Intestinal macrophages display reduced permissiveness to human immunodeficiency virus 1 and decreased surface CCR5

GASTROENTEROLOGY 1999;116:1043–1053

Intestinal Macrophages Display Reduced Permissiveness to Human Immunodeficiency Virus 1 and Decreased Surface CCR5 LING LI,* GANG MENG,* MARTIN F. GRAHAM,‡ GEORGE M. SHAW,*,§ and PHILLIP D. SMITH*,\ *Division of Gastroenterology and Hepatology, Department of Medicine, University of Alabama at Birmingham, and §Howard Hughes Medical Institute, Birmingham, Alabama; \Veterans Administration Medical Center, Birmingham, Alabama; and ‡Department of Pediatrics, Medical College of Virginia, Virginia Commonwealth University, Richmond, Virginia

Background & Aims: Because the role of intestinal mononuclear cells in the pathogenesis of human immunodeficiency virus 1 (HIV-1) disease has not been elucidated, we determined the biological properties of HIV-1 infection in primary intestinal macrophages. Methods: Mucosal macrophages purified from normal human jejunum were infected with well-characterized macrophage-tropic isolates of HIV-1 (ADA, DJV, and Ba-L). Results: Productive HIV-1 infection of intestinal macrophages was demonstrated by the release of p24 antigen, the presence of proviral DNA, and zidovudine inhibition of infection. Surprisingly, the titer of virus needed to establish infection of intestinal macrophages was 100–1000-fold higher than that required to infect peripheral blood derived macrophages. This marked reduction in the permissiveness of intestinal macrophages to HIV-1 was not caused by the isolation procedure or differences in CD4 expression. Instead, intestinal macrophages expressed almost no CCR5, the principal coreceptor for macrophage-tropic HIV-1, compared with blood-derived macrophages, although both cell types contained comparable levels of CCR5 messenger RNA. Exposure of blood-derived but not intestinal macrophages to HIV-1 or gp120 led to increased surface expression of CCR5. Conclusions: Intestinal macrophages express reduced levels of HIV-1, probably because of impaired permissiveness to HIV-1 entry associated with the near absence of cell surface CCR5.

he mucosal surfaces of the gastrointestinal tract are believed to play a pivotal role in the pathogenesis of human immunodeficiency virus 1 (HIV-1) infection. First, these surfaces are the major route by which HIV-1 gains initial access to lymphoid tissue in homosexuals and infants.1,2 Second, the mucosa of the small and large intestines is the largest lymphoid organ3 and the largest source of macrophages4 in the body. Consequently, the mucosa is a potential reservoir for HIV-1–infected mononuclear cells,5 and the activation of these cells is a possible

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source of the high levels of circulating virus in infected persons.6–8 Third, the mucosa may be involved in the initial selection of the genotypic and phenotypic variants that characterize HIV-1 isolated from acutely infected adults and infants.9,10 Fourth, the local and systemic immunosuppression induced by HIV-1 predisposes the gastrointestinal tract mucosa to a complex array of opportunistic infections, causing substantial morbidity in a majority of patients who develop acquired immunodeficiency syndrome (AIDS).11 However, despite the obvious importance of the mucosa in the pathogenesis of HIV-1 infection, the cellular and virological events underlying mucosal HIV-1 disease have received little investigative attention, mostly because of the difficulties associated with the isolation, purification, and culture of mucosal cells. With the lack of information on HIV-1 replication in primary mucosal cells, the immunobiological events involving HIV-1 in the mucosa are presumed to be similar to those that occur in the circulation. However, this assumption may be incorrect because the mucosal immune system is in many ways distinct from the systemic immune system.12 For example, we13 and others14–17 have shown that mucosal macrophages, which are located exclusively in the lamina propria, have phenotypic and functional features that distinguish them from blood monocytes. Consequently, the replication parameters of HIV-1 infection of blood monocytes may Abbreviations used in this paper: AIDS, acquired immunodeficiency syndrome; DMEM, Dulbecco’s modified Eagle medium; DNase, deoxyribonuclease; FITC, fluorescein isothiocyanate; HIV-1, human immunodeficiency virus 1; IL, interleukin; LPS, lipopolysaccharide; LTR, long terminal repeat; MAb, monoclonal antibody; M-CSF, macrophage colony–stimulating factor; TCID50, 50% tissue culture infective dose; RNase, ribonuclease; RT-PCR, reverse-transcription polymerase chain reaction; SIV, simian immunodeficiency virus; SSC, standard saline citrate; STE, sucrose-Tris-EDTA. r 1999 by the American Gastroenterological Association 0016-5085/99/$10.00

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not reflect those of primary lamina propria macrophages. The presence of such a large population of macrophages and the abundance of activation factors in intestinal mucosa contrast with the relatively low prevalence (0.06%) of HIV-1–infected cells in the esophageal mucosa of patients with AIDS.5 Therefore, using our newly developed technique for isolation and purification of large numbers of mucosal macrophages,13 we determined the dose of virus required to establish infection and, once infection was established, characterized the replication kinetics and level of HIV-1 expression by primary lamina propria macrophages purified from normal human intestine.

Materials and Methods Lamina Propria and Monocyte-Derived Macrophages Mucosal macrophages were isolated by neutral protease digestion of intestinal tissue sections from individual donors and then purified by counterflow centrifugal elutriation as described in detail previously.13,18 Briefly, sections of normal human jejunum obtained from healthy subjects undergoing gastrojejunostomy for morbid obesity in an Institutional Review Board–approved protocol were first washed in Ca2⫹and Mg2⫹-free phosphate-buffered saline (PBS; Mediatech, Washington, DC), incubated with 0.2 mol/L EDTA (Fisher Scientific, Norcross, GA) to remove the epithelium, and then minced and treated with the neutral protease, 75 mg/mL (grade I, sp act ⬎6 U/mg; Boehringer Mannheim, Indianapolis, IN), to release the mononuclear cells from the lamina propria. After gradient sedimentation, the mononuclear cells were subjected to counterflow centrifugal elutriation to purify the macrophages.18 The cells isolated by this procedure contained ⬍1% CD3⫹ lymphocytes and displayed the size distribution, morphological features, ultrastructure, and phagocytic activity of macrophages, as reported previously.13 Cells were cultured in Dulbecco’s modified Eagle medium (DMEM; Quality Biologicals, Gaithersburg, MD) plus 50 mg/mL gentamicin, 10% human AB serum (interleukin [IL]-2 below level of detection; Atlanta Biologicals, Atlanta, GA), and 1000 U/mL macrophage colony–stimulating factor (M-CSF; R&D Systems, Minneapolis, MN) for 2 days before inoculation with virus. Viability was typically ⬎98% at the time of inoculation and ⬎80% on day 28 as determined by propidium iodide staining. Peripheral blood monocytes were isolated from leukopaks from normal donors by counterflow centrifugal elutriation,18 allowed to adhere overnight in 24-well tissue culture plates, and then incubated in DMEM plus gentamicin, human AB serum, and M-CSF as above for 5–7 days before virus inoculation. Hereafter, adherent peripheral blood monocytes are referred to as blood-derived macrophages. Only fresh intestinal and blood-derived macrophages were used in the infectivity studies.

