On the 25th of July, disheartening news of the untimely demise of our distinguished colleague and dear friend, John Bienenstock, reached the immunological community and was profoundly felt by mucosal immunologists. Undoubtedly, Dr. Bienenstock is one of the universally acknowledged founders of the discipline of mucosal immunology, together with several current colleagues and the recently deceased Drs. Per Brandtzaeg and Lars Ǻ Hanson. Recognition of the mucosal immune system as an integral, and perhaps the dominant component, of the entire immune system is based on quantitative studies of B and T cells as well as antibody-producing cells in mucosal lymphoid tissues. In the mid 1960's, John Bienenstock, burst into the immunological scene as a mucosal immunologist with a series of groundbreaking studies that convincingly demonstrated the integral interactions of individual components of the mucosal immune system and defined the latter's inductive and effector sites. This culminated a decade later (1974) in a foundational study of mucosal immunity in which Dr. Bienenstock showed that cells in bronchial lymphoid aggregations (BALT) could function in a similar manner to cells in Peyer's patch lymphoid aggregations (GALT) in their ability to repopulate other parts of the mucosal immune system. Thus, for the first time Bienenstock (and ultimately the rest of the immunologic community) could envision a Common Mucosal Immune System (so-named by Bienenstock) that was both a unique and vital part of the immune system as a whole. Even today, almost half a century later, this work is remembered (and cited) in our attempt to understand the immunopathology of the SARS-CoV-2 infection, an infection that initially invades the upper respiratory tract and lung tissue but can spread via routes defined by the migration of cells in the mucosal immune system. However, John Bienenstock's contributions to mucosal immunology didn't stop there. In the ensuing years he and the groups of investigators he led continued to provide impressive studies establishing the structure and production of polymeric secretory IgA, the definition of inductive and effector mucosal sites and their associated histological features, the presence of distinct phenotypes of cells in mucosal compartments, and the induction of immune responses ensuing after a variety of immunization routes. In addition, in recent years the broad yet deep knowledge of mucosal immunity this work encompasses allowed him to pursue difficult questions concerning the neurological regulation of the mucosal immune system and the impact of mucosal microbiota on the mucosal system. Overall, the continued excellence of the research embodied in these studies led to his well-deserved designation as one of the most brilliant and impactful scientists in the field of mucosal immunology. Those of us who were privileged to know John as a close, personal friend will remember his unforgettable charm as well as his witty, entertaining and generous cast of mind. At numerous meetings, he displayed not only an impressive knowledge of immunology but also a deep understanding of a broad spectrum of scientific and cultural conundrums. John's astute intellect generated a continuous stream of insightful questions and solutions as well. For this reason among others, he was an indispensable member of the early and relatively small group of mucosal immunologists establishing the field of study. John's conception of scientific life extended beyond the actual performance of science. This took the form of his prominent role in the organization of many national and international conferences, including those in Canada, the United States, and many European countries. Some of these meetings were sponsored by the Mucosal Immunity Society, and John was in fact an early organizer of the Society and its second President. In addition, he was the co-editor of the “Proceeding of International Meetings of Mucosal Immunologists” published in the Advances of Experimental Medicine and Biology, and more importantly, the co-editor of first two editions of Mucosal Immunology book published by Academic Press/Elsevier. All the while he was the sponsor and advisor to countless graduate students, post-doctoral fellows, and national and international visitors who worked in his laboratory. John was born in Hungary but his family immigrated to England in the late 1930's and John received his college and medical education in that country. After initial scientific training as a post-doctoral Fellow at Harvard, he began his lifelong career as a mucosal immunologist by working at the University of Buffalo under the guidance of Thomas Tomasi, the “father” of mucosal immunology. Then, having already established himself as a leader in mucosal immunology, he moved to MacMaster University where planted his feet and stayed on as one of the intellectual and administrative leaders of this major Canadian educational institution. Among the positions he held at MacMaster was Distinguished University Professor of Pathology and Molecular Medicine, Chair of Pathology and Vice president and Dean of the Faculty of Health Sciences. In addition, in a related position he has recently served as Director of the Brain and Body Institute at St. Joseph's Health Care in Hamilton, Canada. John's productive career and excellence was recognized by many awards including an Honorary Degree of Doctor of Medicine, Goteborg University, Sweden, the MacMaster Community Distinction Award, election to the Canadian Medical Hall of Fame, the Order of Canada from the Canadian government, the designation as a Distinguished University Professor at MacMaster University, and the designation as Distinction as the Fellow of the Royal Society of Canada. John is survived by his wife for over 60 years, Dr. Audrey (Dody) Sanders, herself a remarkable scholar and physician who served as the president of the Royal College of Physicians and Surgeons of Canada. In addition to his exceptional scholarly achievements, we will remember John as a kind, caring, and generous friend who brought pleasure and inspiration to all of us. We shall miss him greatly. The authors declare no competing interests.

