1625

Human Immunodeficiency Virus Type 1 Infection Is Associated with SignificantMucosal Inflammation Characterized by Increased Expression of CCR5,CXCR4, and b-Chemokines

Jenny Olsson,1 Michael Poles,2,3 Anna-Lena Spetz,1

Julie Elliott,2,3 Lance Hultin,3 Janis Giorgi,3,a

Jan Andersson,1 and Peter Anton2,3

1Division of Infectious Diseases, Department of Medicine, HuddingeUniversity Hospital, Karolinska Institute, Huddinge, Sweden; 2Center

for HIV and Digestive Diseases, Division of Digestive Diseases,Department of Medicine, University of California Los Angeles

(UCLA), School of Medicine, and 3UCLA AIDS Institute,Los Angeles, California

Mucosal inflammation is characterized by increased expression of proinflammatory cyto-kines and chemoattractant chemokines, resulting in infiltration of immunocompetent cells.This study compared the degree of mucosal inflammation in human immunodeficiency virustype 1 (HIV-1)–infected gut mucosa with that in tissue samples from subjects with inflam-matory bowel disease (IBD) and from healthy seronegative control subjects. Gut mucosalbiopsy specimens were immunohistochemically stained and were evaluated by in situ imaging.There was significantly increased expression of HIV-1 coreceptors CCR5 and CXCR4, b-chemokine RANTES, and macrophage inflammatory protein (MIP)–1a and MIP-1b, as wellas increased numbers of T cells in lamina propria of HIV-1–infected patients. The results weresimilar in patients with IBD and in HIV-1–infected patients, suggesting increased inflammationin the colon of HIV-1–infected patients. To further investigate the effect of inflammation inHIV-1–infected lamina propria, treatments that reduce immune activation in lamina propriamust be evaluated.

The gastrointestinal tract is a reservoir of a major pool ofCD41 T lymphocytes. Most of these cells are of the memoryphenotype, expressing CD69 and CD45RO, and exhibit func-tional features of activation [1, 2]. In vitro studies have suggestedthat the differentiation of CD41 T cells toward a memory pheno-type is associated with increased susceptibility to human im-munodeficiency virus type 1 (HIV-1) infection [3–6]. This findingsupports the growing evidence that the HIV-1 burden is signif-icantly higher in the intestine and in related lymphoid tissue thanin peripheral blood [7]. A profound loss of CD41 T cells in theintestine has been shown in simian immunodeficiency virus–infected macaques [8]. The gastrointestinal tract is also an im-portant portal for entry and early dissemination of HIV-1 [9],

Received 27 June 2000; revised 17 August 2000; electronically published 23October 2000.

Presented in part: 7th Conference on Retroviruses and Opportunistic In-fections, San Francisco, January 2000 (abstract 116).

Informed consent was obtained from all patients.Financial support: Swedish Medical Research Council (2000-81 and 10850);

Swedish Cancer Foundation (2490BQO-13XCC); Swedish Physicians againstAIDS Foundation; National Institutes of Health (AI-51536, AI-28697, AI-36704, AI-37613, AI-01610, and AI-01668); Macy’s Foundation;OppenheimerFoundation; Glaxo Wellcome Institute for Digestive Health.

a Deceased.Reprints or correspondence: Dr. Jenny Olsson, Division of Infectious Dis-

eases F82, Huddinge University Hospital, 141 86 Huddinge, Sweden (jenny.olsson@medhs.ki.se).

The Journal of Infectious Diseases 2000;182:1625–35q 2000 by the Infectious Diseases Society of America. All rights reserved.0022-1899/2000/18206-0007$02.00

and numerous studies have identified HIV-1 in the intestinal mu-cosa [10, 11], even in patients with undetectable virus loads inplasma (authors’ unpublished data).

HIV-1 uses CD4 plus a coreceptor to infect cells. CXCR4 (CXchemokine receptor 4) and CCR5 (CC chemokine receptor 5)are the principal coreceptors for T cell–tropic/syncytium-inducingor X4 and macrophage-tropic/nonsyncytium-inducing or R5HIV-1 strains, respectively [12–15]. CXCR4 is mainly expressedon naive CD41 cells (CD45RA1), whereas CCR5 is predomi-nantly expressed on memory CD41 cells (CD45RO1) in healthyindividuals [16]. Recent reports also have demonstrated enhancedCXCR4 levels on memory CD41 lymphocytes [17].

In healthy HIV-1–seronegative persons, the expression of CCR5is increased in mucosal mononuclear cells (MMC), compared withthat in peripheral blood mononuclear cells (PBMC), whereasCXCR4 is expressed at similar levels on CD45RO1 T cells in MMCand PBMC [17]. It was recently shown that MMC are more easilyinfected with HIV-1 than are PBMC [17, 18]. Explanations for thehigh susceptibility of MMC to HIV-1 may include the increasedexpression of HIV-1 coreceptors, especially CCR5 [17], as well asthe activation status of the resident cells [15, 19]. The expressionof CCR5 is up-regulated by proinflammatory and Th1 cytokines,whereas Th2 cytokines up-regulate CXCR4 [20, 21]. This suggeststhat the expression of CCR5 and CXCR4 is partly controlled byTh1 or Th2 type cytokines, which are up-regulated in rectalmucosafrom HIV-1–infected patients [22].

RANTES, macrophage inflammatory protein (MIP)–1a, andMIP-1b are the natural ligands for CCR5 [23], whereas stromal-

1626 Olsson et al. JID 2000;182 (December)

derived factor-1 is the ligand for CXCR4 [24]. The physiologicfunction of b-chemokines and their receptors is to direct migra-tion of recruited lymphocyte subsets to sites of inflammation andimmune activation [25, 26]. Blocking of chemokine activity iseffective for inhibiting the migration of certain leukocytes [27].