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Virus Isolates and Titration The HIV-1 isolates used in this study included macrophage-tropic isolates ADA and DJV (kindly provided by H. Gendelman) and Ba-L (ABI, Columbia, MD) and the lymphocyte-tropic isolate IIIB (kindly provided by M. Mulligan). The 50% tissue culture infectious dose (TCID50) of each isolate stock was determined by end point dilution of triplicate serial 2–10-fold dilutions (depending on the isolate) using bloodderived macrophages (5 ⫻ 105 cells/200-µL well) cultured in DMEM containing gentamicin and 10% human AB serum in a 96-well microtiter plate under standard conditions. On day 4, 125 µL of medium was removed and replaced with 150 µL; on day 7, 100 µL of medium was removed and assayed in duplicate for p24 antigen by capture immunoassay (Coulter Immunology, Dupont Medical Products, Miami, FL), which has a lower limit of detection of 7.8 pg/mL.

Analysis of Cell Surface CD4 and CCR5 Blood-derived macrophages and lamina propria macrophages (1 ⫻ 106/mL) were preincubated in PBS plus 10% normal human serum (Coulter Corp., Miami, FL) and 10% normal goat serum (Jackson ImmunoResearch, West Grove, PA) for 15 minutes at room temperature, washed with PBS plus 1% bovine serum albumin (BSA; Sigma Chemical Co., St. Louis, MO) and 0.1% sodium azide, and then incubated separately for 30 minutes at 4°C with fluorescein isothiocyanate (FITC)-conjugated mouse immunoglobulin (Ig) G2 monoclonal antibody (MAb) to CD4 (20 µL/106 cells; Ortho Diagnostics Systems, Raritan, NJ) or with unconjugated mouse IgG2a MAb to CCR5 (1 µg/1 ⫻ 106 cells, the predetermined optimal dose; a kind gift of Charles R. Mackay, LeukoSite, Cambridge, MA).19–21 Cells stained with MAb to CCR5 were washed and then incubated with a 1:50 dilution of FITC goat anti-mouse IgG F(ab8)2 (Jackson ImmunoResearch). For double staining (CCR5 and either HLA-DR or CD13), the cells were washed again (three times), incubated with 10% normal mouse serum (Jackson ImmunoResearch) for 15 minutes at room temperature, and then stained with either phycoerythrinconjugated mouse IgG2 MAbs to HLA-DR or mouse IgG1 MAbs to CD13 (Becton Dickinson). Cells were also stained with control IgG, including either mouse FITC-IgG2 (for CD4 staining) or purified mouse IgG2 followed by FITC-goat anti-mouse IgG F(ab8)2 (for CCR5 staining). After staining, the cells were washed and resuspended in 0.25 mL 1% paraformaldehyde and stored at 4°C until flow cytometric analysis using a FACScan (Becton Dickinson).

Infectivity Studies Purified lamina propria macrophages that had been precultured in M-CSF were resuspended in 200 µL of DMEM containing HIV-1 at the indicated TCID50, incubated for 1 hour, washed three times with 10 mL of PBS, and then cultured in 24-well tissue culture plates in DMEM plus gentamicin and 10% human AB serum at a concentration of 2.5 ⫻ 106 cells/mL per well under standard conditions. For HIV-1 infection of

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blood-derived macrophages, the cells were also preincubated in M-CSF as described above, washed twice with PBS to remove any remaining nonadherent cells (⬍1%), and then incubated with HIV-1 as above. Every 4 days, 500 µL of culture supernatant was removed for p24 determination (stored at ⫺70°C until assayed) and refed with 550 µL of supplemented DMEM without M-CSF.

In Situ Hybridization for HIV-1 RNA Cells were evaluated for HIV-1–specific RNA by in situ hybridization using our previously described protocol5,22 modified for isolated cells and digoxigenin-labeled probes.23 Briefly, air-dried cells on ribonuclease (RNase)-free glass slides were fixed in 3% paraformaldehyde and then washed with 2⫻ standard saline citrate (SSC). After they were rinsed with 0.1 mol/L triethanolamine-HCl (pH 8.0), the slides were acetylated (0.1 mol/L triethanolamine and 0.25% acetic anhydride for 15 minutes at room temperature) and then rinsed with the triethanolamine buffer followed by 2⫻ SSC. The slides were prehybridized for 1 hour with hybridization solution (50% formamide, 4⫻ SSC, Denhardt’s solution, 500 µg/mL heatdenatured [80°C] herring sperm DNA, 250 mg/mL yeast transfer RNA, and 10% dextran sulfate) without probe, rinsed with 2⫻ SSC, and then hybridized overnight at 50°C with hybridization solution containing heat-denatured RNA probe. The probes were synthesized in the antisense (complementary) and sense (noncomplementary) configuration using nucleotide triphosphates from DNA templates from five HIV-1 subclones, each containing two promoters, encompassing 90% of the HIV-1 genome and including the long terminal repeat (LTR)gag, 2.01-kilobase (kb); gag-pol, 2.30 kb; pol, 2.35 kb; pol-env, 2.10 kb; and env, 2.45 kb.5 After shearing by alkaline hydrolysis to yield 200–600-base RNA fragments, the probes were labeled with digoxigenin and titered to optimize the signal-to-background ratio as previously described.23 The slides were washed with 2⫻ SSC, rinsed with sucrose-TrisEDTA (STE; 50 mmol/L NaCl, 20 mmol/L Tris-HCl, and 1 mmol/L EDTA) and treated with RNase A (45 mg/mL STE) to remove nonhybridized probe. After they were washed with SSC, the slides were blocked with 2% BSA and incubated with antidigoxigenin antibody conjugated to alkaline phosphatase (diluted 1:500 in Tris-NaCl [pH 7.5], 1% BSA). After being washed to remove unbound antibody, the slides were incubated with substrate (5-bromo-4-chloro-3-indolyl-phosphate, 4-toluidine salt; Boehringer Mannheim) overnight at 4°C, and the color reaction was stopped by addition of Tris-EDTA (pH 8.0). The purple-staining cells, which corresponded to HIV-1 RNA-expressing cells hybridized with digoxigenin-labeled probe, were evaluated by light microscopy.