Human immunodeficiency virus (HIV) is transmitted primarily sexually across mucosal surfaces. After infection, HIV propagates initially in the lamina propria below the polarized epithelium and causes extensive destruction of mucosal T cells. Immunoglobulin A (IgA) antibodies, produced in the lamina propria and then transcytosed across the mucosal epithelium into the lumen, can be the first line of immune defense against HIV. Here, we used IgA monoclonal antibodies against HIV envelope proteins to investigate the abilities of polarized primate and human epithelial cells to excrete HIV virions from the basolateral to the apical surface via polymeric Ig receptor (pIgR)-mediated binding and the internalization of HIV-IgA immune complexes. African green monkey kidney cells expressing pIgR demonstrated HIV excretion that was dependent on the IgA concentration and the exposure time. Matched IgG antibodies with the same variable regions as the IgA antibodies and IgA antibodies to non-HIV antigens had no HIV excretory function. A mixture of two IgA anti-bodies against gp120 and gp41 showed a synergistic increase in the level of HIV excreted. The capacity for HIV excretion correlated with the ability of IgA antibodies to bind HIV and of the resulting immune complexes to bind pIgR. Consistent with the epithelial transcytosis of HIV-IgA immune complexes, the colocalization of HIV proteins and HIV-specific IgA was detected intracellularly by confocal microscopy. Our results suggest the potential of IgA antibodies to excrete HIV from mucosal lamina propria, thereby decreasing the viral burden, access to susceptible cells, and the chronic activation of the immune system.

Immunoglobulin A (IgA) glycosylation, recognized as an important pathogenic factor in IgA nephropathy (IgAN), is apparently controlled by the polarity of T helper (Th) cytokine responses. To examine the role of cytokine polarity in IgAN, inbred mice were immunized by intraperitoneal priming with inactivated Sendai virus (SeV) emulsified in either complete Freund's adjuvant (CFA) or incomplete Freund's adjuvant (IFA), which promote Th1- or Th2-immune response, respectively, and then boosted identically twice orally with aqueous suspensions of inactivated virus. Next, some mice were challenged intranasally with infectious SeV. Mice primed with CFA or IFA had equal reductions in nasal viral titre relative to non-immune controls, and equally increased serum levels of SeV-specific IgA antibody. Mice primed with CFA showed higher SeV-specific IgG than those with IFA. Splenocytes from mice primed with IFA produced copious amounts of interleukin (IL)-4 and IL-5, but little interferon-gamma and IL-2; those primed with CFA had reciprocal cytokine recall responses. Total serum IgA and especially SeV-specific IgA from mice primed with IFA showed a selective defect in sialylation and galactosylation. Although the frequency and intensity of glomerular deposits and haematuria did not differ, glomerulonephritis in mice primed with IFA and challenged with infectious virus was more severe than in those given CFA, as judged by serum creatinine level. We conclude that the polarity of T cell cytokines controls the pattern of IgA glycosylation and exerts direct or indirect effects on functional glomerular responses to immune complex deposition.

Mucosal immunoglobulin A (IgA) antibodies are synthesized by local plasma cells in the lamina propria and are largely destined for export through the lining epithelium into the luminal secretions. Here, IgA antibodies can bind antigens and exclude them from the body, as has long been appreciated. It is becoming increasingly apparent, though, that passage through mucosal epithelium creates additional opportunities for IgA antibodies to function in host defense. For example, IgA antibodies against viruses can directly counter infections within mucosal epithelium, and immune complexes formed in the lamina propria containing locally produced IgA antibodies can pass through the epithelium via the same route and mechanism as free IgA. Thus, IgA antibodies might first encounter antigens in three anatomic compartments in relation to mucosal epithelium: in the lumen, in the epithelium itself, or in the lamina propria (Lamm, 1997). The nonclassical defense functions of IgA, in which IgA antibodies initially bind antigens in the lamina propria or inside the lining epithelial cells, are the focus of this chapter.

