Impairment of intestinal epithelial barriers contributes to the progression of HIV/SIV infection and leads to generalized HIV-induced immune-cell activation during chronic infection. Rhesus macaques are the major animal model for studying HIV pathogenesis. However, detailed characterization of isolated rhesus epithelial cells (ECs) from intestinal tissues is not well defined. It is also not well documented whether isolated ECs had any other cell contaminants from intestinal tissues during the time of processing that might hamper interpretation of EC preparations or cultures. In this study, we identify and characterize ECs based on flow cytometry and immunohistochemistry methods using various enzymatic and mechanical isolation techniques to enrich ECs from intestinal tissues. This study shows that normal healthy ECs differentially express HLA-DR, CD23, CD27, CD90, CD95 and IL-10R markers. Early apoptosis and upregulation of ICAM-1 and HLA-DR in intestinal ECs are thought to be the key features in SIV mediated enteropathy. The data suggest that intestinal ECs might be playing an important role in mucosal immune responses by regulating the expression of different important regulatory and adhesion molecules and their function.
References
[1]
Shattock RJ, Haynes BF, Pulendran B, Flores J, Esparza J, et al. (2008) Improving defences at the portal of HIV entry: mucosal and innate immunity. PLoS Med 5: e81.
[2]
Wira CR, Grant-Tschudy KS, Crane-Godreau MA (2005) Epithelial cells in the female reproductive tract: a central role as sentinels of immune protection. Am J Reprod Immunol 53: 65–76.
[3]
Vroling AB, Fokkens WJ, van Drunen CM (2008) How epithelial cells detect danger: aiding the immune response. Allergy 63: 1110–1123.
[4]
Brenchley JM, Douek DC (2008) HIV infection and the gastrointestinal immune system. Mucosal Immunol 1: 23–30.
[5]
Miller CJ, Shattock RJ (2003) Target cells in vaginal HIV transmission. Microbes Infect 5: 59–67.
[6]
Hladik F, Hope TJ (2009) HIV infection of the genital mucosa in women. Curr HIV/AIDS Rep 6: 20–28.
[7]
Hladik F, McElrath MJ (2008) Setting the stage: host invasion by HIV. Nat Rev Immunol 8: 447–457.
[8]
Poles MA, Elliott J, Taing P, Anton PA, Chen IS (2001) A preponderance of CCR5(+) CXCR4(+) mononuclear cells enhances gastrointestinal mucosal susceptibility to human immunodeficiency virus type 1 infection. J Virol 75: 8390–8399.
[9]
Bomsel M (1997) Transcytosis of infectious human immunodeficiency virus across a tight human epithelial cell line barrier. Nat Med 3: 42–47.
[10]
Bomsel M, David V (2002) Mucosal gatekeepers: selecting HIV viruses for early infection. Nat Med 8: 114–116.
[11]
Meng G, Wei X, Wu X, Sellers MT, Decker JM, et al. (2002) Primary intestinal epithelial cells selectively transfer R5 HIV-1 to CCR5+ cells. Nat Med 8: 150–156.
[12]
Nazli A, Chan O, Dobson-Belaire WN, Ouellet M, Tremblay MJ, et al. (2010) Exposure to HIV-1 directly impairs mucosal epithelial barrier integrity allowing microbial translocation. PLoS Pathog 6: e1000852.
[13]
MacDonald EM, Savoy A, Gillgrass A, Fernandez S, Smieja M, et al. (2007) Susceptibility of human female primary genital epithelial cells to herpes simplex virus, type-2 and the effect of TLR3 ligand and sex hormones on infection. Biol Reprod 77: 1049–1059.
[14]
Hu SL (2005) Non-human primate models for AIDS vaccine research. Curr Drug Targets Infect Disord 5: 193–201.
[15]
Bhatt RI, Brown MD, Hart CA, Gilmore P, Ramani VA, et al. (2003) Novel method for the isolation and characterisation of the putative prostatic stem cell. Cytometry A 54: 89–99.
[16]
Sankaran S, George MD, Reay E, Guadalupe M, Flamm J, et al. (2008) Rapid onset of intestinal epithelial barrier dysfunction in primary human immunodeficiency virus infection is driven by an imbalance between immune response and mucosal repair and regeneration. J Virol 82: 538–545.
