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Journal of Leukocyte Biology logoLink to Journal of Leukocyte Biology
. 2009 Dec 9;87(4):663–670. doi: 10.1189/jlb.0909605

Dendritic cells transmit HIV-1 through human small intestinal mucosa

Ruizhong Shen *,1, Lesley E Smythies *,1, Ronald H Clements †, Lea Novak ‡, Phillip D Smith *,§,2
PMCID: PMC2858307  PMID: 20007245

Abstract

To dissect the early events in the transmission of HIV-1 from mother to child, we investigated whether DCs participate in HIV-1 entry into human small intestinal mucosa. We isolated human MNLs from jejunal lamina propria and identified a subpopulation of CD11c+HLA-DR+ MNLs that expressed DC-SIGN, CD83, CD86, CD206, and CCR7, indicating a DC phenotype. Jejunal DCs also expressed the HIV-1 receptor CD4 and coreceptors CCR5 and CXCR4 and in suspension rapidly took up cell-free HIV-1. HIV-1 inoculated onto the apical surface of explanted jejunum was transported by lamina propria DCs through the mucosa and transmitted in trans to blood and intestinal lymphocytes. These findings indicate that in addition to intestinal epithelial cells, which we showed previously transcytose infectious HIV-1 to indicator cells, intestinal DCs play an important role in transporting HIV-1 through the intestinal mucosa and the subsequent transmission to T cells.

Keywords: epithelium, trans-infection, lamina propria

Introduction

In the majority of HIV-1 transmissions from mother to child, the upper gastrointestinal tract is the portal through which the virus enters the host [1]. Yet, despite the public health and sociological impact of vertical transmission, particularly in the developing world, the early mucosal events involving HIV-1 entry into the small intestine have received little investigative attention. The paucity of information about this topic has been largely a result of the difficulty in obtaining upper intestinal tissues, isolating intestinal cells, and establishing ex vivo models of intestinal mucosa. Moreover, as DCs may vary between different tissues and mucosal compartments, the phenotype and function of blood-derived and extraintestinal DCs or Langerhans cells cannot be extrapolated to DCs in intestinal mucosa. Therefore, to begin to elucidate the biology of HIV-1 infection in the human intestine, we have developed protocols for the isolation of intestinal mucosal cells [2,3,4] and the establishment of model mucosa with explanted intestinal tissue [4]. Using these protocols, we have shown that jejunal epithelial cells can transcytose infectious HIV-1 to indicator cells [5] and that jejunal CD4+ lymphocytes, not macrophages, are the major mononuclear target cell for the virus that has entered the intestinal lamina propria [4, 6], culminating, as shown by others, in the rapid and profound depletion of intestinal memory CD4+ T cells in acute HIV-1 [7,8,9,10] and macaque SIV [11,12,13,14,15,16,17] infection.

Whether other cells are involved in HIV-1 entry into the intestinal mucosa is not known. However, a role for DCs or Langerhans cells in the translocation of virus across intestinal epithelium is postulated, based on studies of human and macaque vaginal tissue, in which Langerhans cells have been shown to take up HIV-1 and SIV following inoculation of virus onto the vaginal mucosa [18,19,20]. The ability of Langerhans cells to accomplish this function is aided by the cells’ strategic position in the superficial layer of the vaginal (and cervical) epithelium [21, 22]. The possibility that DCs might also participate in HIV-1 transport from the intestinal lumenal surface into the lamina propria to infect subepithelial lymphocytes is suggested further by studies of murine small intestine [23, 24] and colon [25] that demonstrate the presence of transepithelial dendrites capable of sampling luminal microorganisms. In contrast to these findings, others [26] have shown that lamina propria DCs in mouse small intestine lack transepithelial extensions. Whether human small intestinal mucosa contains DCs capable of transporting microorganisms into the mucosa is not known. Therefore, to elucidate pathways of HIV-1 entry into the human intestinal lamina propria, we characterized human intestinal DCs and showed that they are fully capable of capturing and transporting HIV-1 through the mucosa for trans-infection of systemic and intestinal lymphocytes.