In a model of IgA nephropathy (IgAN) induced by Sendai virus (SeV) without Th1/Th2 polarizing immunization, Th2-prone BALB/c mice develop more severe nephritis with acute renal insufficiency than Th1-prone C3H mice. To determine whether Th1 or Th2 predominance influences the severity of experimental IgAN in mice, we employed polarizing immunizations in a SeV-induced IgAN model in Th1-prone C57Bl/6 mice and Th2-prone BALB/c mice. C57Bl/6 mice, immunized with SeV +CFA or +IFA, showed: (1) clear cytokine polarity by splenocytes in recall assays. (2) Total serum IgA and especially SeV-specific IgA from the IFA group showed a selective defect in galactosylation, not seen in the CFA group, and (3) serum creatinine in the IFA group was higher than in the CFA group or nonimmune controls. However, BALB/c mice did not show clear cytokine polarity with CFA/IFA adjuvant. Moreover, spleen cells from naive BALB/c mice produce IFN-gamma (but not IL-2, -4, -5, or -13) upon stimulation with inactivated SeV in vitro. By flow cytometry, IFN-gamma producing cells are CD3(-), CD19(-), CD49b(+) natural killer cells. IFN-gamma production by naive splenocytes is blocked partially by anti-IL12 blocking Abs, and completely by anti-IL18R blocking Abs. In conclusion, C57Bl/6 mice with polarizing priming with SeV showed clear cytokine polarity and distinct kidney injuries. However, BALB/c mice did not show clear cytokine polarity in the same immunizing system, presumably due to the effects of innate responses to SeV upon antigen-specific lymphocytes. Natural IFN-gamma production may influence the risk of renal failure in IgAN.

The hallmark of IgA nephropathy (IgAN), the most common form of glomerulonephritis, is the presence of mesangial deposits containing IgA, specifically the IgA1 subclass, as the most prominent component. The deposited IgA is considered to be part of an immune complex. The family of enzymes known as bacterial IgA proteases exhibits substrate specificity that is essentially limited to the hinge region of IgA1. Here we demonstrate the ability of systemically administered IgA protease to remove glomerular IgA immune complexes, both the antigen and antibody components, in a passive mouse model of IgAN. Thus, IgA protease may have potential as a therapeutic agent for human IgAN.

We show that intraepithelial cell neutralization of HIV by IgA antibodies to internal viral proteins can occur during antibody transcytosis from the basolateral to the apical surface. Polarized epithelial cells expressing the polymeric immunoglobulin receptor (pIgR) were transfected with HIV proviral DNA, and IgA was added to the basolateral side. Transcytosing IgA antibodies against Gag and RT significantly inhibited HIV replication as assessed by infection of HeLa-CD4-LTR/beta-Gal cells and direct p24 assay. Consistent with intracellular neutralization, colocalization of the internal virus proteins and their IgA antibodies was demonstrated by confocal microscopy. Thus, at least in the context of infections of polarized epithelia, antibody-mediated neutralization may not be restricted to viral surface antigens.

This chapter discusses the mucosal immune system of the genitourinary tract. The chapter also discusses the significance of the mucosal immune system in the development of the fetus and neonate, including maternal-fetal interactions and major differences among species. Although the intestinal tract, as a typical locus of mucosal immune system function, and the urogenital system develop in parallel during embryogenesis and maintain anatomical proximity in postnatal life—their immunological characteristics show striking differences. In addition to S-IgA, urogenital IgA contains a considerable percentage of monomers. These features are relevant to inducing humoral immune responses in genital tract secretions. Through the homing properties of lymphocytes in the common mucosal immune system, oral and rectal immunizations yield S-IgA antibodies in most of the external secretions—including those of the female genital tract. For eliciting antibodies in genital secretions, the most effective route of immunization may be intranasal due to distinctive lymphocyte homing receptors and local chemokines involved in the selective population of the genital mucosae by cells originating from nasopharyngeal-associated inductive sites.

BACKGROUND:Recently, we observed that the severity of glomerulonephritis in an experimental model of immunoglobulin A nephropathy (IgAN) induced by Sendai virus differs between C57BL/6 and BALB/c mouse strains. The determinants of differing renal insufficiency are not understood. In the present study, we examine the capacity for mesangial cells to support Sendai viral replication and assess the direct effects of Sendai virus on the production of selected cytokines, chemokines, and eicosanoids by mesangial cells, comparing C57BL/6 to BALB/c mouse strains.METHODS:Sendai virus replication was measured by viral plaque assay using LLCMK2 cells. Production of cytokines [interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-alpha)], chemokines (JE and KC), and eicosanoids [prostaglandin E2 (PGE2) and thromboxane B2 (TxB2)] in culture medium was evaluated by sandwich enzyme-linked immunosorbent assay (ELISA) or competitive enzyme immunoassay (EIA) after 48 hours' incubation with infectious or inactivated Sendai virus.RESULTS:Sendai virus replicates equally well in mesangial cells from both strains, and infection evokes increased IL-6, JE, KC, and PGE2 production in relation to viral dose. BALB/c mesangial cells produce significantly more IL-6 and JE than those from C57BL/6, and the dose response for KC is steeper in BALB/c mesangial cells than those from C57BL/6. Synthesis of PGE2 in BALB/c mesangial cells is higher than that of C57BL/6 mesangial cells, both under basal conditions and in response to infectious Sendai virus, again in a dose-dependent manner. There is no TNF-alpha or thromboxane response to viral stimulation.CONCLUSION:We conclude that different mesangial cell responses to this common mucosal viral pathogen might influence the severity of IgAN in our model system.