[17]
Dorosko SM, Connor RI (2010) Primary human mammary epithelial cells endocytose HIV-1 and facilitate viral infection of CD4+ T lymphocytes. J Virol 84: 10533–10542.
[18]
Desnues B, Ihrig M, Raoult D, Mege JL (2006) Whipple's disease: a macrophage disease. Clin Vaccine Immunol 13: 170–178.
[19]
Nagashima R, Maeda K, Imai Y, Takahashi T (1996) Lamina propria macrophages in the human gastrointestinal mucosa: their distribution, immunohistological phenotype, and function. J Histochem Cytochem 44: 721–731.
[20]
Latza U, Niedobitek G, Schwarting R, Nekarda H, Stein H (1990) Ber-EP4: new monoclonal antibody which distinguishes epithelia from mesothelial. J Clin Pathol 43: 213–219.
[21]
Roche JK (2001) Isolation of a purified epithelial cell population from human colon. Methods Mol Med 50: 15–20.
[22]
Leon F, Roy G (2004) Isolation of human small bowel intraepithelial lymphocytes by annexin V-coated magnetic beads. Lab Invest 84: 804–809.
[23]
Saalbach A, Kraft R, Herrmann K, Haustein UF, Anderegg U (1998) The monoclonal antibody AS02 recognizes a protein on human fibroblasts being highly homologous to Thy-1. Arch Dermatol Res 290: 360–366.
[24]
Saada JI, Pinchuk IV, Barrera CA, Adegboyega PA, Suarez G, et al. (2006) Subepithelial myofibroblasts are novel nonprofessional APCs in the human colonic mucosa. J Immunol 177: 5968–5979.
[25]
Mayer L, Eisenhardt D, Salomon P, Bauer W, Plous R, et al. (1991) Expression of class II molecules on intestinal epithelial cells in humans. Differences between normal and inflammatory bowel disease. Gastroenterology 100: 3–12.
[26]
Das A, Xu H, Wang X, Yau CL, Veazey RS, et al. (2011) Double-positive CD21+CD27+ B cells are highly proliferating memory cells and their distribution differs in mucosal and peripheral tissues. PLoS One 6: e16524.
[27]
Mattapallil JJ, Letvin NL, Roederer M (2004) T-cell dynamics during acute SIV infection. AIDS 18: 13–23.
[28]
Parkos CA, Colgan SP, Diamond MS, Nusrat A, Liang TW, et al. (1996) Expression and polarization of intercellular adhesion molecule-1 on human intestinal epithelia: consequences for CD11b/CD18-mediated interactions with neutrophils. Mol Med 2: 489–505.
[29]
Dippold W, Wittig B, Schwaeble W, Mayet W, Meyer zum Buschenfelde KH (1993) Expression of intercellular adhesion molecule 1 (ICAM-1, CD54) in colonic epithelial cells. Gut 34: 1593–1597.
[30]
Bernstein CN, Sargent M, Gallatin WM, Wilkins J (1996) Beta 2-integrin/intercellular adhesion molecule (ICAM) expression in the normal human intestine. Clin Exp Immunol 106: 160–169.
[31]
Reif AE, Allen JM (1964) The Akr Thymic Antigen and Its Distribution in Leukemias and Nervous Tissues. J Exp Med 120: 413–433.
[32]
Williams AF, Gagnon J (1982) Neuronal cell Thy-1 glycoprotein: homology with immunoglobulin. Science 216: 696–703.
[33]
Haeryfar SM, Hoskin DW (2004) Thy-1: more than a mouse pan-T cell marker. J Immunol 173: 3581–3588.
[34]
Bradley JE, Ramirez G, Hagood JS (2009) Roles and regulation of Thy-1, a context-dependent modulator of cell phenotype. Biofactors 35: 258–265.
[35]
Saalbach A, Wetzel A, Haustein UF, Sticherling M, Simon JC, et al. (2005) Interaction of human Thy-1 (CD 90) with the integrin alphavbeta3 (CD51/CD61): an important mechanism mediating melanoma cell adhesion to activated endothelium. Oncogene 24: 4710–4720.