Nested Polymerase Chain Reaction for Detection of HIV-1 DNA Genomic DNA obtained at 2 hours, 2 days, and 4 days from cultures of lamina propria macrophages that had been mock-inoculated or inoculated with HIV-1 (Ba-L) pretreated with RNase-free deoxyribonuclease (DNase, 5 µg/mL; Sigma)

INTESTINAL MACROPHAGE HIV–1 INFECTION 1045

was subjected to nested polymerase chain reaction (PCR) amplification using primers to detect HIV-1 LTR sequences, as described previously.24,25 In brief, approximately 3 ⫻ 106 cells were lysed in buffer containing guanidine thiocyanate, and the DNA was extracted according to the specifications of the IsoQuick nucleic acid extraction kit (MicroProbe, Bothell, WA). Genomic DNA (2 µg/50 µL) was first amplified (denaturing, 94°C for 1 minute; annealing, 55°C for 1 minute; extension, 72°C for 1 minute; 30 cycles) with an outer set of primers (LTR I, 58-CAAGGATCCTTCCCTGATTGGCAGAACTAC-38; LTR II, 58-TAACCAGAGAGACCCAGTACAGGCAAAAAG-38), and an aliquot (2 µL) of product was reamplified (denaturing, 94°C for 1 minute; annealing, 55°C for 1 minute; extension, 72°C for 1 minute; 25 cycles) with second-round primers (LTR III, 58-GACCTTTGGATGGTGCTACAAGCTA-38; LTR IV, 58-CCTGGAAAGTCCCCAGCGGAAAGTC-38). An additional cycle (72°C for 7 minutes) was performed at the end of each round of amplifications. Amplifications were performed in 50-µL volumes, containing 10 mmol/L Tris-HCl (pH 8.3), 50 mmol/L KCl, 1.5 mmol/L MgCl2, 0.01% gelatin, 200 mmol/L deoxynucleotide triphosphates, 20 pmol of each primer, and 1.25 U of Taq polymerase without oil (Gene Amp PCR system; Perkin Elmer Cetus, Norwalk, CT). A positive control consisting of genomic DNA from HIV-1–infected, phytohemagglutinin-stimulated peripheral blood mononuclear cells and a negative water control were run with each amplification. PCR products were electrophoresed on a 2% agarose gel to analyze for the presence of specific DNA sequences.

Reverse-Transcription PCR for Detection of CCR5 Messenger RNA Fresh blood-derived macrophages and lamina propria macrophages were analyzed for CCR5 messenger RNA (mRNA) using reverse-transcription PCR (RT-PCR) as previously described.26,27 Briefly, total RNA was prepared using the RNeasy Total RNA kit (Qiagen, Chatsworth, CA) according to the manufacturer’s instructions. The extracted RNA was preheated (65°C for 5 minutes) and then reverse transcribed into complementary DNA (cDNA) in a 30-µL reaction mixture containing 200 U of Moloney murine leukemia virus RT (Promega, Madison, WI), 5 µL of oligodeoxythymidylic acid (0.5 mg/mL; Sigma), 1 µL of RNAsin (40 U/mL; Promega), 1.5 µL of 2.5 mmol/L deoxynucleoside triphosphates (Perkin Elmer, Foster City, CA), and 0.6 µL of 100 mmol/L dithiothreitol (GIBCO BRL, Gaithersburg, MD). The resulting cDNA (5 µL) was amplified in a 50-µL reaction containing 0.25 µL of Taq polymerase (5 U/mL; Gene Amp), 4 µL of deoxynucleoside triphosphates (2.5 mmol/L; Promega), 2 µL of 25 mmol/L MgCl2, 5 µL of 10⫻ PCR reaction buffer, and 2.5 µL each of 58-GAAGTTCCTCATTACACCTGCAGCTCTC-38 and 38CTTCTTCTCATTTCGACACCGAAGCAGAG-58 primers (20 mmol/L) corresponding to the second extracellular region of CCR5. The PCR reaction mixture was subjected to amplifications using the following conditions: denaturation at 95°C for 5 minutes, amplification for 5 cycles (95°C for 45 seconds,

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55°C for 45 seconds, and 72°C for 45 seconds) followed by an additional 35 cycles (95°C for 45 seconds, 62°C for 45 seconds, and 72°C for 30 seconds), and extension for 1 cycle (72°C for 5 minutes). The PCR reaction products were detected by electrophoresis on a 2% agarose gel containing ethidium bromide.

Results HIV-1 Infection of Lamina Propria and Monocyte-Derived Macrophages To evaluate HIV-1 infection of mucosal macrophages, cultures of lamina propria macrophages and peripheral blood-derived macrophages were inoculated in parallel with serially diluted macrophage-tropic (ADA, DJV, and Ba-L) or lymphocyte-tropic (IIIB) HIV-1 (1– 10,000 TCID50/2.5 ⫻ 106 cells · mL⫺1) and monitored for p24 production at 4-day intervals for 28 days. Lamina propria and blood-derived macrophages were permissive to all three macrophage-tropic isolates and markedly less permissive to the lymphocyte-tropic isolate (Figure 1). The minimal dose of HIV-1 required to establish infection in the lamina propria macrophages (TCID50, 10,000/ 2.5 ⫻ 106 cells · mL⫺1) was 2–3 logs greater than the dose required to infect blood-derived macrophages (TCID50, 10–100/2.5 ⫻ 106 cells · mL⫺1). In addition, the peak levels of p24 production in the cultures of lamina propria macrophages were 2–3 logs lower than the peak p24 levels in the cultures of macrophages derived from peripheral blood monocytes. Moreover, peak p24 production occurred on day 4 for the lamina propria macrophages, in contrast to day 20 for the blood-derived macrophages. These findings were reproduced in experi-

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ments performed on cells from three different donors. The data suggest that intestinal lamina propria macrophages were markedly less permissive to HIV-1 infection than blood monocytes. p24 in Lamina Propria Macrophage Cultures Reflects New Virus Production As shown in Figure 1, the increase in p24 levels on day 4 compared with day 0 in the cultures of lamina propria macrophages inoculated with macrophage-tropic but not lymphocyte-tropic HIV-1 suggested that productive infection of intestinal macrophages had occurred, albeit with limited virus expression. In addition, in situ hybridization of lamina propria macrophages using an HIV-1–specific RNA probe for gag, pol, and env showed that lamina propria macrophages from HIV-1–inoculated cultures hybridized the antisense but not the sense probe (data not shown). However, both the relatively small amounts of p24 in the culture supernatants and the in situ findings could conceivably be explained by surface binding of virions and subsequent release of virus. Therefore, we tested whether zidovudine, a nucleoside analogue that blocks HIV-1 reverse transcription, could prevent infection and detection of supernatant p24. Virus attachment and release would not be affected by zidovudine. As shown in Figure 2, the 50 µg/mL zidovudine added to both blood-derived and lamina propria macrophage cultures 1 hour before virus inoculation and maintained in the medium throughout the experiment completely inhibited HIV-1 p24 release into the supernatant. Finally, we evaluated the lamina propria macro-

Figure 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 macrophagetropic (ADA, DJV, Ba-L) or lymphocyte-tropic (IIIB) HIV-1 at the indicated TCID50 (TCID50/ 2.5 ⫻ 106 cells · mL⫺1): 1 (:), 10 (䉫), 100 (䊊), 1000 (䉭), and 10,000 (䊏). Culture supernatants were assayed every 4 days for p24 production. Results are from a representative experiment (n ⫽ 3).