The up-regulation of chemokine receptors and of their li-gands is a characteristic correlate of mucosal inflammation [28,29]. Immune activation of resting CD41 T cells triggers viralreplication and dissemination [15, 19]. In this study, we inves-tigated the expression of CCR5, CXCR4, RANTES, MIP-1a,and MIP-1b in colonic mucosal sites in HIV-1–infected patientsand compared the results with those for healthy and inflam-matory (subjects with inflammatory bowel disease [IBD]) con-trol subjects. We quantified the levels of chemokines and chemo-kine receptors in cryopreserved, immunocytochemically stainedtissues by in situ imaging and determined the phenotypic profileof isolated mucosal cells by use of flow cytometry. We inves-tigated whether HIV-1 infection, similar to IBD, is associatedwith mucosal inflammation.

Materials and Methods

Patients. Rectosigmoidal biopsy specimens from 27 HIV-1–seropositive patients with plasma virus loads ranging from !40 cop-ies/mL to 5.7 log10 copies/mL and CD41 cell counts from 50 cells/mL to 967 cells/mL of blood were studied. All patients, except 1,were receiving antiretroviral treatment (table 1). Biopsy specimensfrom 8 active IBD control subjects were used as inflammatory con-trols. These 8 patients were symptomatic, despite treatment with anti-inflammatory drugs (table 1). Biopsy specimens from 14 healthy,HIV-1–seronegative subjects without organic bowel disease served asnegative controls. From these patients, biopsy specimens from 6 HIV-1–infected patients, 5 inflammatory control subjects, and 5 healthycontrol subjects were analyzed by immunohistochemistry (table 1).The cryopreserved tissues were immunohistochemically stained forCD4, CD8, CCR5, CXCR4, RANTES, MIP-1a, and MIP-1b andwere analyzed with in situ imaging. Rectosigmoid biopsy specimensfrom 24 HIV-1–seropositive patients, 3 inflammatory control subjectswith IBD, and 9 healthy control subjects were analyzed by use offlow cytometry (table 1). Biopsy specimens from 3 patients in theHIV-1 cohort were analyzed by both methods. Isolated mucosal cellswere stained for CD4, CD8, CD45, CCR5, and CXCR4 and wereanalyzed by flow cytometry.

Biopsy specimen acquisition and isolation of MMC. MMC wereisolated from 4 endoscopic biopsy specimens from each donor, asreported elsewhere [17]. In brief, a standardized 30-cm site from theanus in the rectosigmoid colon was routinely sampled to avoid po-tentially confounding inflammation resulting from traumatic or in-fectious proctitis. Biopsy specimens were obtained by use of large-cup endoscopic biopsy forceps (Microvasive Radial Jaw no. 1589;Boston Scientific, Boston) with an outside diameter of 3.3 mm. Bi-opsy specimens for immunohistochemistry were immediately cryo-preserved, and tissue samples for flow cytometry were placed in 15mL of RPMI 1640 with 10% fetal calf serum. The biopsy specimenswere maintained at room temperature on a rotating platform for20–60 min before isolation. After rotation, the samples were removed

to a mm2 petri dish containing PBS with 1 mM EDTA and10 3 3550 mM 2-mercaptoethanol. The samples were teased apart using 18-gauge needles.

The disrupted tissue was incubated at 377C for 20 min in a shakingwater bath. After centrifugation, the tissue samples were digestedwith a mixture of collagenase and dispase (no. 269638; Boehringer-Mannheim, Mannheim, Germany; 0.1 mg/mL in RPMI) for 1 h at377C. Further tissue disruption was achieved by sample passagethrough syringes with a series of decreasing needle gauges (18G–21G). Debris was removed using a 70-mm cell strainer (Falcon no.2350; Becton Dickinson Labware, Franklin Lakes, NJ). The resultingcells were resuspended in RPMI containing 10% fetal calf serum.MMC, which primarily include epithelial cells and leukocytes, werecounted visually using a hemocytometer, and the proportion of mon-onuclear cells that were leukocytes was estimated. About 20% of thetotal MMC were leukocytes from a mean yield of 1.3 3 106 5

( ) from 4 biopsy specimens. Viability, as determined1.1 3 106 n p 6by the exclusion of trypan blue, was 190%.

Flow cytometry. Monoclonal antibodies (MAbs) used in thisstudy included CD4–fluorescein isothiocyanate (clone SK3) andCD45–peridinin chlorophyll protein (clone 2D1; both from BectonDickinson, San Jose, CA), CD8-allophycocyanin (clone SK1; Cal-tag Laboratories, Burlingame, CA), and anti–CXCR4-R-phyco-erythrin (PE; clone 12G5; PharMingen, San Diego). Anti-CCR5(clone 2D7) was provided by Walter Newman (Leukosite, Cam-bridge, MA) and was prepared as a 1:1 conjugate with PE byKenneth Davis and Noel Warner (Becton Dickinson). A FACS-Calibur flow cytometer was used with Cell Quest software (bothfrom Becton Dickinson).

Initial gating on the isolated MMC was done using side-scatterand CD45 fluorescence. A well-defined and separate population ofmucosal leukocytes was identified as CD45bright and represented∼10%–50% of the initial mononuclear sample population. Of these,20%–40% were CD41 T cells. Despite variability in yield amongstudy subjects, parallel isolations from the same subject (4 biopsyspecimens each) resulted in similar data (data not shown). To es-timate the number of CCR5 molecules per lymphocyte, the ob-served CCR5 relative fluorescence intensity (RFI) was amplifiedby a calibration factor (44), as described elsewhere [31]. This cali-bration factor is the number of PE molecules present on the CCR5-specific MAb detected per RFI channel number. For MAbs pre-pared as a 1:1 conjugate with PE, the RFI channel number canbe multiplied by the calibration number to estimate the number ofMAbs bound per cell.