MATERIALS AND METHODS

Isolation and purification of mucosal cells and blood lymphocytes

Human intestinal (jejunal) tissue and blood were obtained from otherwise healthy donors undergoing gastric bypass for obesity in compliance with Institutional Review Board-approved protocols. Lamina propria MNLs were isolated from tissue segments by enzyme digestion, as described previously [2,3,4]. Briefly, segments of fresh intestinal tissue were washed and dissected to remove the submucosa. After removing residual mucus with DTT and the epithelium with EDTA, the remaining lamina propria was minced and digested with collagenase (1 mg/mL; Sigma-Aldrich, St. Louis, MO, USA) to release the MNLs, which were then subjected to gradient sedimentation to remove debris and nonmononuclear cells. PBLs were purified by gradient sedimentation followed by magnetic cell sorting using anti-CD3 bead isolation, according to the manufacturer’s manual (Miltenyi Biotec, Auburn, CA, USA). The PBLs were cultured in RPMI with 10% human AB serum, PHA (5 μg/mL; Sigma-Aldrich), and IL-2 (20 U/mL; R&D Systems, Minneapolis, MN, USA) for 4–7 days prior to infection studies.

HIV-1 molecular clones and viruses

A replication-competent clone of YU2 [27] was transfected into 293T cells by Fugene 6 (Roche, Indianapolis, IN, USA), according to the manufacturer’s protocol. After 60 h, the supernatants were harvested, clarified by low-speed centrifugation (1000 g, 10 min), filtered through a 0.45-μm filter, aliquoted, and stored at –80°C. Virus was titrated by p24 ELISA (Perkin-Elmer, Boston, MA, USA). To generate noninfectious GFP-tagged viral particles, the pLR2PHIVgag-GFP plasmid was transfected with pCMV-ENV/HIV-YU2 (R5) into subconfluent monolayer cultures of 293T cells by Fugene 6 (Roche), as described previously [28]. Supernatants were harvested after 60 h, clarified by low-speed centrifugation, filtered through a 0.45-μm pore-size filter, and ultracentrifuged to concentrate the GFP-labeled, virus-like particles. The pellets were resuspended in DMEM, aliquoted, and frozen at –80°C.

Flow cytometric analysis

To phenotype the intestinal DCs, intestinal lamina propria MNLs were purified as described above and then incubated with optimal concentrations of PE-, allophycocyanin-, PerCP-, or FITC-conjugated antibodies to CD11c, HLA-DR, DC-SIGN, CD13, CD83, CD86, CD206, CCR7, CD4, CCR5, and CXCR4 (BD PharMingen, San Diego, CA, USA) at 4°C for 20 min. Isotype control antibodies were included in each experiment. After staining, cells were washed with PBS, fixed with 1% paraformaldehyde, and analyzed by flow cytometry. Data were analyzed with FlowJo software (Tree Star, Inc., Ashland, OR, USA).

Analysis of HIV-1 uptake by isolated lamina propria MNLs

To assure a high level of HIV-1 exposure, suspensions of 5 × 105 MNLs isolated from intestinal lamina propria were inoculated with 3.75 × 108 GFP-tagged YU2 virions representing ∼40 ng p24 and cultured in RPMI plus 10% human AB serum at 37°C. The mixtures were transferred to ice at 0, 15, 30, and 120 min after inoculation and stained with CD11c-allophycocyanin or CD3-PE (BD PharMingen) to identify DCs and lymphocytes that contained HIV-1-GFP virions.

Colocalization of DCs and HIV-1 virions in intact intestinal mucosa

Intestinal tissue explants were constructed as described previously [4]. After warming to 37°C, 7.5 × 108, GFP-tagged YU2 virions were inoculated onto the apical surface of the intestinal tissue in 50 μL RPMI. After 30 min, the virus-containing media were aspirated, and the explants were washed twice with trypsin to remove residual virus still present on the epithelial surface. The tissue then was snap-frozen in optimal cutting temperature compound (Sakura Finetek, Torrance, CA, USA), cut into 5 μm sections, and stored at –80°C. Immunofluorescence and confocal microscopic analysis were performed according to our protocol described previously [4]. Control explants were treated identically but were inoculated with media alone. Sections were also stained with anti-GFP Far Red (Alexa 647 1:100; Invitrogen Molecular Probes, Carlsbad, CA, USA) to confirm that the GFP signal corresponded to GFP+ virions and not background autofluorescence. Imaging was performed using a laser-scanning confocal microscope equipped with UV, argon, krypton, and helium/neon lasers.

