Transepithelial transport and mucosal defence I: the role of M cells

How do cells of the immune system encounter the majority of antigens that enter the body through the gut and airways? The epithelia lining these systems contain a remarkable cell type, the M cell, that delivers antigens across the epithelium to lymphocytes and macrophages. In this article, Marian Neutra and Jean-Pierre Kraehenbuhl describe the structure of the M cell, its function in promoting the immune response and its exploitation by invading pathogens. In the next issue of Trends in Cell Biology, these authors will review the other immunological function of epithelia, secretion of polymeric IgA.     

Intracellular transport of transferrin- and asialoorosomucoid-colloidal gold conjugates to lysosomes after receptor-mediated endocytosis

Proteins coupled to colloidal gold particles have been widely used to visualize the uptake and intracellular transport of specific ligands by receptor-mediated endocytosis. The intracellular route of lysosome-directed ligands such as asialoglycoproteins (ASGP) are apparently unaltered by conjugation to gold, but the pathway of transferrin, a ligand that normally recycles to the cell surface, was reported to be altered by conjugation to 15-20 nm gold. In this study, we sought to determine whether a smaller transferrin-gold probe would recycle, and whether it might enter the same endosomal and lysosomal compartments as does a larger, lysosome-directed ASGP gold probe by visualizing their simultaneous uptake in human hepatoma (HepG2) cells. In the same cells, endocytosis of fluid-phase protein was followed using the soluble tracer native ferritin; lysosomal compartments were identified by acid phosphatase cytochemistry; and cell surfaces were labeled with ruthenium red or cationized ferritin. During the first 10 min of uptake at 37 degrees C, specific receptor-bound ferrotransferrin (FeTf)-8 nm gold and asialoorosomucoid (ASOR)-20 nm gold were clustered together in coated pits and entered the same coated vesicles, smooth vesicles, and tubules in the peripheral cytoplasm. At later times, however, transferrin-gold did not return to the cell surface; unlike native transferrin, this gold probe accompanied ASOR-gold into multivesicular bodies (MVB). The MVBs that contained probes were at first devoid of acid phosphatase activity, but at 30 min, enzyme activity was detected in a few MVBs. Native ferritin was present, along with gold probes, in all compartments of the endocytic pathway. We conclude that the normal intracellular pathway of transferrin is altered by its association with a colloidal gold particle.     

Lipoxygenase metabolites of arachidonic acid do not induce mucus secretion from rabbit intestinal goblet cells in vitro

Lipoxygenase metabolites of arachidonic acid are effective mucus secretagogues in the respiratory tract but their efficacy in the intestinal tract was unknown. Mucosal explants and sheets of epithelial cells isolated from rabbit small and large intestine were exposed to leukotrienes B4, C4, and D4 and monohydroxyeicosatetraenoic acids 5-HETE, 12-HETE, and 15-HETE. Light and electron microscopic inspection of goblet cells in treated tissues failed to detect evidence of recent compound exocytosis of mucin granules or other morphological evidence of secretory activity. These results indicate that lipoxygenase metabolites are not directly responsible for the increased mucus secretion observed in ulcerative colitis.     

Membrane domains of intestinal epithelial cells: distribution of Na+,K+- ATPase and the membrane skeleton in adult rat intestine during fetal development and after epithelial isolation

The organization of the basolateral membrane domain of highly polarized intestinal absorptive cells was studied in adult rat intestinal mucosa, during development of polarity in fetal intestine, and in isolated epithelial sheets. Semi-thin frozen sections of these tissues were stained with a monoclonal antibody (mAb 4C4) directed against Na+,K+-ATPase, and with other reagents to visualize distributions of the membrane skeleton (fodrin), an epithelial cell adhesion molecule (uvomorulin), an apical membrane enzyme (aminopeptidase), and filamentous actin. In intact adult epithelium, Na+,K+-ATPase, membrane-associated fodrin, and uvomorulin were concentrated in the lateral, but not basal, subdomain. In the stratified epithelium of fetal intestine, both fodrin and uvomorulin were localized in areas of cell-cell contact at 16 and 17 d gestation, a stage when Na+,K+-ATPase was not yet expressed. These molecules were excluded from apical domains and from cell surfaces in contact with basal lamina. When Na+,K+-ATPase appeared at 18-19 d, it was codistributed with fodrin. Detachment of epithelial sheets from adult intestinal mucosa did not disrupt intercellular junctions or lateral cell contacts, but cytoplasmic blebs appeared at basal cell surfaces, and a diffuse pool of fodrin and actin accumulated in them. At the same time, Na+,K+-ATPase moved into the basal membrane subdomain, and extensive endocytosis of basolateral membrane, including Na+,K+-ATPase, occurred. Endocytosis of uvomorulin was not detected and no fodrin was associated with endocytic vesicles. Uvomorulin, along with some membrane-associated fodrin and some Na+,K+-ATPase, remained in the lateral membrane as long as intercellular contacts were maintained. Thus, in this polarized epithelium, interaction of lateral cell-cell adhesion molecules as well as basal cell-substrate interactions are required for maintaining the stability of the lateral membrane skeleton and the position of resident membrane proteins concentrated in the lateral membrane domain.     