Immunohistochemistry. Cryopreserved colon biopsy specimensembedded in OCT compound (Tissue-TEK, Mites, Elkhart, IN)were cut into 8 mm–thick sections, mounted on glass slides (HTC;Novakemi, Stockholm), and were fixed with 2% formaldehyde(Sigma, Stockholm). Slides were washed with balanced salt solution(Gibco, Paisley, UK) and were stored at 2207C until stained. Thestaining procedure, which was used to identify cell markers, HIV-1 coreceptors, and soluble mediators of inflammation at the single-cell level, has been described elsewhere [32, 33]. In brief, cells werepermeabilized with 0.1% Saponin in balanced salt solution. Afterperoxidase quenching, blocking with 1% fetal calf serum, and avi-din and biotin blocking, using a blocking kit (Vector Laboratories,Burlingame, CA), the sections were incubated overnight at roomtemperature with primary antibodies.

Table 1. CD4 cells in blood, plasma virus load, Centers for Disease Control and Prevention (CDC) classification,and medications for all patients.

Study subjectsCD4 cells/mL

in bloodPlasma

virus loadCDC

classificationa Medications

HIV-1–seropositivepatients

H1b,c 715 2000 C1 Saquinavir, zidovudin, lamivudine, ritonavirH2b 750 ND B2 Lamivudine, zidovudin, indinavirH3b,c 504 !40 C2 Stavudin, nelfinavir, nevirapinH4b,c 900 !40 B1 Lamivudine, stavudin, indinavirH5b 420 523,000 B1 No medicationsH6b

!50 250,000 B3 Saquinavir, abacavir, nelfinavirH7c 160 !50 Stavudin, didanosin, ritonavir, saquinavir, hydroxyureaH8c 180 !500 Stavudin, saquinavir, ritonavirH9c 550 !50 Stavudin, lamivudine, ritonavir, saquinavir, nevirapinH10c 743 !50 B3 Stavudin, saquinavir, ritonavirH11c 798 900 Ritonavir, zidovudin, lamivudineH12c 410 !50 Lamivudine, ritonavir, saquinavir, stavudinH13c 680 !400 Lamivudine, ritonavir, indinavir, nevirapinH14c 967 !50 Didanosin, nevirapin, stavudinH15c 285 27,000 C1 Didanosin, stavudin, nelfinavirH16c 612 13,719 C1 Lamivudine, nelfinavir, stavudinH17c 506 64,233 B2 Stavudin, hydroxyurea, amprenavirH18c 272 19,200 A2 Indinavir, lamivudine, ritonavir, stavudinH19c 629 8453 A3 Lamivudine, zidovudinH20c 228 3844 B2 Abacavir, nevirapin, amprenavirH21c 416 15,463 B2 Amprenavir, abacavir, stavudinH22c 441 14,399 A3 Indinavir, ritonavir, didanosin, stavudinH23c 236 75,408 B3 Lamivudine, saquinavir, stavudinH24c 154 63,707 C3 Lamivudine, saquinavirH25c 467 9800 C3 Hydroxyurea, didanosin, nevirapin, stavudinH26c 177 293,864 C3 Amprenavir, lamivudine, ritonavir, zidovudinH27c 184 13,357 C3 Hydroxyurea, didanosin, nevirapin, stavudin

Inflammatory controlsubjectsd

I1b MesalamineI2b 6-mercaptopurine, mesalamine, fish oilI3b MesalamineI4b 6-mercaptopurine, mesalamine, fish oil, psylliumI5b Folic acid, prednisone, azathioprine, mesalamineI6c UnknownI7c UnknownI8c Unknown

Healthy controlsubjectse

N1b Glipizide, metformin, atorvastatin, estrogen, calcium,dioctyl sodium sulfosuccinate

N2b Prochlorperazine, diphenoxylate-atropine, naproxynN3b AmitriptylinN4b

N5b Hydromorphone, hyoscyamine sulfateN6c

N7c

N8c

N9c

N10c

N11c

N12c

N13c

N14c

NOTE. ND, not determined.a Classification of severity of HIV disease, as suggested by CDC in 1993 [30].b Analyzed by in situ imaging.c Analyzed by flow cytometry.d All patients had ulcerative colitis, except patients I3 and I7 who had Crohn’s disease.e Patients N2, N3, and N5 had functional bowel symptoms.

1628 Olsson et al. JID 2000;182 (December)

After an additional blocking with serum, secondary, biotinylatedantibodies were incubated at room temperature, followed by incu-bation with an avidin–biotin–horseradish peroxidase complex (Vec-tastain elite kit; Vector Laboratories). Color reaction was developedby 3′-diaminobenzidine tetrahydrochloride (Vector Laboratories),and the tissue samples were counterstained with hematoxylin. Thefollowing antibodies were used: monoclonal CCR5 (mixture of M,455449.111; K, 45531.111) and CXCR4 (44717.111); polyclonal af-finity-purified, biotinylated RANTES (BAF 278); MIP-1a (BAF270); MIP-1b (BAF 271; all from R&D Systems, Minneapolis); mon-oclonal CD4 (SK3) and CD8 (SK1; both from Becton Dickinson);secondary, biotinylated affinity-purified goat anti–mouse IgG1 andgoat anti–mouse IgG2b (both from Caltag Laboratories); and don-key anti-goat (Jackson ImmunoResearch Laboratories, West Grove,PA). Control stainings, without the primary antibody, were done onthe sections to control for nonspecific background. For antibodieswith subtype IgG1 (CD4 and CD8), an irrelevant mouse IgG1 wasused to control for subtype-specific nonspecific staining.