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INTESTINAL MACROPHAGE HIV–1 INFECTION 1047

Exposure to Neutral Protease, Reduced Cell Viability, or Intracellular Sequestration of Virus Does Not Account for Reduced p24 Production by HIV-1–Infected Lamina Propria Macrophages

Figure 2. Inhibition of p24 production by (A) HIV-1–infected bloodderived 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).

phages exposed to HIV-1 for viral DNA because reverse transcription of viral RNA into DNA occurs only in infected cells. Nested PCR analysis for DNA sequences corresponding to the HIV-1 LTR showed that cells from cultures inoculated with HIV-1 but not from mockinfected cultures contained viral DNA (Figure 3). Viral DNA was not detected 2 hours after inoculation, indicating that the HIV-1 PCR products observed in 2- and 4-day cultures were not caused by carryover of viral DNA in the virus inoculum. Together, these findings indicate that primary mucosal macrophages isolated and purified from normal intestine support HIV-1 infection and replication, but far less efficiently than do macrophages derived from peripheral blood monocytes.

Figure 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).

Because the mucosal macrophages, but not the blood-derived macrophages, had been exposed to neutral protease during the isolation procedure, we examined whether treatment with this enzyme might have altered the ability of mononuclear phagocytes to support HIV-1 replication. Elutriated blood monocytes were therefore treated with either dispase according to the macrophage isolation procedure or media alone and then preincubated in M-CSF and infected with HIV-1 as above. As shown in Figure 4, HIV-1–infected blood-derived macrophages that had been treated with dispase expressed the same levels of p24 as cells that had not been exposed to the enzyme from day 12 through peak infection on day 20, indicating that the reduced p24 production by the lamina propria macrophages was not caused by the cells’ prior exposure to dispase. The depletion of CD4⫹ T cells is a hallmark of HIV-1 disease progression, and recent evidence suggests that such depletion occurs earlier and preferentially in intestinal mucosa.28 Therefore, to determine whether HIV-1 might disproportionately reduce lamina propria macrophage viability and, consequently, viral replication, we examined mock-infected and HIV-1–infected lamina propria macrophages stained with propidium iodide at 5-day intervals during a 15-day culture period and analyzed the cells for viability by flow cytometry. As shown in Figure 5, flow cytometric analysis of ungated cells (insets) showed that viability on days 5 and 10 was the same (94%–95%) for both HIV-1–infected and

Figure 4. Treatment of blood-derived macrophages with neutral protease does not alter p24 production. Blood-derived macrophages treated with dispase (䊏) according to the isolation protocol or media (C) 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).

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Figure 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).

mock-infected lamina propria macrophages (Figure 5, left and middle). By day 15, viability had decreased to 76% in the HIV-1–infected macrophages compared with 92% in the mock-infected cells (Figure 5, right). However, because peak p24 production occurred on day 4 (Figure 1), it is unlikely that reduced viability, which was not detected until day 15, is the basis for the 100-fold differences in HIV-1 infection and replication in blood– vs. intestinal tissue–derived macrophages. In contrast to the assembly of HIV-1 exclusively at the cell surface in lymphocytes, the assembly of HIV-1 in growth factor–treated macrophages occurs at the surface as well as in pleomorphic, smooth-surfaced cytoplasmic vacuoles, where virus accumulates as mature virions.29 To evaluate whether sequestration of virus in the lamina propria macrophages might account for the limited detection of p24 in the culture supernatants, lamina propria macrophages were infected with HIV-1; on day 4, the cells were fractured by freeze-thawing and the culture supernatants analyzed for p24. As shown in Table 1, fracturing of the cells resulted in only a 2–4-fold increase in the level of p24 in the culture supernatants. Thus Table 1. Effect of Cell Fracturing on Release of HIV-1 p24 by Lamina Propria Macrophages Inoculation dose TCID50 , 1000

TCID50 , 10,000

Source of p24 Culture supernatant Culture supernatant ⫹ fractured cellsb Culture supernatant Culture supernatant ⫹ fractured cells

⫾ SD. were frozen (⫺70°C) and thawed three times.

aMean bCells

(pg/mL)a

p24 (n ⫽ 3)

27 ⫾ 2 120 ⫾ 5 466 ⫾ 8 1075 ⫾ 21

intracellular sequestration of HIV-1 cannot explain the differences observed in virus replication. Identification of CD4 on Lamina Propria Macrophages Because CD4 is the primary receptor for HIV-1 entry into mononuclear cells,30–32 probably acting through its ability to increase the efficiency of gp120-CCR5 interaction,33 we analyzed lamina propria macrophages for CD4 expression. As shown in Table 2, the percentage (mean ⫾ SD) of lamina propria macrophages that expressed CD4 (20% ⫾ 11%) was not significantly different from that of dispase-treated monocytes (38% ⫾ 6%; P ⫽ 0.06). However, the density (relative mean fluorescence intensity) of surface CD4 on the lamina propria macrophages (1.5 ⫾ 0.2) was significantly less than that of dispase-treated monocytes (2.3 ⫾ 0.3; P ⫽ 0.02; Table 2). Treatment of monocytes with dispase according to the isolation protocol did not significantly affect CD4 expression on the monocytes (Table 2). Marked Reduction in the Expression of CCR5 on Lamina Propria Macrophages The principal cellular coreceptor for gp120mediated entry of macrophage-tropic HIV-1 strains is CCR5.34–38 This coreceptor is expressed on blood monocytes36–38 and microglial cells20 and at low levels on CD4⫹ lymphocytes,21 but the level of CCR5 expression on mucosal macrophages is not known. Therefore, we used flow cytometry to analyze lamina propria macrophages for CCR5 and determined whether coreceptor expression could be modulated by HIV-1, gp120, or LPS. We first analyzed the cells for HLA-DR/CCR5 and