Proteolytic processing of reovirus is required for adherence to intestinal M cells.

Reovirus adheres specifically to apical membranes of mouse intestinal M cells and exploits M-cell transepithelial transport activity to enter Peyer's patch mucosa, where replication occurs. Proteolytic conversion of native reovirus to intermediate subviral particles (ISVPs) occurs in the intestine, but it is not known whether conversion is essential for interaction of virus with M cells. We tested the capacity of native virions, ISVPs, and cores (that lack outer capsid proteins) to bind to intestinal epithelial cells in vivo and found that only ISVPs adhered to M cells. Thus, intraluminal conversion of native reovirus to ISVPs is a prerequisite for M-cell adherence, and outer capsid proteins unique to ISVPs (either sigma 1 or products of mu 1) mediate interaction of virus with M-cell apical membranes.     

The pericentriolar material in Chinese hamster ovary cells nucleates microtubule formation

The structure and function of the centrosomes from Chinese hamster ovary (CHO) cells were investigated by electron microscopy of negatively stained wholemount preparations of cell lysates. Cells were trypsinized from culture dishes, lysed with Triton X-100, sedimented onto ionized, carbon-coated grids, and negatively stained with phosphotungstate. The centrosomes from both interphase and dividing cells consisted of pairs of centrioles, a fibrous pericentriolar material, and a group of virus-like particles which were characteristic of the CHO cells and which served as markers for the pericentriolar material. Interphase centrosomes anchored up to two dozen microtubules when cells were lysed under conditions which preserved native microtubules. When Colcemid-blocked mitotic cells, initially devoid of microtubules, were allowed to recover for 10 min, microtubules formed at the pericentriolar material, but not at the centrioles. When lysates of Colcemid-blocked cells were incubated in vitro with micotubule protein purified from porcine brain tissue, up to 250 microtubules assembled at the centrosomes, similar to the number of microtubules that would normally form at the centrosome during cell division. A few microtubules could also be assembled in vitro onto the ends of isolated centrioles from which the pericentriolar material had been removed, forming characteristic axoneme- like bundles. In addition, microtubules; were assembled onto fragments of densely staining, fibrous material which was tentatively identified as periocentriolar material by its association of CHO can initiate and anchor microtubules both in vivo and in vitro.     

Role of immunoglobulin A in protection against reovirus entry into Murine Peyer's patches

Reovirus type 1 Lang (T1L) infects the mouse intestinal mucosa by adhering specifically to epithelial M cells and exploiting M-cell transport to enter the Peyer's patches. Oral inoculation of adult mice has been shown to elicit cellular and humoral immune responses that clear the infection within 10 days. This study was designed to determine whether adult mice that have cleared a primary infection are protected against viral entry upon oral rechallenge and, if so, whether antireovirus secretory immunoglobulin A (S-IgA) is a necessary component of protection. Adult BALB/c mice that were orally inoculated on day 0 with reovirus T1L produced antiviral S-IgA in feces and IgG in serum directed primarily against the reovirus sigma1 attachment protein. Eight hours after oral reovirus challenge on day 21, the Peyer's patches of previously exposed mice contained no detectable virus whereas Peyer's patches of naive controls contained up to 2,300 PFU of reovirus/mg of tissue. Orally inoculated IgA knockout (IgA(-/-)) mice cleared the initial infection as effectively as wild-type mice and produced higher levels of reovirus-specific serum IgG and secretory IgM than C57BL/6 wild-type mice. When IgA(-/-) mice were rechallenged on day 21, however, their Peyer's patches became infected. These results indicate that intestinal S-IgA is an essential component of immune protection against reovirus entry into Peyer's patch mucosa.     

A newborn mouse model for the study of intestinal pathogenesis of shigellosis

Shigella infection is characterized by the induction of acute inflammation, which is responsible for the massive tissue destruction of the intestinal mucosa. A murine model would be a valuable tool for gaining a better understanding of the physiopathology of shigellosis and the host immune response to Shigella infection, but adult mice do not develop disease upon oral inoculation. We therefore attempted to develop a model of infection in newborn mice. Four-day-old mice inoculated with 50 microl of 5 x 10(9) invasive wild-type Shigella flexneri 5a were susceptible to bacterial infection, but mice inoculated with the non-invasive strain BS176 were not. Histologically, 4-day-old mice infected with the invasive strain presented intestinal lesions and inflammation similar to those described in patients with shigellosis. Moreover, cytokine and chemokine responses consistent with inflammation were observed. Lower bacterial inocula induced less severe intestinal damage. In contrast, 5-day-old mice inoculated with either the invasive or the non-invasive strain were not infected. We have thus established a mouse model that is suitable for the study of the pathogenesis of intestinal Shigella infection.     

