INTRODUCTION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

HIV-infected patients may present with respiratory complications related to alterations in pulmonary immune regulation. Early in disease, HIV may cause an interstitial lung disease (ILD) associated with a marked infiltration of T cells in the lung interstitium and alveolar spaces (1). Lymphocyte alveolitis is mainly due to the compartmentalization of cytotoxic T lymphocytes (CTL), which account for the major effector arm of the lung immune system against HIV: they have anti-HIV activity and prevent viral spread into the surrounding microenvironment (2, 3). Nevertheless, over time the effects of HIV on lung immunocompetence include a progressive decline in pulmonary CTL response; the starvation of tissue CTL activity favors the appearance of opportunistic infections (2).

The mechanisms accounting for the accumulation of HIV-specific CTL in the respiratory tract have been recently investigated (4, 5). Compelling evidence suggests that cytokine networks are involved in the development of T-cell alveolitis; in particular, macrophage-derived cytokines such as IL-15 and TNF- define regulatory interactions, which upregulate the expression of molecules involved in antigen-presenting cell (APC)/T-cell contact and initiate cell cycle progression in resident pulmonary lymphocytes. A second mechanism that accounts for T-cell alveolitis is the enlisting of T cells into the lung in response to not yet characterized chemoattractant molecules.

Candidate factors that regulate CTL migration in the lung of HIV-infected patients include chemokines, which are cytokines that direct leukocyte migration (6). The non-ERL CXC-chemokines (CXC chemokines without the Glu-Leu-Arg [ERL] motif before the CXC motif) are most likely to be involved since they attract activated T lymphocytes (10). The expression of non-ERL CXC-chemokines such as interferon- (IFN-)-inducible protein (IP-10) monokine induced by IFN- (Mig) and IFN--inducible T-cell-chemoattractant (I-TAC) is dramatically enhanced by IFN-, a cytokine that is upregulated in the lungs of HIV-infected persons (11). In addition, these selective chemoattractants share a common receptor, CXCR3, which is highly expressed by activated lymphocytes (12), including CTL and natural killer (NK) cells. Finally, in several ILDs associated with an upregulation of IFN- production, including sarcoidosis (13) and tuberculosis (14), CXCR3-ligands have recently been shown to play a role in the recruitment of activated CXCR3+ T cells into inflamed pulmonary tissues.

To investigate the mechanisms favoring infiltration of CTL in the lung of HIV-infected persons, we evaluated the expression of CXCR3 on lung T cells from HIV-infected patients, and evaluated the contribution of IP-10 and Mig to local T-cell recruitment. Our data suggest that the local production of ligands that bind to CXCR3 is involved in the pathophysiology of HIV-associated T-cell alveolitis.

	METHODS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Study Populations

Eleven HIV-seropositive patients (nine men and two women; average age, 35.5 ± 5.5 yr; 10 nonsmokers, one smoker) underwent bronchoalveolar lavage (BAL) evaluation. According to CDC classification, four patients were previously asymptomatic patients who during their follow-up showed clinical pulmonary symptoms and/or signs of ILD other than, or in addition to, lymphoadenopathy (Patients 1 to 4 in Table 1). Two patients were classified in Category B of the CDC classification and had previously received antiretroviral therapy (Patients 5 and 6 in Table 1). Five patients were classified in Category C and had full-blown AIDS and were receiving antiretroviral therapy (Patients 7 to 11 in Table 1); all of them had less than 150 peripheral blood CD4+ cells/mm³ and less than 400 peripheral blood CD8+ cells/mm³.

Seven healthy HIV-seronegative adult control subjects were selected (five men and two women; average age, 31.5 ± 4.1 yr; two nonsmoking healthy adults and five nonsmoking subjects evaluated for complaints of cough without lung disease). They showed normal physical examinations, chest radiographs, lung function tests, and BAL cell numbers.

Preparation of Cell Suspensions

The BAL was performed according to the technical recommendations and guidelines for the standardization of BAL procedures (15). Briefly, a total of 200 ml of saline solution was injected in 25-ml aliquots via fiberoptic bronchoscopy, with immediate vacuum aspiration after each aliquot. Immediately after the BAL, the fluid was filtered through gauze and the volume was measured. A volume of 100 to 200 ml of BAL recovery and a sample of 50% of the instilled volume with a minimum of 50 ml was considered acceptable. The percentage of BAL recovery was 53.7 ± 4.2% and 55.1 ± 3.7% of the injected fluid in patients with HIV-1 infection and control subjects, respectively. Cells recovered from the BAL were washed three times with PBS, resuspended in endotoxin-tested RPMI 1640 (Sigma Chemical Co., St. Louis, MO) supplemented with 20 mM HEPES and L-glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin, and 10% FCS (ICN Biochemicals, Costa Mesa, CA) and then counted. Macrophages, lymphocytes, neutrophils, and eosinophils were differentially counted in a total count of 300 cells according to morphologic criteria in cytocentrifuged smears stained with Wright-Giemsa.