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INTESTINAL MACROPHAGE HIV–1 INFECTION 1049

Table 2. CD4 Expression on Blood Monocytes and Lamina Propria Macrophages Experiment

Monocytes

Monocytes ⫹ dispase

Lamina propria macrophages

1 2 3 Mean ⫾ SD

38 (2.2)a 39 (3.3) 34 (3.3) 37 ⫾ 3 (2.9 ⫾ 0.6)

44 (2.7) 39 (2.1) 32 (2.1) 38 ⫾ 6b (2.3 ⫾ 0.3)c

32 (1.4) 17 (1.7) 10 (1.3) 20 ⫾ 11b (1.5 ⫾ 0.2)c

aPercent

of cells (relative mean fluorescence intensity). in percent of monocytes treated with dispase vs. lamina propria macrophages that express CD4: P ⫽ 0.06. cDifference in relative mean fluorescence intensity of surface CD4 on monocytes treated with dispase vs. lamina propria macrophages: P ⫽ 0.02. bDifference

CD13/CCR5. As shown in Figure 6A, the majority of both blood-derived macrophages and lamina propria macrophages expressed HLA-DR and CD13, which is consistent with a mononuclear phagocyte phenotype and indicates that these surface glycoproteins had not been removed by the isolation procedure. However, 12.6% of blood-derived macrophages also expressed CCR5, but ⬍1% of lamina propria macrophages expressed this coreceptor (Figure 6A). In control experiments (n ⫽ 2), dispase treatment did not diminish CCR5 expression on blood-derived macrophages, indicating that the near absence of CCR5 on the lamina propria macrophages also was not caused by the isolation procedure. Despite the difference in CCR5 surface expression on blood-derived macrophages and lamina propria macrophages, both cell types expressed mRNA for CCR5 (Figure 6B). Regarding CCR5 message expression, two patterns of PCR products were detected in the mononuclear phagocytes used in our studies: (1) a 174–base pair (bp) band (Figure 6B, donors 1 and 3), which is present in persons homozygous for wild-type CCR5 (⫹/⫹)27; and (2) a 174- plus 142-bp band pattern (Figure 6B, donors 2 and 4) present in persons heterozygous for the defective CCR5 allele (⫹/D32).27 In none of the subjects from whom samples were derived for the present studies did we detect the 142-bp single band pattern that is characteristic of persons who are homozygous for the defective CCR5 (D32/D32) allele and thus resistant to HIV-1 infection.27 We next determined whether CCR5 expression could be induced in the lamina propria macrophages. Exposure of blood-derived macrophages to HIV-1 (strain Ba-L; TCID50, 1000/mL) or gp120 (1 µg/mL; National Institutes of Health AIDS Research and Reference Reagent Program) caused significant increases in the percentage (mean ⫾ SD) of cells that expressed CCR5 (9.8% ⫾ 4.2% to 16.2% ⫾ 4.1% and 16.4% ⫾ 7.7%, respectively; P ⬍ 0.02), but exposure of lamina propria macrophages to either HIV-1 or gp120 had no effect on the percentage of mucosal macrophages that expressed the coreceptor (Figure 6C). In contrast, exposure of blood-derived macrophages to lipopolysaccharide (LPS, 10 µg/mL for 24 hours) caused an 85% reduction in the

percentage of cells that expressed CCR5 (9.8% ⫾ 4.2% to 1.5% ⫾ 1.0%) but had no effect on the nearly undetectable level of surface CCR5 present on lamina propria macrophages (Figure 6C).

Discussion The results of this study show that macrophages isolated from normal human intestinal mucosa can be productively infected by macrophage-tropic HIV-1. Unexpectedly, the amount of virus required to establish infection in mucosal macrophages was 3 logs greater than that required to establish infection in macrophages derived from peripheral blood monocytes, indicating that mucosal macrophages are substantially less permissive to infection by HIV-1 than blood monocytes. This reduced permissiveness was not caused by exposure of the mucosal cells to neutral protease during their isolation or a significant reduction in the percentage of intestinal macrophages that express CD4, although the density of the CD4 on mucosa-derived macrophages was significantly reduced. Instead, ⬍1% of lamina propria macrophages expressed CCR5, a level that was not increased by exposure to HIV-1 or gp120. This near absence of detectable CCR5 on intestinal macrophages is similar to the marked reduction or absence of certain other surface receptors on intestinal macrophages, including CD14,13–15 CD16,39 CD80,40 and CD89.41 The level of viral production in the mucosal macrophages was also markedly reduced compared with that of blood-derived macrophages. This reduction was shown not to be a consequence of HIV-1–induced macrophage death or intracellular sequestration of virus. Rather, the reduced expression of virus probably was caused by impaired permissiveness of the mucosal macrophages to HIV-1 infection. The intestinal macrophages used in our study were isolated from normal jejunum. Because colonic macrophages are also potential targets of HIV-1 infection, it will be important to determine their infectivity as well. However, we have not yet been able to purify isolated colonic macrophages to the same level as jejunal macrophages.

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A

B

Figure 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).

As the largest lymphoid organ in the body, the mucosa is a potentially important reservoir for HIV-1–infected cells. One group of investigators has detected higher levels of p24 antigen in homogenized intestinal biopsy specimens than in serum from HIV-1–infected patients.42

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However, we believe our study is the first to compare directly the infectivity of purified intestinal macrophages with that of purified blood monocytes (blood-derived macrophages). The reduced permissiveness of intestinal macrophages to HIV-1 infection and replication suggests that other mucosal cells, such as CD4⫹ lymphocytes or dendritic cells, may be more important target cells for HIV-1 than resident macrophages.43,44 In the genital tract mucosa of simian immunodeficiency virus (SIV)infected macaques, dendritic cells appear to be the first cells to become infected with virus after genital inoculation.45 Also, the inability of cytokines, such as M-CSF, granulocyte macrophage–CSF, IL-6, tumor necrosis factor ␣, and interferon gamma, to up-regulate p24 production by HIV-1–infected lamina propria macrophages (Meng and Smith, manuscript in preparation) suggests that besides showing reduced permissiveness to HIV-1, mucosal macrophages are also less responsive to factors that normally enhance virus production by monocytes. Experiments are in progress to determine whether dual infection of lamina propria macrophages by HIV-1 and opportunistic pathogens can up-regulate virus expression, as recently shown for HIV-1–infected lymph node macrophages coinfected with either Mycobacterium avium complex or Pneumocystis carinii.8 Study of SIV infection of rhesus macaques suggests that the initial and predominant target cell in the gastrointestinal mucosa is a CD4⫹ T cell.46,47 Moreover, SIV-infected intestinal macrophages are rare during the first 2 weeks of infection,47 although such cells have been detected 2 weeks after SIV inoculation48 and may increase in percentage after 3 weeks as the local CD4⫹ T-cell population decreases.47 The reduced HIV-1 permissiveness of resident intestinal macrophages we report now is consistent with the low frequency of SIV-infected macrophages in early SIV infection. In addition, the near absence of CCR5⫹ human intestinal macrophages is consistent with the near absence (⬍1%) of rectal macrophages that express CCR5 in immunohistochemical staining studies of rhesus macaque tissues.49 However, the expression of CCR5 in 3% of colon macrophages from the same animals suggests differential coreceptor expression at different anatomic sites.49 After SIV infection and the associated inflammation are established,50 the recruitment or random distribution of virus-infected blood monocytes to sites of mucosal inflammation could contribute to the subsequent increase in the local prevalence of infected macrophages. Even during late-stage human disease, however, the prevalence of mucosal macrophages actively replicating HIV-1 is low (0.06% of lamina propria mononuclear cells).13 The present findings, together with observations that

