence of IgA, IgM and IgG1 immunoglobulin-containing cells in local intestinal mucosal immune responses to Hymenolepis citelli, H. diminuta and H. microstoma infections in mice. In the intestinal lamina propria of H. citelli and H. diminuta infected mice there was no increase in the mean numbers of immunoglobulin-containing cells when compared with uninfected control mice, but there was in H. microstoma infected mice. The numbers of IgG1- positive cells in both infected and uninfected mice were very small relative to IgA and IgM immunocytes. The distribution of immunocytes in the lamina propria of infected and uninfected mice was essentially similar and the localization of isotypes in duodenal sections showed no immunoglobulins in the villous epithelial cells. There was also no marked difference between primary and secondary infections indicating that immunoglobulin-containing cells play no major role in functional immunity against hymenolepid infections in the mouse. The presence of IgA and IgM was also demonstrated on the tegument of the tapeworms, although the distribution was patchy and more abundant on H. microstoma than on H. diminuta or H. citelli. The time of appearance of both isotypes was latest on H. citelli.