Translocation of HIV-1 across explanted intestinal mucosa

YU2 viruses containing 40 ng p24 were inoculated onto the apical surface of intestinal tissue explants in 50 μL RPMI. After 2 h, cells that had migrated through the tissue and into the basolateral chamber were harvested and analyzed by flow cytometry to determine whether migrated cells carried HIV-1. CD11c-allophycocyanin, CD13-allophycocyanin, CD3-PE (BD PharMingen), and KC57-FITC (Beckman Coulter, Fullerton, CA, USA) were used to identify DCs, CD13+ cells, lymphocytes, and HIV-1 virions, respectively.

Infection of TZMbl cells, blood lymphocytes, and intestinal lamina propria lymphocytes

YU2 HIV-1 containing 40 ng p24 was inoculated onto the apical surface of intestinal tissue explants in 50 μL RPMI. After 2 h, migrated cells in the lower chamber were harvested and added to TZMbl cells [29], PBLs, or isolated lamina propria MNLs. Migrated cells from media-inoculated explants or cell-depleted supernatant from the lower chamber of the YU2-inoculated explants were added in parallel as controls. The migrated cells (or lower chamber supernatant) were cultured with blood lymphocytes or lamina propria MNLs in RPMI plus 10% human AB serum. After a 2-day incubation, cells were analyzed for HIV-1 infection by staining for β–gal+ cells, as described previously [5], or by flow cytometry for infection of blood lymphocytes and lamina propria MNLs. For these experiments, CD3-PE (BD PharMingen) and KC57-FITC (Beckman Coulter) were used to identify lymphocytes and HIV-1 virions, respectively. To confirm further the trans-infection of HIV-1 by intestinal DCs to blood and lamina propria lymphocytes, the migrated cells were cultured with PBLs or lamina propria MNLs in RPMI plus 10% human AB serum. Supernatant was collected on Days 2, 4, 6, and 8 after inoculation, and the level of viral replication after trans-infection was determined by ELISA.

RESULTS

Human intestinal lamina propria contains mononuclear cells with a myeloid DC phenotype

To characterize intestinal DCs, lamina propria MNLs were isolated from normal human jejunum using our established protocol and analyzed by flow cytometry [2, 3]. Among the MNLs, 1.6 ± 0.7% of the cells expressed the myeloid DC marker CD11c (n=5), indicating a myeloid DC subpopulation; only 0.3% of the intestinal MNLs expressed the plasmacytoid marker CD123 (data not shown). Therefore, in this report, we focused on myeloid DCs. As shown in Figure 1, >90% of the CD11c+ cells among lamina propria MNLs from five donors expressed the myeloid marker CD13 (aminopeptidase N) and the MHC II antigen HLA-DR. The majority of CD11c+ cells also expressed DC-SIGN and the marker of mature DCs CD83, and a substantial proportion expressed the costimulatory molecule CD86 and the C-type lectin receptor CD206, which recognizes terminally mannosylated molecules. A subpopulation expressed CCR7, the chemokine receptor involved in DC homing to lymph nodes. Thus, intestinal CD11c+ cells expressed markers and innate response receptors characteristic of DCs, and hereafter, in this report, lamina propria CD11c+ MNLs are designated intestinal DCs.

Figure 1.

Figure 1.

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Phenotype of intestinal CD11c+ DCs. Lamina propria MNLs were isolated from normal human jejunum, stained with fluorescence-conjugated antibodies to CD11c and the indicated markers, and analyzed by flow cytometry by gating on the CD11c+ population. Profiles are representative of cells from a single donor. The percent-positive cells are the mean from five separate donors. FL16-A/1-A/2-A, Fluorescence 16-A/1-A/2-A; APC=allophycocyanin.

Intestinal DCs express HIV-1 receptor and coreceptors

We next analyzed intestinal DCs for the expression of the HIV-1 receptor CD4 and the coreceptors CCR5 and CXCR4. As shown in Figure 2, jejunal DCs expressed CD4 (60.6%), CCR5 (44.8%), and CXCR4 (62.7%). Importantly, a substantial proportion of intestinal DCs coexpressed CD4 and CCR5 (29.2%) and CD4 and CXCR4 (47.5%). The proportion of intestinal DCs that coexpressed these receptors was strikingly higher than the proportion of intestinal macrophages (≤0.5%) that expressed them [4]. Thus, human intestinal DCs express CD4/CCR5, DC-SIGN, and CD206, receptors that bind HIV-1 and mediate cis- and trans-infection of T cells [30,31,32,33,34,35].