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LPS down-modulates monocyte C-C chemokine receptors51 and stimulates chemokine production,52 offer potential insights into mucosal events in HIV-1 infection. These findings suggest the following sequence of events. As CCR5⫹ CD14⫹ blood monocytes traffic through the mucosa, they encounter local inflammatory or chemotactic signals that direct their migration into the lamina propria. During migration, the monocytes also encounter bacterial products, such as LPS, in the local microenvironment.53 The LPS binds to surface CD14, the receptor for complexes of LPS and LPS-binding protein,54,55 which activates the cells and causes downmodulation of C-C chemokine receptors, including CCR5.51 The monocytes then take up residence in the lamina propria and differentiate into macrophages, loosing their surface CD1413–15 as well, by yet unknown mechanisms. Although terminal differentiation does not alter macrophage permissiveness to HIV-1,56,57 the LPSinduced down-modulation of CCR5 expression would reduce resident macrophage permissiveness to HIV-1 that had entered the lamina propria by transcytosis across the epithelium.58 In persons already infected with HIV-1, virus-infected CCR5⫹ CD14⫹ monocytes migrating from the microcirculation into the lamina propria would also encounter LPS, which could then down-modulate HIV-1 expression, as reported for primary blood monocytes,59–61 possibly through the release of C-C chemokines capable of suppressing HIV-1 replication.52 Thus, LPS-induced down-modulation of both CCR5 and HIV-1 expression probably contributes to the relatively low prevalence (0.06%) of HIV-1 mRNA-expressing cells among lamina propria mononuclear cells,5 which may in turn contribute to the relatively low frequency of virus transmission (⬍3%) that occurs during sexual encounters among homosexuals.62 Further elucidation of these mucosal events will advance our understanding of the pathogenesis of HIV-1 disease and could suggest novel therapeutic strategies.

References 1. Smith PD, Wahl SM. Immunobiology of mucosal HIV-1 infection. In: Ogra PL, Mestecky J, Lamm ME, Strober W, Bienenstock J, McGhee JR, eds. Mucosal immunology. San Diego, CA: Academic, 1999:977–989. 2. Li L, Smith PD. Immunobiology of mucosal HIV-1 infection. Curr Opin Gastroenterol 1996;12:560–563. 3. Brandtzaeg P. Overview of the mucosal immune system. Curr Top Microbiol Immunol 1989;146:13–25. 4. Lee SH, Starkey PM, Gordon S. Quantitative analysis of total macrophage content in adult mouse tissues: immunochemical studies with monoclonal antibody F4/80. J Exp Med 1984;161: 475–489. 5. Smith PD, Fox CH, Masur H, Winter HS, Alling DW. Quantitative analysis of mononuclear cells expressing human immunodeficiency virus type 1 RNA in esophageal mucosa. J Exp Med 1994;180:1541–1546.

INTESTINAL MACROPHAGE HIV–1 INFECTION 1051

6. Wei X, Ghosh SK, Taylor ME, Johnson VA, Emini EA, Deutsch P, Lifson JD, Bonhoeffer S, Nowak MA, Hahn BA, Saag MS, Shaw GM. Viral dynamics in human immunodeficiency virus type 1 infection. Nature 1995;373:117–122. 7. Ho DD, Neumann AU, Perelson AS, Chen W, Leonard JM, Markowitz M. Rapid turnover of plasma virions and CD4 lymphocytes in HIV-1 infection. Nature 1995;373:123–126. 8. Orenstein JM, Fox C, Wahl SM. Macrophages as a source of HIV during opportunistic infections. Science 1997;276:1857–1861. 9. Zhu T, Mo H, Wang N, Nam DS, Cao Y, Koup RA, Ho DD. Genotypic and phenotypic characterization of HIV-1 in patients with primary infection. Science 1993;261:1179–1181. 10. Wolinsky SM, Wike CM, Korber BTM, Hutto C, Parks WP, Rosenblum LL, Kunstman KJ, Furtado MR, Munoz JL. Selective transmission of human immunodeficiency virus type-1 variants from mothers to infants. Science 1992;255:1134–1136. 11. Smith PD. Intestinal infections in HIV-1 disease. In: Blaser MJ, Smith PD, Ravdin JI, Greenberg HB, Guerrant RL, eds. Infections of the gastrointestinal tract. New York: Raven, 1995:483–498. 12. Conley ME, Delacroix DL. Intravascular and mucosal immunoglobulin A: two separate but related systems of immune defense. Ann Intern Med 1987;106:892–899. 13. Smith PD, Janoff EN, Moseteller-Barnum M, Merger M, Orenstein JM, Kearney JF, Graham MF. Isolation and purification of CD14negative mucosal macrophages from normal human small intestine. J Immunol Methods 1997;202:1–11. 14. Grimm MC, Pavli P, Doe WF. Evidence for a CD14⫹ population of monocytes in inflammatory bowel disease mucosa—implications for pathogenesis. Clin Exp Immunol 1995;100:291–297. 15. Rugtveit J, Haraldsen G, Hogasen AK, Bakka A, Brandtzaeg P, Scott H. Respiratory burst of intestinal macrophages in inflammatory bowel disease is mainly caused by CD14⫹ L1⫹ monocyte derived cells. Gut 1995;37:367–373. 16. Fais S, Pallone F. Inability of normal human intestinal macrophages to form multinucleated giant cells in response to cytokines. Gut 1995;37:798–801. 17. Rugtveit J, Nilsen EM, Bakka A, Carlsen H, Brandtzaeg P, Scott H. Cytokine profiles differ in newly recruited and resident subsets of mucosal macrophages from inflammatory bowel disease. Gastroenterology 1997;112:1493–1505. 18. Wahl LM, Smith PD. Isolation of macrophages/monocytes from human peripheral blood and tissues. Curr Protocols Immunol 1991;7.6.1-7.6.8. 19. Choe H, Farzan M, Sun Y, Sullivan N, Rollins B, Ponath PD, Wu L, Mackay CR, LaRosa G, Newman W, Gerard N, Gerard C, Sodroski J. The b-chemokine receptors CCR3 and CCR5 facilitate infection by primary HIV-1 isolates. Cell 1996;85:1135–1148. 20. He J, Chen Y, Farzan M, Choe H, Ohagen A, Gartner S, Busciglio J, Yang X, Hofmann W, Newman W, Mackay C, Sodroski J, Gabuzda D. CCR3 and CCR5 are co-receptors for HIV-1 infection of microglia. Nature 1997;385:645–649. 21. Bleul CC, Wu L, Hoxie JA, Springer TA, Mackay CR. The HIV coreceptors CXCR4 and CCR5 are differentially expressed and regulated on human T lymphocytes. Proc Natl Acad Sci USA 1997;94:1925–1930. 22. Smith PD, Saini SS, Raffeld M, Manischewitz JF, Wahl SM. Cytomegalovirus induction of tumor necrosis factor-␣ by human monocytes and mucosal macrophages. J Clin Invest 1992;90: 1642–1648. 23. Bucy RP, Panoskaltsis-Mortari A, Huang G, Li J, Karr L, Ross M, Russell JH, Murphy KM, Weaver CT. Heterogeneity of single cell cytokine gene expression in clonal T cell populations. J Exp Med 1994;180:1251–1262. 24. Allan JS, Short M, Taylor ME, Su S, Hirsch VM, Johnson PR, Shaw GM, Hahn BH. Species-specific diversity among simian immunodeficiency viruses from African green monkeys. J Virol 1991;65: 2816–2828.