Quantification of chemokines, chemokine receptors, and cellularityby in situ imaging. Digital images of stained samples were trans-ferred from a DMR-X microscope (Leica, Wetzlar, Germany) intoa computerized image-analysis system (Quantimet 550IW; Leica,Cambridge, UK), which allowed for the detection of 16.7 milliondifferent colors. Two different methods of analysis were used. Thefirst method determined the percentage of positive area in the totalarea: 2–8 fields, depending on the size of the biopsy, with a totalmean area of mm2, were assessed for positive-stained area52.2 3 10and for the total area of hematoxylin-positive cells present. Theintestinal epithelium, including the crypts, was excluded. The fre-quency of positively stained area was measured in a semiquanti-tative way by use of a specialized software program [33], and resultswere expressed as the percentage of positive area of total tissuearea. Limiting dilution of cDNA-transfected cells injected into con-trol tonsils indicated a sensitivity of the assay of >1 positive cellper 1000 events [33]. This method was used for CCR5, CXCR4,RANTES, MIP-1a, and MIP-1b. The results achieved by this tech-nique will reach lower percentages than the results achieved as thepercentage of positive cells, since this technique only identifies theactual positively stained part of the whole cell.

The second method of analysis determined the percentage ofpositive cells and cellularity. In this method, the positive and neg-ative cells in the digital image were marked manually and werecounted by a specialized in situ imaging computer program (Quan-timet 550 IW; Leica, Cambridge, UK; program sequence used inthese analyses made by Ola Noren). Results were given as thepercentage of positive cells of all cells. Cellularity was reported asthe number of cells per square millimeter. The epithelium and co-lonic crypts were excluded from the area measured. This methodwas used for the quantification of CD4 and CD8 cells and of CCR5.Multiplying the frequency of positive cells by the number of cellsper square millimeter generated the absolute number of CD41 andCD81 cells.

Statistical analysis. The Mann-Whitney U test was used forstatistical analysis for comparison between study cohorts. Spear-man rank correlations were used to measure the correlation of CD4cell counts in blood and tissue. Comparisons yielding wereP ! .05considered significant.

Results

CD81 cells were significantly increased, and CD41 cells re-mained unchanged in number in gut lamina propria of HIV-1–infected patients. To define the degree of cellular inflamma-tion characterized by the change in numbers of CD41 andCD81 cells in HIV-1–infected gut lamina propria, biopsy spec-imens were immunohistochemically stained for CD4 and CD8cells and were evaluated by in situ imaging. We analyzed biopsyspecimens from 6 HIV-1–seropositive patients, 5 seronegativehealthy control subjects, and 5 seronegative inflammatory con-trol subjects with IBD (table 1). The frequency of CD41 cellswas significantly increased in the lamina propria of inflam-matory control subjects ( ), compared with that47.9% 5 20.0%in both HIV-1–infected patients ( ) and healthy26.6% 5 5.7%control subjects ( ; ). No statistical differ-23.3% 5 5.9% P ! .05ence in frequency of CD41 cells could be shown between HIV-1–infected patients and healthy control subjects.

There was a 4-fold mean increase in the number of CD81

cells in the gut lamina propria of HIV-1–infected subjects( ), compared with that in healthy control sub-19.4% 5 13.0%jects ( ; ). Inflammatory control subjects4.6% 5 1.8% P ! .005had a 2-fold increase in CD81 cells ( ), compared9.5% 5 3.4%with the increase for healthy control subjects ( ). TheP ! .05HIV-1–infected patients showed a higher mean number ofCD81 cells than did inflammatory control subjects, but no sta-tistical difference could be shown.

To evaluate the absolute cellular infiltration of the tissue, weanalyzed the total number of cells per area by in situ imaging.The total cellularity in the lamina propria was presented as cellsper square millimeter. The mean cellularity in inflammatory con-trol subjects ( cells/mm2) was ∼50% higher than11,430 5 4140that for noninflammatory healthy control subjects (7130 5

cells/mm2). HIV-1–infected patients showed a mean non-1412significant increase of 20% ( cells/mm2), compared8565 5 620with that of healthy control subjects. The absolute number ofCD41 plus CD81 cells per area was calculated by multiplyingthe percentage of CD41 plus CD81 cells by the total number ofcells per square millimeter, resulting in T cellularity. The meanT cellularity in HIV-infected patients ( cells/mm2)3990 5 1687was increased ∼2-fold, compared with that in healthy controlsubjects ( cells/mm2; ). Inflammatory control1990 5 585 P ! .05subjects had a mean 3.5-fold increase ( cells/mm2),6898 5 5030compared with that of healthy control subjects ( ; figureP ! .011). No statistical differences in the total T cellularity could befound between HIV-1–infected patients and inflammatory con-trol subjects.

CCR5 and CXCR4 expression was increased in the gut laminapropria of HIV-1–infected patients. To quantify the expressionof chemokine receptor expression in colon, we evaluated (by insitu imaging) biopsy specimens from 6 HIV-1–seropositive pa-tients, 5 HIV-1–seronegative inflammatory control subjects withIBD, and 5 HIV-1–seronegative healthy control subjects (table1). The expression of CCR5 in HIV-1–infected tissue reached a

JID 2000;182 (December) HIV-1 Chemokine Receptors and Ligands in Gut 1629

Figure 1. Combined T cellularity in colonic mucosa in human im-munodeficiency virus type 1 (HIV-1)–infected patients (mean, 3990 5

), inflammatory bowel disease (IBD) control subjects (1687 6898 5

), and healthy control subjects ( ), expressed as the num-5030 1990 5 585ber of T cells/mm2. T cellularity was calculated by multiplying the totalno. of cells/mm2 with the percentage of cells positive for CD4 and CD8,as determined by in situ imaging. HIV-1–infected patients had a mean2-fold increase, compared with that for healthy control subjects (P !