Figure 2.

Figure 2.

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HIV-1 receptor and coreceptor expression on intestinal DCs. Gradient sedimentation-purified jejunal MNLs were stained with the indicated fluorescence-conjugated antibodies and analyzed by FACS by gating on the CD11c+ population. Profiles are analyses from a single donor, and the percent-positive cells are the mean values from five separate donors.

DCs take up HIV-1 rapidly in isolated lamina propria MNLs

We next determined whether intestinal DCs were capable of taking up HIV-1. Cultures of isolated intestinal MNLs were inoculated with GFP-tagged YU2 (HIV-1-GFP), and at the indicated time-points, cells were harvested and analyzed by flow cytometry for CD11c, CD3, and GFP. As early as 15 min after inoculation, 5.1% of CD11c+ cells contained HIV-1-GFP, and by 2 h postinoculation, the proportion of HIV-1-GFP-containing cells increased to 31% (Fig. 3, upper panels). In contrast, CD3+ T cells, 21.1% of which were CD4+ (n=12), contained barely detectable HIV-1-GFP, increasing from 0.22% at virus inoculation to 0.44% of CD3+ cells at 2 h (Fig. 3, lower panels). These findings indicate that HIV-1 binds to or enters intestinal DCs within the first 2 h of exposure to the virus.

Figure 3.

Figure 3.

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HIV-1 uptake by intestinal DCs in isolated mucosal mononuclear cells. Cultures of lamina propria MNLs isolated from normal human jejunum were inoculated with GFP-tagged YU2 and incubated at 37°C. At the indicated time-points, cells were removed, stained with anti-CD11c or anti-CD3, and analyzed by flow cytometry by gating on the CD11c population (upper panel) or CD3 population (lower panel). Results are representative of cells isolated from four separate donors.

Intestinal DCs take up HIV-1 in explanted mucosal tissue

Having shown that suspensions of intestinal DCs were capable of taking up HIV-1, DCs in intestinal tissue were evaluated for the ability to take up virus in situ. GFP-tagged YU2 was inoculated onto the apical surface of intestinal tissue explants, and 30 min later, the explant was harvested, and tissue sections were analyzed by confocal microscopy for CD83+ cells that contained HIV-1-GFP particles. As shown in Figure 4, HIV-1-GFP particles were detected in CD83+ cells in the lamina propria of the intestinal mucosa. HIV-1-GFP was not observed in CD3+ T cells in the mucosa, but occasionally, a cell-free GFP-tagged virus was detected in the lamina propria (data not shown). Intestinal explants inoculated with media alone contained no detectable GFP+ particles. Thus, isolated intestinal DCs and lamina propria DCs in intact intestinal mucosa were capable of taking up HIV-1.

Figure 4.

Figure 4.

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Colocalization of DCs and HIV-1 in intestinal mucosa. GFP-tagged YU2 virions were inoculated onto the apical surface of jejunal tissue explants, and after 30 min, the explants were harvested, sectioned, stained, and analyzed by confocal microscopy for CD83+ DCs containing GFP-tagged YU2. GFP-tagged virions were stained with anti-GFP Alexa 488 to amplify the GFP signal, DCs with anti-CD83-Alexa Fluor 594, and cell nuclei with 4′,6-diamidino-2-phenylindole. Sections are representative of experiments with intestinal tissue explants from two separate donors; original magnification, ×630.

DCs translocate HIV-1 through intestinal tissue

Having shown that DCs in explanted intestinal mucosa were capable of taking up HIV-1 inoculated onto the apical surface, we next investigated whether lamina propria DCs could transport virus through the mucosa. Intestinal explants were established as above and inoculated with YU2, an R5 virus, or media for control, and 2 h later, the cells that had migrated through the tissue into the lower chamber were collected and analyzed by flow cytometry to determine whether migrated cells carried HIV-1. Among the total population of cells that migrated through the mucosa (Fig. 5, top panels), slightly over 1% contained HIV-1, but ∼23% of the migrated DCs contained HIV-1. Less than 1% of migrated CD13+ cells, which were predominantly macrophages, contained virus (Fig. 5, middle panels). Among the CD3+ lymphocytes that migrated through explants inoculated with HIV-1, none contained detectable HIV-1 (Fig. 5, bottom panels). These findings indicate that the cells that take up HVI-1 and transport the virus rapidly through the lamina propria are predominantly CD11c+ DCs.