1052 LI ET AL.

25. Gao F, Yue L, Craig S, Thornton CL, Robertson DL, McCutchan FE, Bradac JA, Shart PM, Hahn BH, WHO Network for HIV Isolation and Characterization. Genetic variation of HIV type-1 in four World Health Organization–sponsored vaccine evaluation sites: generation of functional envelope (glycoprotein 160) clones representative of sequence subtypes A, B, C, and E. AIDS Res Hum Retro 1994;10:1359–1368. 26. Wilcox CM, Harris PR, Redman TR, Kawabata S, Hiroi T, Kiyono H, Smith PD. High mucosal levels of tumor necrosis factor-␣ messenger RNA in AIDS-associated cytomegalovirus-induced esophagitis. Gastroenterology 1998;114:77–82. 27. Wu L, Paxton WA, Kassam N, Ruffing N, Rottman JB, Sullivan N, Choe H, Sodroski J, Newman W, Koup RA, Mackay CR. CCR5 levels and expression pattern correlate with infectability by macrophage-tropic HIV-1, in vitro. J Exp Med 1997;185:1681– 1691. 28. Schneider T, Jahn HU, Schmidt W, Riechken EO, Zeitz M, Ullrich R, Berlin Diarrhea/Wasting Syndrome Study Group. Loss of CD4⫹ T lymphocytes in patients infected with human immunodeficiency virus type 1 is more pronounced in the duodenal mucosa than in the peripheral blood. Gut 1995;37:524–529. 29. Orenstein JM, Meltzer MS, Phipps T, Gendelman HE. Cytoplasmic assembly and accumulation of human immunodeficiency virus types 1 and 2 in recombinant human colony-stimulating factor-1– treated human monocytes: an ultrastructural study. J Virol 1988; 62:2578–2586. 30. Maddon PJ, Dalgleish AG, McDougal JS, Clapham PR, Weiss RA, Axell R. The T4 gene encodes the AIDS virus receptor and is expressed in the immune system and the brain. Cell 1986;47: 333–348. 31. Lasky LA, Nakamura G, Smith DH, Fennie C, Shimasaki C, Patzer E, Berman P, Gregory T, Capon DJ. Delineation of a region of the human immunodeficiency virus type 1 gp120 glycoprotein critical for interaction with the CD4 receptor. Cell 1987;50:975–985. 32. Finbloom DS, Hoover DL, Meltzer MS. Binding of recombinant HIV coat protein gp120 to human monocytes. J Immunol 1991;146: 1316–1321. 33. Trkola A, Dragic T, Arthos J, Binley JM, Olson WC, Allaway GP, Cheng-Meyer C, Robinson J, Maddon PJ. CD4-dependent, antibodysensitive interactions between HIV-1 and its co-receptor CCR5. Nature 1996;384:184–187. 34. Deng H, Liu R, Ellmeir W, Choe S, Unutmaz D, Burkhart M, DiMarzio P, Marmon S, Sutton RE, Hill CM, Davis CB, Peiper SC, Schall TJ, Littman DR, Landau NR. Identification of a major co-receptor for primary isolates of HIV-1. Nature 1996;381:661– 666. 35. Dragic T, Litwin V, Allaway GP, Martin SR, Huang Y, Nagashima KA, Cayanan C, Maddon PJ, Koup RA, Moore JP, Paxton WA. HIV-1 entry into CD4⫹ cells is mediated by the chemokine receptor CC-CKR-5. Nature 1996;381:667–673. 36. Alkhatib G, Combardiere C, Broder CC, Feng Y, Kennedy PE, Murphy PM, Berger EA. CC CKR5: A RANTES, MIP-1a, MIP-1b receptor as a fusion cofactor for macrophage-tropic HIV-1. Science 1996;272:1955–1958. 37. Choe H, Farzan M, Sun Y, Sullivan N, Rollins B, Ponath PD, Wu L, Mackay CR, LaRosa G, Newman W, Gerard N, Gerard C, Sodroski J. The beta-chemokine receptors CCR3 and CCR5 facilitate infection by primary HIV-1 isolates. Cell 1996;85:1135–1148. 38. Doranz BJ, Rucker J, Yi Y, Smyth RJ, Samson M, Peiper SC, Parmentier M, Collman RG, Doms RW. A dual-tropic primary HIV-1 isolate that uses fusin and the beta-chemokine receptors CKR-5, CKR-3, and CKR-2b as fusin cofactors. Cell 1996;85:1149– 1158. 39. Rogler G, Hausmann M, Vogl D, Aschenbrenner E, Andus T, Falk W, Andreeesen R, Scholmerich J, Gross V. Isolation and phenotypic characterization of colonic macrophages. Clin Exp Immunol 1998;112:205–215.

