Infection Of Human Epithelial Cells Research Articles

Persistent chlamydial envelope antigens in antibiotic-exposed infected cells trigger neutrophil chemotaxis.

An in vitro coculture model system was used to explore conditions that trigger neutrophil chemotaxis to Chlamydia trachomatis infected human epithelial cells (HEC-1B). Polarized HEC-1B monolayers growing on extracellular matrix (ECM) were infected with C. trachomatis serovar E. By 36 h, coincident with the secretion of chlamydial lipopolysaccharide and major outer membrane protein to the surfaces of infected cells, human polymorphonuclear neutrophils (PMNL) loaded with azithromycin migrated through the ECM and infiltrated the HEC-1B monolayer. Bioreactive azithromycin was delivered by the chemotactic PMNL to infected epithelial cells in concentrations sufficient to kill intracellular chlamydiae. However, residual chlamydial envelopes persisted for 4 weeks, and PMNL chemotaxis was triggered to epithelial cells containing residual envelopes. Infected endometrial cells demonstrated up-regulation of ENA-78 and GCP-2 chemokine mRNA. Thus, despite appropriate antimicrobial therapy, residual chlamydial envelope antigens may persist in infected tissues of culture-negative women and provide one source for sustained inflammation.

Infection of epithelial cells by pathogenic neisseriae reduces the levels of multiple lysosomal constituents.

Members of our group reported recently that neisseria infection of human epithelial cells results in accelerated degradation of the major lysosomal integral membrane protein LAMP1 and that this is due to hydrolysis of this glycoprotein at its immunoglobulin A1 (IgA1)-like hinge by the neisseria type 2 IgA1 protease (L. Lin et al., Mol. Microbiol. 24:1083-1094, 1997). We also reported that the IgA1 protease plays a major role in the ability of the pathogenic neisseriae to survive within epithelial cells and hypothesized that this is due to alteration of lysosomes as a result of protease-mediated LAMP1 degradation. In this study, we tested the hypothesis that neisseria infection leads to multiple changes in lysosomes. Here, we report that neisseria infection also reduces the levels of three other lysosomal markers: LAMP2, lysosomal acid phosphatase (LAP), and CD63. In contrast, neither the epidermal growth factor receptor level nor the beta-tubulin level is affected. A detailed examination of LAMP2 indicated that the reduced LAMP2 levels are not the result of an altered biosynthetic rate or of cleavage by the IgA1 protease. Nevertheless, the protease plays a role in reducing LAMP2 and LAP activity levels, as these are partially restored in cells infected with an iga mutant. We conclude that neisseria infection results in multiple changes to the lysosomes of infected epithelial cells and that these changes are likely an indirect result of IgA1 protease-mediated cleavage of LAMP1.

The Neisseria type 2 IgA1 protease cleaves LAMP1 and promotes survival of bacteria within epithelial cells.

Infection of human epithelial cells by Neisseria meningitidis (MC) and Neisseria gonorrhoeae (GC) increases the rate of degradation of LAMP1, a major integral membrane glycoprotein of late endosomes and lysosomes. Several lines of evidence indicate that the neisserial IgA1 protease is directly responsible for this LAMP1 degradation. LAMP1 contains an IgA1-like hinge region with potential cleavage sites for the neisserial type 1 and type 2 IgA1 proteases. Neisserial type 2 IgA1 protease cleaves purified LAMP1 in vitro. Unlike its wild-type isogenic parent, an iga mutant of N. gonorrhoeae cannot affect LAMP1 turnover and its growth in epithelial cells is dramatically reduced. Thus, IgA1 protease cleavage of LAMP1 promotes intracellular survival of pathogenic Neisseria spp.

Epithelial cell polarization is a determinant in the infectious outcome of immunoglobulin A-mediated entry by Epstein-Barr virus.

Diseases of the nasopharyngeal epithelium due to Epstein-Barr virus (EBV) infection typically occur in chronic virus carriers with preexisting virus-specific antibodies. In vitro studies have shown that EBV-specific immunoglobulin A (IgA) promotes infection of human epithelial cells, otherwise refractory to EBV, via the polymeric immunoglobulin receptor (pIgR). To determine if EBV similarly exploits IgA transport mechanisms in vivo, we examined the fate of IgA-EBV complexes in the blood of mice, where pIgR-mediated transcytosis of IgA immune complexes through hepatocytes eliminates exogenous antigens from the circulation. By PCR analysis we showed hepatobiliary transport of IgA-EBV in viremic mice, but without detectable hepatocellular infection by immunostaining. Because efficient transport of EBV immune complexes might avert an infectious outcome, we modulated the transcytotic pathway in polarized Madin-Darby canine kidney (MDCK) cells transfected with pIgR to determine the effect on viral antigen expression. Like hepatocytes in vivo, MDCK cells in polarized monolayers translocated IgA-EBV from the basal cell face into apical medium without evidence for infection. However, when exposed to IgA-EBV as unpolarized single-cell suspensions, MDCK cells expressed EBV immediate-early and early antigens. These results suggest that pIgR-mediated transcytosis of pIgA-EBV through epithelium facilitates endogenous spread of EBV in long-term virus carriers, with infection being confined to cells with altered polarity from prior cytopathology.