Figure 5.

Figure 5.

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Translocation of HIV-1 by migrated cells across intestinal explants. YU2 virions were inoculated onto the apical surface of intestinal tissue explants, and after 2 h, migrated cells in the lower chamber were harvested and analyzed by flow cytometry. YU2 was stained with KC57-FITC, DCs with CD11c-allophycocyanin, myeloid cells with CD13-allophycocyanin, and lymphocytes with CD3-PE. Data are representative of explants from four separate donors. Differences in the percentage of CD11c+GFP+ and CD13+GFP+ cells harvested from the lower chamber of control explants and YU2-inoculated explants were significant (P<0.03).

Intestinal DCs transmit HIV-1 to T cells in trans

To explore the capacity of intestinal DCs to transmit HIV-1 to T cells, we first applied YU2 HIV-1 to explanted jejunal tissue, as described above, and tested whether the cells that migrated through the mucosa into the lower chamber contained infectious virions. As shown in Figure 6A, migrated cells from explants inoculated with YU2, but not media, infected TZMbl cells, demonstrating that HIV-1 transported through the mucosa by DCs was infectious. Next, we evaluated whether migrated DCs transmitted HIV-1 to lymphocytes. Cells that migrated into the lower chamber of explants inoculated with YU2, but not the lower chamber supernatant or cells from media-inoculated explants, infected PBLs (Fig. 6B), as well as lamina propria MNLs (Fig. 6C) in trans. To confirm further that intestinal DCs transmit infectious HIV-1 to blood and lamina propria lymphocytes, we also showed that viruses transmitted to PBLs (Fig. 6D) and lamina propria MNLs (Fig. 6E) by intestinal DCs resulted in progressive p24 production. Thus, infectious R5 virus inoculated onto the apical surface of explanted intestinal tissue was taken up by lamina propria DCs, transported through the mucosa, and able to trans-infect blood and lamina propria mononuclear target cells.

Figure 6.

Figure 6.

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Intestinal DCs deliver HIV-1 for trans-infection of mononuclear target cells. YU2 HIV-1 or media were inoculated onto the apical surface of intestinal tissue explants, and 2 h later, cells that had migrated into the lower chambers of media- or virus-inoculated explants were cultured for 2 days with TZMbl cells and analyzed by β-gal staining (A). Cells that had migrated into the lower chamber or lower chamber supernatant from media- or YU2-inoculated explants were cocultured with PBLs or lamina propria MNLs. After culture for 2 days, the PBLs (B) and lamina propria (LP) MNLs (C) were analyzed by flow cytometry for HIV-1. CD3-PE and KC57-FITC were used to identify lymphocytes and HIV-1 virions, respectively. Data are representative of three experiments with explanted jejunum from separate donors. In addition, the kinetics of viral replication in the PBLs (D) and lamina propria MNLs (E) after trans-infection was determined by measuring p24 production by ELISA (n=2).

DISCUSSION

We investigated the role of DCs in HIV-1 entry into human small intestinal mucosa. CD11c+ cells constituted <2% of the lamina propria MNLs isolated from normal jejunal mucosa. The majority of the CD11c+ cells expressed HLA-DR, CD83, and DC-SIGN, and a large proportion expressed CD86 and CD206, consistent with a DC phenotype. Importantly, the CD11c+ DCs also expressed the HIV-1 receptor CD4 and coreceptors CCR5 and CXCR4. Intestinal DCs in suspension and in explanted jejunal mucosa rapidly captured HIV-1, inoculated into the suspension or onto the mucosal surface. DCs in jejunal explants transported captured virus through the mucosa and transmitted it to PBLs and intestinal lymphocytes in trans, and transmitted HIV-1 was infectious in TZMbl cells. Thus, in addition to intestinal epithelial cells [5], local DCs in the human small intestine mediate the translocation of infectious HIV-1 into and through the mucosa.