, Mann Whitney U test), whereas IBD control subjects showed a mean.053.5-fold increase, compared with that for healthy control subjects (P !

, Mann-Whitney U test). No statistical difference in total T cellularity.01was found between HIV-1–infected patients and IBD control subjects.Each symbol represents 1 patient.

mean of positively stained area of the total cell8.6% 5 6.3%area, corresponding to a ∼6-fold increase, compared with thatfor healthy control subjects ( ). The expression of CCR5P ! .01in inflammatory control subjects reached a mean of 9.1% 5

positively stained area of the total area, corresponding to9.2%a ∼7-fold increase, compared with that for healthy control sub-jects ( ; figures 2and 3A). Noninflammatory control sub-P ! .05jects had a mean of positively stained area.1.3% 5 1.2%

Additional computer-assisted manual counting analyses weredone on the same samples to detect the percentage of positivecells of all lamina propria cells. The frequency of CCR5-express-ing cells in HIV-1–infected tissue reached a mean of 63% 5

of all cells, corresponding to a ∼3-fold increase compared12.7%with that for healthy control subjects ( ). The frequencyP p .01of CCR5-expressing cells in inflammatory control subjectsreached a mean of of all cells, corresponding to a58% 5 34.2%∼2.5-fold increase, compared with that for healthy control sub-jects. Noninflammatory control subjects had a mean of 23% 5

positive cells (data not shown).13.3%The expression of CXCR4 in HIV-1–infected tissue reached

a mean of positive area of the total area, corre-8.5% 5 5.7%sponding to a ∼9-fold increase compared with that for healthycontrol subjects ( ). The expression of CXCR4 in inflam-P ! .01matory control subjects reached a mean of pos-10.6% 5 11.1%itive area, corresponding to an ∼11-fold increase, comparedwith that for healthy control subjects ( ; figure 3B).P ! .05

No significant correlation between CCR5 or CXCR4 expres-sion and virus load could be shown (data not shown). However,the 2 HIV-1–infected patients with the highest expression levels

of CCR5 and CXCR4 were the only patients with preserved CD4cells and virus loads 12000 copies/mL (HIV-1–infected patientsH1 and H5 in table 1).

Chemokine receptor expression in CD451 T lymphocytes incolonic mucosa. To characterize differences in the phenotypicexpression profiles of the chemokine receptors CCR5 andCXCR4 on CD41 and CD81 cells in the colonic mucosa, weused flow cytometry to characterize isolated MMC from colonbiopsy specimens from HIV-1 infected samples, healthy sero-negative control subjects, and inflammatory control subjectswith IBD (table 1). Initial gating to separate the lymphocyteswas done using side-scatter and panCD45 fluorescence. Thefrequency of CD41, CD81, CCR51, and CXCR41 cells wasdetermined. Triple labeling was performed for CD45/CD4/CCR5, CD45/CD8/CCR5, and CD45/CD4/CXCR4.

Flow cytometry assessment (HIV-1–infected patients H1, H3,H4, and H7–H15 in table 1) showed that the total frequency ofCD451 cells expressing CCR5 in gut lamina propria was similarin all 3 groups, with a mean of (range, 25%–92%)57.4% 5 17.5%positive cells (data not shown). No significant correlation to virusload could be shown (data not shown). Triple labeling for CD45/CD4/CCR5 (all HIV-1–infected patients in table 1) showed thatHIV-1–infected patients had a decrease in CD451CD41 cells ex-pressing CCR5 in the colonic mucosa, compared with that forboth healthy control subjects and inflammatory control subjects( for both). No significant correlation to virus load couldP ! .05be shown (data not shown). The mean incidence of CD451CD41

cells expressing CCR5 was in HIV-1–infected32.0% 5 18.2%patients, in inflammatory control subjects, and65.9% 5 23.2%

in healthy control subjects (figure 3C). In ad-53.6% 5 13.2%dition, there was a significant decrease in the mean number ofCCR5 molecules/CD451CD41 T cells in HIV-1–infected patients( molecules/cell), compared with that in healthy con-3202 5 1578trol subjects ( molecules/cell; ). The mean6854 5 4182 P ! .01number of CCR5 molecules/CD451CD41 cells in inflammatorycontrol subjects was molecules/cell (figure 3D). In3419 5 1250contrast to CCR5 expression on CD451CD41 cells, no statisticaldifference could be shown between cells from HIV-1–infectedbiopsy specimens and healthy controls when evaluatingCD451CD81 cells expressing CCR5 (mean, ; fig-91.0% 5 16.3%ure 3E) or CD451CD41 cells expressing CXCR4 (mean,

; data not shown). No significant correlation be-67.4% 5 12.9%tween CXCR4 expression and virus load could be shown (datanot shown).

