Volume 116, Issue 5, Pages 1043-1053 (May 1999) Intestinal macrophages display reduced permissiveness to human immunodeficiency virus 1 and decreased surface CCR5  Ling Li *, Gang Meng *, Martin F. Graham‡, George M. Shaw*,§, Phillip D. Smith*,∥  Gastroenterology  Volume 116, Issue 5, Pages 1043-1053 (May 1999) DOI: 10.1016/S0016-5085(99)70007-7 Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 1 Kinetics and levels of p24 production by HIV-1–infected blood-derived macrophages and lamina propria macrophages. (A) Primary blood-derived macrophages and (B) lamina propria macrophages were isolated, purified, and cultured as described previously13 and then inoculated with macrophage-tropic (ADA, DJV, Ba-L) or lymphocyte-tropic (IIIB) HIV-1 at the indicated TCID50 (TCID50/2.5 × 106 cells · mL−1): 1 (★), 10 (♢), 100 (○), 1000 (▵), and 10,000 (■). Culture supernatants were assayed every 4 days for p24 production. Results are from a representative experiment (n = 3). Gastroenterology 1999 116, 1043-1053DOI: (10.1016/S0016-5085(99)70007-7) Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 2 Inhibition of p24 production by (A) HIV-1–infected blood-derived macrophages and (B) lamina propria macrophages by zidovudine (AZT). Cultures of blood-derived macrophages and lamina propria macrophages cultured in media alone or media plus 50 μg/mL AZT were inoculated with HIV-1Ba-L and then assayed for p24 production as described. Bars correspond to mean peak p24 levels for blood-derived macrophages (day 16) and lamina propria macrophages (day 4) from a representative experiment (n = 2). Gastroenterology 1999 116, 1043-1053DOI: (10.1016/S0016-5085(99)70007-7) Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 3 Detection of DNA sequences corresponding to the HIV-1 LTR in lamina propria (L.p.) macrophages. Cultures of lamina propria macrophages were analyzed by nested PCR for the presence of DNA sequences corresponding to the HIV-1 LTR at 2 hours and 2 and 4 days after inoculation with HIV-1. Mock-infected lamina propria macrophages and HIV-1–infected peripheral blood mononuclear cells (PBMCs) and a tube containing water served as controls. Results are from a representative experiment (n = 2). Gastroenterology 1999 116, 1043-1053DOI: (10.1016/S0016-5085(99)70007-7) Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 4 Treatment of blood-derived macrophages with neutral protease does not alter p24 production. Blood-derived macrophages treated with dispase (■) according to the isolation protocol or media (▨) were analyzed for p24 production 8–20 days after inoculation with HIV-1. Bars correspond to the mean p24 values for duplicate determinations. Results are from a representative experiment (n = 2). Gastroenterology 1999 116, 1043-1053DOI: (10.1016/S0016-5085(99)70007-7) Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 5 Effect of HIV-1 infection on lamina propria macrophage viability. Mock-infected (upper panel) and HIV-1–infected (lower panel) lamina propria macrophages were analyzed ungated (insets) by flow cytometry for propidium iodide fluorescence on days 5, 10, and 15. Propidium iodide intercalates into the DNA of dead cells, causing them to fluoresce. Percentages indicate the proportion of cells that did not fluoresce, corresponding to viable cells. Results are from a representative experiment (n = 3). Gastroenterology 1999 116, 1043-1053DOI: (10.1016/S0016-5085(99)70007-7) Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 6 Discordant expression of CCR5 coreceptor on blood-derived macrophages and lamina propria macrophages. Fresh blood-derived macrophages and lamina propria macrophages were analyzed (A) by dual fluorescence flow cytometry for CCR5/CD13 and CCR5/HLA-DR (representative contour plots, n = 5), (B) by RT-PCR for CCR5 mRNA expression (agarose gel electrophoretic patterns of PCR products from 4 of 6 donors), and (C) by flow cytometry after a 24-hour incubation with medium or LPS (10 μg/mL), HIV-1 (Ba-L 1000 TCID50/2.5 × 106 cells · mL−1), or gp120 (1 μg/mL). Bars represent mean ± SD percent of CCR5-positive cells for 3 independent experiments. Insets in the upper panels of A show the contour plots for the isotype-matched control antibody (mouse IgG2a followed by FITC-goat anti-mouse IgG). Gastroenterology 1999 116, 1043-1053DOI: (10.1016/S0016-5085(99)70007-7) Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 6 Discordant expression of CCR5 coreceptor on blood-derived macrophages and lamina propria macrophages. Fresh blood-derived macrophages and lamina propria macrophages were analyzed (A) by dual fluorescence flow cytometry for CCR5/CD13 and CCR5/HLA-DR (representative contour plots, n = 5), (B) by RT-PCR for CCR5 mRNA expression (agarose gel electrophoretic patterns of PCR products from 4 of 6 donors), and (C) by flow cytometry after a 24-hour incubation with medium or LPS (10 μg/mL), HIV-1 (Ba-L 1000 TCID50/2.5 × 106 cells · mL−1), or gp120 (1 μg/mL). Bars represent mean ± SD percent of CCR5-positive cells for 3 independent experiments. Insets in the upper panels of A show the contour plots for the isotype-matched control antibody (mouse IgG2a followed by FITC-goat anti-mouse IgG). Gastroenterology 1999 116, 1043-1053DOI: (10.1016/S0016-5085(99)70007-7) Copyright © 1999 American Gastroenterological Association Terms and Conditions