[36]
van der Flier LG, Clevers H (2009) Stem cells, self-renewal, and differentiation in the intestinal epithelium. Annu Rev Physiol 71: 241–260.
[37]
Koretzky GA, Picus J, Thomas ML, Weiss A (1990) Tyrosine phosphatase CD45 is essential for coupling T-cell antigen receptor to the phosphatidyl inositol pathway. Nature 346: 66–68.
[38]
Kishihara K, Penninger J, Wallace VA, Kundig TM, Kawai K, et al. (1993) Normal B lymphocyte development but impaired T cell maturation in CD45-exon6 protein tyrosine phosphatase-deficient mice. Cell 74: 143–156.
[39]
Gomperts BN, Belperio JA, Rao PN, Randell SH, Fishbein MC, et al. (2006) Circulating progenitor epithelial cells traffic via CXCR4/CXCL12 in response to airway injury. J Immunol 176: 1916–1927.
[40]
McDonald GB, Jewell DP (1987) Class II antigen (HLA-DR) expression by intestinal epithelial cells in inflammatory diseases of colon. J Clin Pathol 40: 312–317.
[41]
Hershberg RM, Framson PE, Cho DH, Lee LY, Kovats S, et al. (1997) Intestinal epithelial cells use two distinct pathways for HLA class II antigen processing. J Clin Invest 100: 204–215.
[42]
Tokugawa Y, Koyama M, Silver J (1997) A molecular basis for species differences in Thy-1 expression patterns. Mol Immunol 34: 1263–1272.
[43]
Park SM, Chen L, Zhang M, Ashton-Rickardt P, Turner JR, et al. (2010) CD95 is cytoprotective for intestinal epithelial cells in colitis. Inflamm Bowel Dis 16: 1063–1070.
[44]
Moller P, Koretz K, Leithauser F, Bruderlein S, Henne C, et al. (1994) Expression of APO-1 (CD95), a member of the NGF/TNF receptor superfamily, in normal and neoplastic colon epithelium. Int J Cancer 57: 371–377.
[45]
Kaiserlian D, Lachaux A, Grosjean I, Graber P, Bonnefoy JY (1993) Intestinal epithelial cells express the CD23/Fc epsilon RII molecule: enhanced expression in enteropathies. Immunology 80: 90–95.
[46]
Aubry JP, Pochon S, Graber P, Jansen KU, Bonnefoy JY (1992) CD21 is a ligand for CD23 and regulates IgE production. Nature 358: 505–507.
[47]
Pochon S, Graber P, Yeager M, Jansen K, Bernard AR, et al. (1992) Demonstration of a second ligand for the low affinity receptor for immunoglobulin E (CD23) using recombinant CD23 reconstituted into fluorescent liposomes. J Exp Med 176: 389–397.
[48]
Altmann GG, Quaroni A (1990) Behavior of fetal intestinal organ culture explanted onto a collagen substratum. Development 110: 353–370.
[49]
Whitehead RH, Brown A, Bhathal PS (1987) A method for the isolation and culture of human colonic crypts in collagen gels. In Vitro Cell Dev Biol 23: 436–442.
[50]
Quaroni A, Wands J, Trelstad RL, Isselbacher KJ (1979) Epithelioid cell cultures from rat small intestine. Characterization by morphologic and immunologic criteria. J Cell Biol 80: 248–265.
[51]
Fonti R, Latella G, Bises G, Magliocca F, Nobili F, et al. (1994) Human colonocytes in primary culture: a model to study epithelial growth, metabolism and differentiation. Int J Colorectal Dis 9: 13–22.
[52]
Baten A, Sakamoto K, Shamsuddin AM (1992) Long-term culture of normal human colonic epithelial cells in vitro. FASEB J 6: 2726–2734.
[53]
Pahar B, Lackner AA, Piatak M Jr, Lifson JD, Wang X, et al. (2009) Control of viremia and maintenance of intestinal CD4(+) memory T cells in SHIV(162P3) infected macaques after pathogenic SIV(MAC251) challenge. Virology 387: 273–284.