The total frequency of CD451CD41 cells in HIV-1–infectedindividuals reached a mean of , corresponding to12.7% 5 4.6%a ∼2-fold decrease compared with that for healthy control sub-jects ( ; ). The frequency of CD451CD4128.2% 5 4.6% P ! .0001cells in inflammatory control subjects reached a mean of

, which was not significantly different from per-23.7% 5 19.7%centages seen in healthy control subjects ( ). The28.2% 5 4.6%frequency of CD41 cells in the mucosa of HIV-1–infected indi-viduals was correlated significantly with the CD4 cell count in

Figure 2. Top, Micrograph illustrating immunohistochemical staining for CCR5 in cryopreserved colonic mucosa of human immunodeficiencyvirus type 1 (HIV-1)–infected patient. Positive area was stained brown by 3′-diaminobenzidine tetrahydrochloride (DAB); all cells were counter-stained with hematoxylin. A, Crypts and epithelial cells were encircled (blue line) for exclusion, allowing analysis of the lamina propria area only.B, Detection of positively stained (yellow) area. C, Detection of the total cellular area (encircled with red lines). D, Composite graph of totalcellular area (red) and CCR5-positive area (green) revealed. Results achieved from this field: total area measured was 22891.39 mm2, cell areameasured was 14858.98 mm2, percentage of positive area in the total area was 11.01%. Bottom, Immunohistochemically stained CCR5-expressingcells (stained brown with DAB and counterstained with hematoxylin) in gut lamina propria from HIV-1–infected tissue sample (E), inflammatorybowel disease tissue sample (F), and healthy control tissue sample (G). Original magnification, 3220. Bar corresponds to 20 mm (E–G).

JID 2000;182 (December) HIV-1 Chemokine Receptors and Ligands in Gut 1631

Figure 3. Expression of human immunodeficiency virus type 1 (HIV-1) coreceptors in gut mucosa evaluated by in situ imaging of immuno-histochemically stained biopsy specimens (A and B), and by flow cytometry (C–E). Results in A and B are expressed as percentage of positivelystained area in the total area and in C–E as percentage of positively stained cells of the gated cells. A, Total expression of CCR5 in the gut laminapropria (mean expression in HIV-1–infected patients, ; in inflammatory control subjects [inflammatory bowel disease, IBD],8.6% 5 6.3%

; and in healthy control subjects, ). Statistical difference (Mann-Whitney U test) was achieved between HIV-1–infected9.1% 5 9.2% 1.3% 5 1.2%patients and healthy control subjects ( ) and IBD control subjects and healthy control subjects ( ). Each symbol represents 1 patient.P ! .01 P ! .05B, Total expression of CXCR4 in the lamina propria (mean expression in HIV-1–infected patients, ; in IBD control subjects,8.5% 5 5.7%

; and in healthy control subjects, ). Statistical difference (Mann-Whitney U test) was achieved between HIV-1–infected10.6% 5 11.1% 0.9% 5 0.4%patients and healthy control subjects ( ) and IBD control subjects and healthy control subjects ( ). Each symbol represents 1 patient.P ! .01 P ! .05C, Frequency of CD451CD41 cells expressing CCR5 (mean expression in HIV-1–infected patients, ; in IBD control subjects,32.0% 5 18.2%

; and in healthy control subjects, ). Statistical significance (Mann-Whitney U test) was achieved between HIV-65.9% 5 23.2% 53.6% 5 13.2%1–infected patients and healthy control subjects and IBD control subjects ( for both). D, Mean number of CCR5 molecules/P ! .05CD451CD41CCR51 cells for HIV-1–infected patients, IBD control subjects, and healthy control subjects. HIV-1–infected patients had a significantdecrease ( molecules/cell), compared with that of healthy control subjects ( molecules/cell; , Mann-Whitney U3202 5 1578 6854 5 4182 P ! .01test). Mean number of CCR5 molecules/CD451CD41CCR51 cells in IBD control subjects was . E, Frequency of CD451CD81 cells3419 5 1250expressing CCR5. There was no statistical difference among cohorts.

blood ( , ). The frequency of CD451CD81 cellsP ! .05 r p .406in HIV-1–infected individuals reached a mean of 46.1% 5

, a significant increase compared with that for healthy con-15.6%trol subjects ( ; ). The frequency of29.4% 5 10.2% P ! .05CD451CD81 cells in inflammatory control subjects reached amean of , which was not statistically different37.9% 5 22.1%from that for healthy control subjects.

The expression of RANTES, MIP-1a, and MIP-1b is in-creased in gut lamina propria from HIV-1–infected patients.To quantify the expression of the chemokine receptor ligandsin colon, in situ imaging was performed on biopsy specimensfrom 6 HIV-1–seropositive patients, 5 HIV-1–seronegative in-flammatory control subjects with IBD, and 5 HIV-1–seroneg-ative healthy control subjects (table 1). b-Chemokine RANTEShad a mean expression of positive lamina propria8.3% 5 3.6%area in HIV-1–infected subjects, corresponding to a ∼3-foldincrease, compared with that for healthy control subjects( ). The expression of RANTES in inflammatory controlP ! .05subjects showed a nonsignificant increase, compared with thatfor noninflammatory control subjects ( ), with a meanP p .22

of (figure 4A). The mean RANTES expression in4.4% 5 0.7%noninflammatory control subjects reached .2.6% 5 2.2%

MIP-1a in HIV-1–infected subjects was expressed over amean area of , corresponding to a ∼3-fold increase,1.0% 5 0.3%compared with that for healthy control subjects ( ; figureP ! .014B). Inflammatory and healthy control subjects showed a meanexpression of and , respectively.0.5% 5 0.4% 0.2% 5 0.2%

MIP-1b–positive cells had a mean expression of 5.7% 5

positive lamina propria area in HIV-1–infected patients,1.6%corresponding to a ∼30-fold increase, compared with that forhealthy control subjects ( ). MIP-1b had a mean expres-P ! .01sion of in inflammatory control subjects, corre-2.5% 5 1.3%sponding to a ∼24-fold increase, compared with that in healthycontrol subjects ( ), and the healthy control subjects hadP ! .01a mean MIP-1b expression of (figure 4C).0.2% 5 0.2%

Discussion

Here we showed that the total expression of both CCR5 andCXCR4 was significantly increased in HIV-1–infected lamina