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40. Rugtveit J, Bakka A, Brandtzaeg P. Differential distribution of B7.1 (CD80) and B7.2 (CD86) costimulatory molecules on mucosal macrophage subsets in human inflammatory bowel disease (IBD). Clin Exp Immunol 1997;110:104–113. 41. Merger M, Shimada T, Kubagawa H, Smith PD. Human lamina propria macrophages lack expression of CD14 and CD89 (Fc␣R) (abstr). Gastroenterology 1996;110:A966. 42. Fackler OT, Schafer W, Schmidt T, Zippel W, Heise W, Schneider T, Zeitz M, Riecken EO. Mueller-Lantzsch N, Ullrich R. HIV-1 p24 but not proviral load is increased in the intestinal mucosa compared with the peripheral blood in HIV-infected patients. AIDS 1998;12: 139–146. 43. Frankel SS, Wenig BM, Burke AP, Mannan P, Thompson LDR, Abbondanzo SL, Nelson AM, Pope M, Steinman RM. Replication of HIV-1 in dendritic cell-derived syncytia at the mucosal surface of the adenoid. Science 1996;72:115–117. 44. Pope M, Elmore E, Ho D, Marx P. Dendritic cell-T cell mixtures, isolated from the skin and mucosae of macaques, support the replication of SIV. AIDS Res Hum Retrovir 1997;13:819–827. 45. Spira AI, Marx PA, Patterson BK, Mahoney J, Koup RA, Wolinsky SM, Ho DD. Cellular targets of infection and route of viral dissemination after an intravaginal inoculation of simian immunodeficiency virus into rhesus macaques. J Exp Med 1996;183:215– 225. 46. Heise C, Vogel P, Miller CJ, Halsted CH, Dandekar S. Simian immunodeficiency virus infection of the gastrointestinal tract of rhesus macaques. Am J Pathol 1993;142:1759–1771. 47. Veazey RS, DeMaria M, Chalifoux LV, Shvetz DE, Pauley DR, Knight HL, Rosenzweig M, Johnson RP, Desrosiers RC, Lackner AA. Gastrointestinal tract as a major site of CD4⫹ T cell depletion and viral replication in SIV infection. Science 1998;280:427– 431. 48. Heise C, Miller CJ, Lackner A, Sandekar S. Primary acute simian immunodeficiency virus infection of intestinal lymphoid tissue is associated with gastrointestinal dysfunction. J Infect Dis 1994; 169:1116–1120. 49. Zhang L, He T, Talal A, Wang G, Frankel SS, Ho DD. In vivo distribution of the human immunodeficiency virus/simian immunodeficiency virus coreceptors: CXCR4, CCR3, and CCR5. J Virol 1998;72:5035–5045. 50. Sasseville VG, Du Z, Chalifoux LV, Pauley DR, Young HL, Sehgal PK, Desrosiers RC, Lackner AA. Induction of lymphocyte proliferation and severe gastrointestinal disease in macaques by a nef gene variant of SIVmac239. Am J Pathol 1996;149:163–176. 51. Sica A, Saccani A, Borsatti A, Power CA, Wells TNC, Luini W, Polentarutti N, Sozzani S, Mantovani A. Bacterial lipopolysaccharide rapidly inhibits expression of C-C chemokine receptors in human monocytes. J Exp Med 1997;185:969–974. 52. Verani A, Scarlatti G, Comar M, Tresoldi E, Polo S, Giacca M, Lusso P, Siccardi AG, Vercelli D. C-C chemokines released by lipopolysaccharide (LPS)-stimulated human macrophages suppress HIV-1 infection in both macrophages and T cells. J Exp Med 1997;185:805–816. 53. Simon G, Gorbach SL. Intestinal flora in health and disease. Gastroenterology 1984;86:174–193. 54. Schumann RR, Leong SR, Flaggs GW, Gray PW, Wright SD, Mathison JC, Tobias PS, Ulevitch RJ. Structure and function of lipopolysaccharide binding protein. Science 1990;249:1429– 1431. 55. Wright SD, Ramos RA, Tobias PS, Ulevitch RJ, Mathison JC. CD14, a receptor for complexes of lipopolysaccharide (LPS) and LPS binding protein. Science 1990;249:1431–1433. 56. Heizinger NK, Bukrinsky MI, Haggerty SA, Ragland AM, Kewalramani V, Lee MA, Gendelman HE, Ratner L, Stevenson M,

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57.

58.

59.

60.

Emerman M. The Vpr protein of human immunodeficiency virus type 1 influences nuclear localization of viral nucleic acids in nondividing host cells. Proc Natl Acad Sci USA 1994;91:7311– 7315. Fletcher TM, Brichacek B, Sharova N, Newman MA, Stivahtis G, Sharp PM, Emerman M, Hahn BH, Stevenson M. Nuclear import and cell cycle arrest function of the HIV-1 Vpr protein are encoded by two separate genes in HIV-2/SIVSM. EMBO J 1996;15:6155– 6165. Bomsel M. Transcytosis of infectious human immunodeficiency virus across a tight human epithelial cell line barrier. Nat Med 1997;3:42–47. Kornbluth RS, Oh PS, Munis JR, Cleveland PH, Richman DD. Inter ferons and bacterial lipopolysaccharide protect macrophages from productive infection by human immunodeficiency virus in vitro. J Exp Med 1989;169:1137–1151. Bernstein MS, Tong-Starksen SE, Locksley RM. Activation of human monocyte-derived macrophages with lipopolysaccharide

INTESTINAL MACROPHAGE HIV–1 INFECTION 1053

decreases human immunodeficiency virus replication in vitro at the level of gene expression. J Clin Invest 1991;88:540–545. 61. Nottet H, de Graaf L, de Vos NM, Bakker LJ, van Strijp JAG, Visser MR, Verhoef J. Down-regulation of human immunodeficiency virus type 1 (HIV-1) production after stimulation of monocyte-derived macrophages infected with HIV-1. J Infect Dis 1993;167:810– 817. 62. Koopman JS, Jacquez IMJ. Assessing risk factors for transmission of infection. Am J Epidemiol 1991;133:1199–1209.

Received May 19, 1998. Accepted January 19, 1999. Address requests for reprints to: Phillip D. Smith, M.D., Division of Gastroenterology and Hepatology, UAB Station, 703 South 19th Street, Birmingham, Alabama 35294. Fax: (205) 934-8493. Supported by the National Institutes of Health (DK-47322, AI-41530, DK-34151, DE-72621, AI-45218) and the Research Service of the Veterans Administration.