Heat Stress or Rotavirus Infection of Human Epithelial Cells Generates a Distinct Hyperphosphorylated Form of Keratin 8

The two major intermediate filament (IF) phosphoglycoproteins of human simple epithelia are keratins 8 and 18 (K8/18). Previous studies showed that heat stress and, in some cases, viral infection of cultured cells can be associated with alteration in IF organization. Here we show that heat stress of the human colonic cell line HT29 increased K8 and K18 phosphorylation and glycosylation and generated a distinct hyperphosphorylated form of K8 (HK8) that was previously noted upon G2/M arrest of epithelial cells. In contrast, rotavirus infection of HT29 cells resulted in phosphorylation changes similar to those induced by heat stress but did not alter K8/18 glycosylation. The identity of HK8 was determined using phosphatase treatment, tryptic phosphopeptide mapping, and K8-specific antibodies. A time course of heat stress showed that the increased phosphorylation and glycosylation of K8/18 occurred several hours after induction of heat shock protein 70 expression. Rotavirus altered the organization of the K8/18 network in most infected cells, whereas the effect of heat stress on K8/18 organization was less prominent. However, in vitro filament assembly of purified K8/18 was not significantly altered after isolation from heat-stressed or rotavirus-infected cells, despite having increased solubility and hyperphosphorylation. Our results indicate that increased glycosylation and/or phosphorylation of K8 and K18, in association with heat stress or rotavirus infection, does not significantly alter in vitro keratin filament assembly, whereas the association of these modifications with filament reorganization in cells is more dramatic. Generation of HK8 appears to be a late event and to occur in common with several forms of stress including heat, viral infection, and arrest of cells in G2/M.

Effects of Listeria monocytogenes and Yersinia enterocolitica on cytokine gene expression and release from human polymorphonuclear granulocytes and epithelial (HEp-2) cells.

The gene expression and cytokine release of the proinflammatory cytokines interleukin-1 beta (IL-1 beta), IL-6, and tumor necrosis factor alpha (TNF-alpha) after infection of human epithelial cells (HEp-2 cells) and polymorphonuclear granulocytes (PMNs) were investigated by using isogenic pairs of Listeria monocytogenes and Yersinia enterocolitica strains. By polymerase chain reaction-assisted mRNA amplification and RNA dot blot analysis, we showed that PMNs and HEp-2 cells expressed enhanced levels of mRNA encoding IL-1 beta, IL-6, and TNF-alpha after bacterial infection. Concomitant with the enhanced mRNA level, an increased secretion rate of IL-1 beta, IL-6, and TNF-alpha from PMNs as assessed by enzyme-linked immunosorbent assay was observed. HEp-2 cells after infection also released IL-6 and TNF-alpha into the cell supernatant, while no IL-1 beta release was detected. Cellular coincubation experiments were carried out with Transwell chambers. Our studies revealed that the coculture of PMNs and HEp-2 cells led to an increased IL-1 beta and IL-6 release. In contrast, after infection with the invasive bacteria, reduced levels of TNF-alpha were measured. Our data show that PMNs secrete the proinflammatory cytokines IL-1 beta, IL-6, and TNF-alpha within some hours after infection with L. monocytogenes and Y. enterocolitica and that cellular interactions with epithelial cells alone via soluble mediators influence the net amount of released proinflammatory cytokines.

Infection of human epithelial cells by Epstein-Barr Virus (EBV): II. Biochemical characterization of EBV-determined proteins synthesized in epithelial cells

Primary cultures of epithelial cells were grown from tonsils of patients with diseases not related to EBV. The cells were implanted with EBV receptors and exposed to EBV of the transforming (B95-8, AG-876) and nontransforming (P3HR-1) strains. The EBV-infected and control cells were pulsed with [ 35S]methionine at 18–24 h after infection, and cell extracts were prepared for immunoprecipitation with anti-EBV sera and analysis by gel electrophoresis and autoradiography. About 20 EBV-determined proteins ranging from 22 to 185 kDa were detected in P3HR-1 virus-infected epithelial cells. Only a few polypeptides were detected in extracts of cells infected with AG-876 virus while no EBV-specific proteins were immunoprecipitated from extracts of B95-8 virus-infected cells. These results demonstrate that the system of EBV receptor-implanted normal human epithelial cells can be used for direct biochemical analysis of EBV infection in the epithelial tissue.

Inhibiting herpes simplex virus type 2 infection in human epithelial cells by gossypol, a potent spermicidal and contraceptive agent

Gossypol, a male contraceptive, has an antifertility effect of 99.9%. It has also been reported to possess antiviral activities, and to inhibit herpes simplex virus infection in mice. Dorsett and Kerstine found that gossypol inactivated the infectivity of the enveloped virus parainfluenza type 3 and herpes simplex for HEp-2 carcinoma cells but had no effect on the infectivity of the nonenveloped poliovirus. The authors studied the effect of gossypol on infection by herpes simplex virus type 2 (HSV-2), the causative agent of herpes genitalis. In a series of experiments, gossypol and HSV-2 were added sequentially to confluent cultures of human amniotic epithelial cells and observed daily for cytopathic changes. No cytotoxicity was observed. Gossypol exerted a pronounced antiviral effect at 100 mcM; it totally inhibited the infection by 104 plaque-forming units (PFU); 10 mcM inhibited 100 PFU, and even 3 mcM caused some inhibition of HSV. Gossypol's antiviral effect is dose-dependent. The results clearly demonstrate the inhibitory effect of gossypol on HSV-2 infection; the doses used are comparable to or lower than the minimum effective antispermatozoal concentration of gossypol. Studies have reported the lack of toxicity of gossypol on normal cells and tissues, and its inhibiting effect on the growth of gonococci. Further studies should determine whether the antimicrobial effects of gossypol should be considered a favorable side effect in its use as a contraceptive, or whether gossypol has a value as an antiviral agent alone.