[54]
Das A, Veazey RS, Wang X, Lackner AA, Xu H, et al. (2011) Simian immunodeficiency virus infection in rhesus macaques induces selective tissue specific B cell defects in double positive CD21+CD27+ memory B cells. Clin Immunol 140: 223–228.
[55]
Veazey RS, Rosenzweig M, Shvetz DE, Pauley DR, DeMaria M, et al. (1997) Characterization of gut-associated lymphoid tissue (GALT) of normal rhesus macaques. Clin Immunol Immunopathol 82: 230–242.
[56]
Bloom S, Simmons D, Jewell DP (1995) Adhesion molecules intercellular adhesion molecule-1 (ICAM-1), ICAM-3 and B7 are not expressed by epithelium in normal or inflamed colon. Clin Exp Immunol 101: 157–163.
[57]
Sanderson IR, Ouellette AJ, Carter EA, Walker WA, Harmatz PR (1993) Differential regulation of B7 mRNA in enterocytes and lymphoid cells. Immunology 79: 434–438.
[58]
Salik E, Tyorkin M, Mohan S, George I, Becker K, et al. (1999) Antigen trafficking and accessory cell function in respiratory epithelial cells. Am J Respir Cell Mol Biol 21: 365–379.
[59]
Han DC, Huang GT, Lin LM, Warner NA, Gim JS, et al. (2003) Expression of MHC Class II, CD70, CD80, CD86 and pro-inflammatory cytokines is differentially regulated in oral epithelial cells following bacterial challenge. Oral Microbiol Immunol 18: 350–358.
[60]
Ding Y, Qin L, Zamarin D, Kotenko SV, Pestka S, et al. (2001) Differential IL-10R1 expression plays a critical role in IL-10-mediated immune regulation. J Immunol 167: 6884–6892.
[61]
Lai CF, Ripperger J, Morella KK, Jurlander J, Hawley TS, et al. (1996) Receptors for interleukin (IL)-10 and IL-6-type cytokines use similar signaling mechanisms for inducing transcription through IL-6 response elements. J Biol Chem 271: 13968–13975.
[62]
Takasugi K, Yamamura M, Iwahashi M, Otsuka F, Yamana J, et al. (2006) Induction of tumour necrosis factor receptor-expressing macrophages by interleukin-10 and macrophage colony-stimulating factor in rheumatoid arthritis. Arthritis Res Ther 8: R126.
[63]
Du Z, Kelly E, Mecklenbrauker I, Agle L, Herrero C, et al. (2006) Selective regulation of IL-10 signaling and function by zymosan. J Immunol 176: 4785–4792.
[64]
Elbim C, Reglier H, Fay M, Delarche C, Andrieu V, et al. (2001) Intracellular pool of IL-10 receptors in specific granules of human neutrophils: differential mobilization by proinflammatory mediators. J Immunol 166: 5201–5207.
[65]
Denning TL, Campbell NA, Song F, Garofalo RP, Klimpel GR, et al. (2000) Expression of IL-10 receptors on epithelial cells from the murine small and large intestine. Int Immunol 12: 133–139.
[66]
Li Q, Estes JD, Duan L, Jessurun J, Pambuccian S, et al. (2008) Simian immunodeficiency virus-induced intestinal cell apoptosis is the underlying mechanism of the regenerative enteropathy of early infection. J Infect Dis 197: 420–429.
[67]
George MD, Reay E, Sankaran S, Dandekar S (2005) Early antiretroviral therapy for simian immunodeficiency virus infection leads to mucosal CD4+ T-cell restoration and enhanced gene expression regulating mucosal repair and regeneration. J Virol 79: 2709–2719.
[68]
George MD, Sankaran S, Reay E, Gelli AC, Dandekar S (2003) High-throughput gene expression profiling indicates dysregulation of intestinal cell cycle mediators and growth factors during primary simian immunodeficiency virus infection. Virology 312: 84–94.
[69]
Pearce-Pratt R, Phillips DM (1996) Sulfated polysaccharides inhibit lymphocyte-to-epithelial transmission of human immunodeficiency virus-1. Biol Reprod 54: 173–182.