1632 Olsson et al. JID 2000;182 (December)

Figure 4. Total expression of b-chemokines in lamina propria eval-uated by in situ imaging on immunohistochemically stained biopsy sec-tions. Results are expressed as percentage of positively stained area inthe total area. A, RANTES (mean expression in HIV-1–infected patients,

; in inflammatory control subjects [inflammatory bowel dis-8.3% 5 3.6%ease, IBD, 4.45 0.7%; and in healthy control subjects, ).2.6% 5 2.2%Significant difference (Mann-Whitney U test) was shown between HIV-1–infected patients and healthy control subjects ( ). Each symbolP ! .05represents 1 patient. B, Macrophage inflammatory protein (MIP)–1a

(mean expression in HIV-1–infected patients, ; in IBD con-1.0% 5 0.3%trol subjects, ; and in healthy control subjects,0.5 5 0.4% 0.2% 5

). A significant difference (Mann-Whitney U test) was shown be-0.2%tween HIV-1–infected patients and healthy control subjects ( ).P ! .005Each symbol represents 1 patient. C, MIP-1b (mean expression in HIV-1–infected patients, ; in IBD control subjects,5.7% 5 1.6% 2.5% 5

; and in healthy control subjects, ). A significant dif-1.3% 0.2% 5 0.2%ference (Mann-Whitney U test) was shown between HIV-1–infected pa-tients and healthy control subjects ( ) and between IBD controlP ! .005subjects and healthy control subjects ( ). Each symbol representsP ! .011 patient.

propria, compared with that in healthy control tissue samples.The pattern was similar in inflammatory control and HIV-1–in-fected tissues. These high expression levels suggest that HIV-1,in line with active IBD, is associated with a strong inflammatoryresponse in the gut lamina propria. Proinflammatory (tumor ne-crosis factor–a and interleukin-12) and type 1 (interferon-g andinterleukin-2) cytokines up-regulate HIV-1 coreceptor CCR5

mRNA expression, whereas type 2 cytokines up-regulate CXCR4[20, 21], suggesting that CCR5 and CXCR4 expression may dif-fer, depending on cytokine expression of the local milieu. In-creased CCR5 expression is correlated with inflammatory con-ditions [34]. In the cervical mucosa, both infectious andnoninfectious inflammation up-regulate CCR5 [35]. High levelsof CCR5 may have an impact on the prognosis of HIV-1–infectedpatients [36].

In this study, patients with plasma virus loads 12000 copies/mL and stable CD4 cell counts had the highest expression levelsof CCR5 and CXCR4 in the gut lamina propria (HIV-1–infectedpatients H1 and H5 in table 1). This suggests that mucosal in-flammation may facilitate viral replication and thereby worsenthe prognosis for HIV-1–infected individuals. However, no dif-ferences in CCR51 expression frequencies on total lymphocytes(CD45bright) between HIV-1–infected patients and inflammatoryand healthy control subjects could be detected by flow cytometry.Levels of CD41 T cells expressing CCR5 (the most importanttargets for HIV-1) were decreased in HIV-1–infected patients,compared with that of healthy and inflammatory control subjects( for both). An increased turnover of CD41 and CD81P ! .05cells has been suggested in HIV-1 infection [37–39]; however, therehave been arguments against this suggestion [40, 41]. These re-sults suggest a selective loss of the CD41 T cells expressing CCR5in HIV-1–infected lamina propria. Our findings of a selective lossof CD41CCR51 T cells combined with the increased T cellularitywould suggest an increased turnover of these cells at least withinthe intestinal mucosa.

There are several explanations for the apparent discrepanciesbetween results obtained from in situ imaging and those ob-tained from flow cytometry. First, flow cytometry was per-formed on cells obtained from mechanically and chemicallydisrupted tissues, whereas in situ imaging was done on intact,cryopreserved, tissue samples. Computer-assisted manualcounting of the immunohistochemically stained sections wasdone for CD4, CD8, and CCR5 cells. The results showed thatthe incidence of positive cells was much higher than the per-centage of positive area. This difference is simply due to thefact that the whole cell surface of a positive cell is not stained.The relative difference observed among the study cohorts re-mained, however, significant.

In the 3 patients analyzed by both methods, the percentagesof CD41 and CCR51 cells obtained by in situ imaging werehigher than those obtained by flow cytometry, whereas fre-quencies for CD81 cells were slightly lower. Immunohisto-chemistry and in situ imaging detect the total expression ofthese molecules in tissue, whereas only gated CD451 cells wereanalyzed by flow cytometry. One explanation is that cells otherthan T cells, such as macrophages and dendritic cells, expressCD4 and CCR5 molecules. This would give higher percentagesby in situ imaging. Nevertheless, as long as the chemokines andchemokine receptors were expressed in the mucosa, they wouldstill contribute to the inflammatory response. Another possible

JID 2000;182 (December) HIV-1 Chemokine Receptors and Ligands in Gut 1633

explanation may be that preapoptotic T cells were lost duringthe isolation procedure before flow cytometric analysis. Fur-thermore, preapoptotic cells in HIV-1 samples may predomi-nantly be the CD41 cells expressing CCR5, which we showedare decreased in HIV-1–infected colonic mucosa. Immunohisto-chemistry can be considered as an optional way to analyze totalexpression, whereas flow cytometry is used for subgroupanalyses.

Another explanation for the differences in expression of CD4,CD8, CCR5, and CXCR4 is that different patient materialswere used for the 2 experiments. However, the similarities re-garding plasma virus load and CD4 cell count between thecohorts strongly contradicts this explanation.