Protective immunoglobulin A and G antibodies bind to overlapping intersubunit epitopes in the head domain of type 1 reovirus adhesin sigma1

Nonfusogenic mammalian orthoreovirus (reovirus) is an enteric pathogen of mice and a useful model for studies of how an enteric virus crosses the mucosal barrier of its host and is subject to control by the mucosal immune system. We recently generated and characterized a new murine immunoglobulin A (IgA)-class monoclonal antibody (MAb), 1E1, that binds to the adhesin fiber, sigma1, of reovirus type 1 Lang (T1L) and thereby neutralizes the infectivity of that strain in cell culture. 1E1 is produced in hybridoma cultures as a mixture of monomers, dimers, and higher polymers and is protective against peroral challenges with T1L either when the MAb is passively administered or when it is secreted into the intestines of mice bearing subcutaneous hybridoma tumors. In the present study, selection and analysis of mutants resistant to neutralization by 1E1 identified the region of T1L sigma1 to which the MAb binds. The region bound by a previously characterized type 1 sigma1-specific neutralizing IgG MAb, 5C6, was identified in the same way. Each of the 15 mutants isolated and analyzed was found to be much less sensitive to neutralization by either 1E1 or 5C6, suggesting the two MAbs bind to largely overlapping regions of sigma1. The tested mutants retained the capacity to recognize specific glycoconjugate receptors on rabbit M cells and cultured epithelial cells, even though viral binding to epithelial cells was inhibited by both MAbs. S1 sequence determinations for 12 of the mutants identified sigma1 mutations at four positions between residues 415 and 447, which contribute to forming the receptor-binding head domain. When aligned with the sigma1 sequence of reovirus type 3 Dearing (T3D) and mapped onto the previously reported crystal structure of the T3D sigma1 trimer, the four positions cluster on the side of the sigma1 head, across the interface between two subunits. Three such interface-spanning epitopes are thus present per sigma1 trimer and require the intact quaternary structure of the head domain for MAb binding. Identification of these intersubunit epitopes on sigma1 opens the way for further studies of the mechanisms of antibody-based neutralization and protection with type 1 reoviruses.     

Porcine reproductive and respiratory syndrome virus (PRRSV) infection spreads by cell-to-cell transfer in cultured MARC-145 cells, is dependent on an intact cytoskeleton, and is suppressed by drug-targeting of cell permissiveness to virus infection

Background

Porcine reproductive and respiratory syndrome virus (PRRSV) is the etiologic agent of PRRS, causing widespread chronic infections which are largely uncontrolled by currently available vaccines or other antiviral measures. Cultured monkey kidney (MARC-145) cells provide an important tool for the study of PRRSV replication. For the present study, flow cytometric and fluorescence antibody (FA) analyses of PRRSV infection of cultured MARC-145 cells were carried out in experiments designed to clarify viral dynamics and the mechanism of viral spread. The roles of viral permissiveness and the cytoskeleton in PRRSV infection and transmission were examined in conjunction with antiviral and cytotoxic drugs.

Results

Flow cytometric and FA analyses of PRRSV antigen expression revealed distinct primary and secondary phases of MARC-145 cell infection. PRRSV antigen was randomly expressed in a few percent of cells during the primary phase of infection (up to about 20–22 h p.i.), but the logarithmic infection phase (days 2–3 p.i.), was characterized by secondary spread to clusters of infected cells. The formation of secondary clusters of PRRSV-infected cells preceded the development of CPE in MARC-145 cells, and both primary and secondary PRRSV infection were inhibited by colchicine and cytochalasin D, demonstrating a critical role of the cytoskeleton in viral permissiveness as well as cell-to-cell transmission from a subpopulation of cells permissive for free virus to secondary targets. Cellular expression of actin also appeared to correlate with PRRSV resistance, suggesting a second role of the actin cytoskeleton as a potential barrier to cell-to-cell transmission. PRRSV infection and cell-to-cell transmission were efficiently suppressed by interferon-γ (IFN-γ), as well as the more-potent experimental antiviral agent AK-2.

Conclusion

The results demonstrate two distinct mechanisms of PRRSV infection: primary infection of a relatively small subpopulation of innately PRRSV-permissive cells, and secondary cell-to-cell transmission to contiguous cells which appear non-permissive to free virus. The results also indicate that an intact cytoskeleton is critical for PRRSV infection, and that viral permissiveness is a highly efficient drug target to control PRRSV infection. The data from this experimental system have important implications for the mechanisms of PRRSV persistence and pathology, as well as for a better understanding of arterivirus regulation.