The intestinal DCs that we characterized were mature and activated, based on high CD83 and moderate CD86 expression, and displayed receptors (CD4/CCR5, DC-SIGN, and CD206) known to mediate HIV-1 attachment. That ∼30% of the CD11c+ DCs did not express detectable CD83 raises the possibility that CD11c+CD83– intestinal DCs took up the virus and then matured into CD11c+CD83+ DCs, characteristic of DCs that encounter microbes [36], en route through the mucosa. Nevertheless, our characterization of primary intestinal DCs, rather than monocye-derived DCs, is particularly noteworthy, as studies from our laboratory have shown that intestinal extracellular matrix down-regulates receptors on other APCs, specifically, blood monocytes recruited to the jejunal lamina propria [37, 38]. Characteristic of mucosal DCs, the majority of human intestinal DCs expressed DC-SIGN, a C-type lectin receptor that binds the HIV-1 envelope gp120, independent of CD4 [39], and enhances trans-infection of T cells [30, 31]. The presence of DC-SIGN on intestinal lamina propria DCs is similar to its expression on vaginal lamina propria DCs and stands in contrast to vaginal epithelial Langerhans cells, which lack DC-SIGN [33]. In addition, human jejunal DCs displayed the mannose receptor CD206, another C-type lectin receptor expressed on dermal DCs and monocyte-derived DCs that has been shown in blocking studies to bind HIV-1 gp120 [32, 33] by binding mannose residues on HIV-1 gp120 [40]. The presence of the mannose receptor, along with DC-SIGN and CD4/CCR5, on mature intestinal DCs is consistent with the increased expression of these receptors on monocyte-derived DCs during in vitro culture [32]. Thus, human intestinal DCs express at least three receptors—CD4/CCR5, DC-SIGN, and CD206—shown to mediate binding to HIV-1. Investigations are currently under way to determine the relative contribution of these receptors to the transmission of virus to T cells. Finally, we also show here that a substantial proportion of intestinal DCs expresses CCR7, the chemokine receptor that directs DC trafficking to CCL19 and CCL21 on stromal cells in the lymph node T cell zone [41]. The expression of this key chemokine receptor on intestinal DCs would facilitate their recruitment to lymph nodes for the trans-infection of systemic T cells.

Importantly, our analysis of the early interaction between HIV-1 and intestinal MNLs in suspension and in explanted mucosa showed that DCs, rather than lymphocytes, were the predominant cell type that took up HIV-1 initially and transported it through the mucosa. These findings do not preclude HIV-1 binding to, or entry into, intestinal T cells and subsequent replication in the T cells. HIV-1 uptake by intestinal lymphocytes at the early time-points studied here may occur but below the level of detection in our assays. In this regard, recent evidence indicates that a single virus is sufficient to induce productive infection [42]. Also, the low proportion of intestinal CD13+ cells, the majority of which are macrophages [37, 38], which carried HIV-1 out of the tissue, does not preclude a role for macrophages in the translocation of HIV-1 through intestinal mucosa, although intestinal macrophages do not support HIV-1 replication [4, 6]. The findings presented here implicate lamina propria DCs as an early target cell in HIV-1 infection in the intestinal mucosa. These studies cannot distinguish whether DCs directly capture HIV-1, inoculated onto the apical mucosal surface via dendrites that extend across the epithelium, or whether DCs take up HIV-1 following an initial step of transcytosis by epithelial cells, followed by release of virus into the lamina propria. Studies are currently under way to define further these two potential pathways of DC involvement in mucosal HIV-1 infection.

A clear understanding of the immunobiology of mucosal HIV-1 infection in the upper gastrointestinal tract is critical for devising effective strategies for the prevention of mother-to-child HIV-1 transmission. The involvement of intestinal DCs in the early uptake of HIV-1 and the subsequent rapid transport of the virus through small intestinal mucosa, as we show here, underscore the importance of defining this entry pathway to identify candidate molecules for vaccine antigens.

AUTHORSHIP

R. Shen contributed conceptually to the project, performed the experiments, evaluated the data, constructed the figures, and participated in writing the manuscript. L. E. Smythies provided critical intellectual expertise about DC biology, helped with the tissue and explant experiments, and critiqued the manuscript. R. H. Clements provided the intestinal specimens and guidance regarding the manipulation of the tissues. L. Novak provided microscopic expertise and performed the confocal microscopic analysis. P. D. Smith contributed conceptually to the project, evaluation of the data, and writing of the manuscript.

ACKNOWLEDGMENTS

This work was supported by National Institutes of Health grants DK-47322, DK-54495, AI-83027, AI-83539, AI-74438, and RR-20136, the Mucosal HIV and Immunobiology Center (DK-64400), and the Research Service of the Veterans Administration.

Footnotes

Abbreviations: DC=dendritic cell, MNL=mononuclear leukocyte, SIGN=specific ICAM-3-grabbing nonintegrin

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