[70]
Taguchi M, Sampath D, Koga T, Castro M, Look DC, et al. (1998) Patterns for RANTES secretion and intercellular adhesion molecule 1 expression mediate transepithelial T cell traffic based on analyses in vitro and in vivo. J Exp Med 187: 1927–1940.
[71]
van der Linden PJ, de Goeij AF, Dunselman GA, van der Linden EP, Ramaekers FC, et al. (1994) Expression of integrins and E-cadherin in cells from menstrual effluent, endometrium, peritoneal fluid, peritoneum, and endometriosis. Fertil Steril 61: 85–90.
[72]
Dustin ML, Rothlein R, Bhan AK, Dinarello CA, Springer TA (1986) Induction by IL 1 and interferon-gamma: tissue distribution, biochemistry, and function of a natural adherence molecule (ICAM-1). J Immunol 137: 245–254.
[73]
Rothlein R, Dustin ML, Marlin SD, Springer TA (1986) A human intercellular adhesion molecule (ICAM-1) distinct from LFA-1. J Immunol 137: 1270–1274.
[74]
Reiss Y, Hoch G, Deutsch U, Engelhardt B (1998) T cell interaction with ICAM-1-deficient endothelium in vitro: essential role for ICAM-1 and ICAM-2 in transendothelial migration of T cells. Eur J Immunol 28: 3086–3099.
[75]
Greenwood J, Wang Y, Calder VL (1995) Lymphocyte adhesion and transendothelial migration in the central nervous system: the role of LFA-1, ICAM-1, VLA-4 and VCAM-1. off. Immunology 86: 408–415.
[76]
Oppenheimer-Marks N, Davis LS, Bogue DT, Ramberg J, Lipsky PE (1991) Differential utilization of ICAM-1 and VCAM-1 during the adhesion and transendothelial migration of human T lymphocytes. J Immunol 147: 2913–2921.
[77]
Frick AG, Joseph TD, Pang L, Rabe AM, St Geme JW 3rd, et al. (2000) Haemophilus influenzae stimulates ICAM-1 expression on respiratory epithelial cells. J Immunol 164: 4185–4196.
[78]
Maingat F, Halloran B, Acharjee S, van Marle G, Church D, et al. (2011) Inflammation and epithelial cell injury in AIDS enteropathy: involvement of endoplasmic reticulum stress. FASEB J 25: 2211–2220.
[79]
Ramesh G, Alvarez X, Borda JT, Aye PP, Lackner AA, et al. (2005) Visualizing cytokine-secreting cells in situ in the rhesus macaque model of chronic gut inflammation. Clin Diagn Lab Immunol 12: 192–197.
[80]
Zhao W, Pahar B, Borda JT, Alvarez X, Sestak K (2007) A decline in CCL3-5 chemokine gene expression during primary simian-human immunodeficiency virus infection. PLoS One 2: e726.
[81]
Wang X, Das A, Lackner AA, Veazey RS, Pahar B (2008) Intestinal double-positive CD4+CD8+ T cells of neonatal rhesus macaques are proliferating, activated memory cells and primary targets for SIVMAC251 infection. Blood 112: 4981–4990.
[82]
Yoshino N, Ami Y, Terao K, Tashiro F, Honda M (2000) Upgrading of flow cytometric analysis for absolute counts, cytokines and other antigenic molecules of cynomolgus monkeys (Macaca fascicularis) by using anti-human cross-reactive antibodies. Exp Anim 49: 97–110.
[83]
Vugmeyster Y, Howell K, Bakshi A, Flores C, Hwang O, et al. (2004) B-cell subsets in blood and lymphoid organs in Macaca fascicularis. Cytometry A 61: 69–75.
[84]
Pahar B, Lackner AA, Veazey RS (2006) Intestinal double-positive CD4+CD8+ T cells are highly activated memory cells with an increased capacity to produce cytokines. Eur J Immunol 36: 583–592.
[85]
Xu H, Wang X, Pahar B, Moroney-Rasmussen T, Alvarez X, et al. (2010) Increased B7-H1 expression on dendritic cells correlates with programmed death 1 expression on T cells in simian immunodeficiency virus-infected macaques and may contribute to T cell dysfunction and disease progression. J Immunol 185: 7340–7348.