Because of different fixation protocols and the fact that noneof the primary antibodies generated significant signals in bothimmunohistochemistry and flow cytometry, different primary an-tibodies had to be used with each technique. This may havecontributed to some differences in results. The anti-CCR5 anti-bodies used for immunohistochemistry bind to the second loopof G-coupled proteins of the CCR5 molecule, which is affectedneither by virus nor by chemokine binding. A high binding ofvirus or chemokines to the receptor might block binding of theanti-CCR5 antibody used in flow cytometry, causing false lowresults. In this study, the CCR5 density on CD451CD41 cellswas decreased in HIV-1 patients, compared with that in healthycontrol subjects ( ), suggesting that high levels of b-chemo-P ! .01kines or virus may have down-regulated CCR5 expression [18,42, 43]. Nevertheless, the number of receptors per cell remainsabove the level identified for maximal infection in vitro [44].

Complex patterns of chemokine expression have been cor-related with many human inflammatory diseases [25, 29, 45].Triggering of inflammation in the upper respiratory tract causedincreased levels of RANTES and MIP-1a [46, 47]. Here weshowed that the expression levels of RANTES, MIP-1a, andMIP-1b were significantly increased in HIV-1–infected laminapropria, compared with that for healthy control subjects. Ofnote, the expression of b-chemokines was equal to, or evenhigher, in HIV-infected lamina propria than in active IBD sam-ples. The strong increase of b-chemokines suggests that HIV-1, similar to IBD, is associated with increased mucosal inflam-mation and immune activation.

MIP-1a, MIP-1b, and especially RANTES inhibit HIV-1 in-fection in vitro [18, 42, 43]. Hence, high levels may indicate aprotective effort against initial HIV-1 infection or spread. Theinitial trials that examined this were in vitro studies on isolatedcells in suspension and with supraphysiologic levels of RANTES.One recent vaccine study reported protective correlation of lowvirus load and high levels of b-chemokines in plasma [48]. Recentstudies addressing the in vivo function did not show any cor-relation between tissue virus load and the RANTES expressionin lymphoid tissue in already infected macaques and humans [49,50]. The high expression of RANTES and MIP-1b in HIV-in-fected tissue may rather, through its chemoattractant effects,

cause an increased influx of immunocompetent cells and resultin activation of CD41 and CD81 T cells, increasing potentialtargets for HIV-1 infection and possibly apoptosis. This supportsinterpretations that increased levels, despite being a natural re-sponse to inflammatory stimuli, may enhance rather than preventHIV-1 spread. In this study, all patients with a virus load 12000copies/mL showed high levels of RANTES (HIV-1–infected pa-tients H1, H5, and H6 in table 1), whereas the patients with lowervirus loads had a broader spectrum of expression. This wouldsuggest a correlation between high levels of RANTES and highplasma virus load, although our cohort was too small to assessthis.

Increased levels of chemoattractant b-chemokines, soluble me-diators of inflammation, would probably contribute to an in-creased cellular component of inflammation in the tissue. In-creased cellularity is also a strong sign of immune activation inthe gut mucosa. A significantly increased total cellularity wasdetected in inflammatory control subjects ( ). In HIV-1–P ! .05infected tissue samples, we showed an increased mean cellularitythat approached significance ( ), and, of importance, thereP p .1was no reduction in cell numbers. Other reports have not revealedany difference in cellularity between HIV-1 patients receivinghighly active antiretroviral therapy and in various disease stagesand healthy control subjects [51]. However, when selectively eval-uating the absolute number of T cells, we showed a significantincrease in both HIV-1–infected patients ( ) and inflam-P ! .05matory control subjects ( ), compared with that in healthyP ! .01control subjects, suggesting cellular inflammation in gut mucosain HIV-1–infected individuals.

These results suggest that the increased levels of b-chemo-kines are attracting cells to the gut lamina propria in both HIV-1–infected patients and inflammatory control subjects. Sincethe CD81 cells are increased and the CD41 T cells are decreasedor unchanged, a selective increased apoptosis among the CD41

cells could be hypothesized [4, 37]. The most likely explanationfor the overall observation is a combination of increased influxof total cells coupled with an increased turnover of CD41 cells.This hypothesis is further supported by the fact that the fre-quency of CD451CD41 cells expressing CCR5, the most im-portant targets for HIV-1, was selectively lower in HIV-1 pa-tients than in inflammatory and healthy control subjects whenevaluated by flow cytometry. Human gut mucosa may, as hasbeen demonstrated in macaque models [8], be an important sitefor CD41 T cell loss.

Here we showed that HIV-1 infection was associated with sig-nificantly increased expression of the chemoattractant chemo-kines (RANTES, MIP-1a, and MIP-1b), suggesting continuouslyincreased soluble mediators of inflammation in the lamina pro-pria of the colon. The increased T cellularity in HIV-1–infectedpatients shows that there is also cellular inflammation in the gutlamina propria. As in IBD, HIV-1 may cause mucosal inflam-mation. This inflammatory response may worsen the prognosisfor HIV-1 patients by causing recruitment of additional potential

1634 Olsson et al. JID 2000;182 (December)

targets for HIV-1 infection and thereby contribute to the spreadof HIV-1 and further CD4 T cell loss. To further investigate thiseffect in HIV-1 mucosal pathogenesis, therapies that reduce in-flammation in the intestinal mucosa must be evaluated.

Acknowledgments

We thank Lena Radler (Division of Infectious Diseases, KarolinskaInstitute, Stockholm) for expert technical assistance and Marie Fuerst(University of California, Los Angeles, Center for HIV and DigestiveDiseases) for assistance in clinical data collation. We also thank MonicaTsang (R&D Systems, Minneapolis) for the kind gift of monoclonalantibodies, the University of California Los Angeles Center for AIDSResearch Core Laboratories of Mucosal Immunology and Flow Cyto-metry, Alan Dine (Proctor and Gamble Pharmaceuticals, Cincinnati),and Procter and Gamble Pharmaceuticals.

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