Purification of Alveolar Macrophages and T Cells

Alveolar macrophages (AMs) were enriched from the BAL cell suspensions by rosetting with neuraminidase-treated SRBC followed by Ficoll/Hypaque (F/H) gradient separations (5). AMs were further enriched by removing residual CD3⁺, CD16⁺, and CD56⁺ lymphocytes using high-gradient magnetic separation columns (Mini MACS; Miltenyi Biotec, Bergisch Gladbach, Germany), as previously described (5). After this multistep selection procedure, more than 95% of the above cells were viable, as judged by the trypan blue exclusion test. The staining with mAb showed that more than 98% of BAL cells expressed the AM-associated CD68 antigen.

The cell suspension of peripheral blood mononuclear cells was depleted of adherent cells by incubation for 45 min in plastic Petri dishes at 37° C in an atmosphere of 95% air and 5% CO₂. T cells were enriched from the resulting cell suspensions by rosetting with neuraminidase-treated SRBC followed by F/H gradient separations, as previously described (5).

Monoclonal Antibodies and Cytokines

The commercially available conjugated or unconjugated mAbs used belonged to the Becton-Dickinson and PharMingen series and included: CD3, CD4, CD8, CD28, CD45R0, CD45RA, and isotype-matched controls. Anti-IL-15 M110 (IgG₁) and anti-IL-15 receptor (IL-15R) (IgG₁) mAbs were kindly provided by Dr. A. Troutt (Immunex Co., Seattle, WA); anti-TNF- (MAB11), anti-IL-4 (8D4-8), and anti-IFN- (4S.B3) mAbs were purchased from PharMingen (San Diego, CA). Purified rabbit antihuman IP-10 polyclonal antibody, anti-Mig mAb (R&D Systems Inc., Minneapolis, MN) and anti-hCXCR3 mAb (1C6; Leukocyte Inc., Cambridge, MA) were also used.

IL-15 was kindly provided by Dr. A. Troutt (Immunex Co). IFN-, rhIP-10, and rhMig chemokines were purchased from R&D Systems.

Phenotypic Evaluation of BAL Cells

The frequency of BAL cells positive for the above reagents was determined by overlaying the flow cytometry histograms of the samples stained with the different reagents as previously reported (5). Cells were scored using a FACScan analyzer (Becton-Dickinson, Rutherford, NJ) and data were processed using the Macintosh CELLQuest software program (Becton-Dickinson). The expression of cytoplasmic cytokine was evaluated after permeabilization of cell membranes using 1:2 diluted PermeaFix (Ortho, Raritan, NJ) for 40 min. After permeabilization procedures anti-IL-15, anti-IL-4, anti-IFN-, anti-IP-10, and anti-Mig antibodies were added. Because pulmonary cells bore cytoplasmic cytokine in a unimodal expression pattern, indicating that the entire cell population exhibits relatively homogeneous fluorescence, the percentage of positive cells does not represent the most accurate way of enumerating positive cells. For this reason, the mean fluorescence intensity (MFI) was used to compare the positivity of these specific antigens on different cell populations. To evaluate whether the shift of the positive cell peak was statistically significant, the Kolmogorov-Smirnov test for analysis of histograms was used according to the Macintosh CELLQuest software user's guide (Becton-Dickinson).

For immunofluorescence analysis, control IgG₁ and IgG_2a and IgG_2b were obtained from Becton-Dickinson; control rat antiserum consisted of ascites containing an irrelevant rat IgG_2b (kindly provided by Dr. A. Rosato, Padova, Italy); control rabbit antiserum consisted of rabbit IgG (purified protein) purchased from Serotec (Serotec, UK); goat antirabbit IgG and goat F(ab')2 antirat IgG were obtained from Immunotech (Marseille, France).

Generation of Macrophage Supernatants

To verify the ability of AMs to release the chemokine, unstimulated AMs (1 × 10⁶/ml) were isolated from both HIV-infected patients and healthy subjects, resuspended in RPMI medium, and cultured for 24 h in 24-well plates at 37° C in 5% CO₂. In separate experiments AMs were stimulated with IFN- (100 U/ml), PMA (10 ng/ml), and LPS (10 µg/ml) (Difco Laboratories, Detroit, MI). After the incubation period, supernatants were harvested, filtered through a 0.45-µm Millipore filter, and immediately stored at 80° C. At the end of the culture time AM viability was always greater than 95%. Chemotactic activity of supernatants was determined as reported below.

Migration Activity of Pulmonary T Cells in Response to CXCR Chemokines

T-cell migration was measured in a 48-well modified Boyden chamber (AC48; Neuroprobe, Bethesda, MD). The chamber is made of two sections: different chemotactic stimuli were loaded in the bottom section and cells were added in the top compartment. Polyvinylpyrrolidone-free polycarbonate membranes with 3- to 5-µm pores (for HIV patients and cell lines, respectively) (Osmonics, Livermore, CA) and coated with fibronectin were placed between the two chamber parts. Only the bottom face of the filters was pretreated with fibronectin in order to increase the adherence of migrated cells. Before use, fibronectin-treated filters were extensively washed in order to avoid the shedding of fibronectin. In preliminary experiments we demonstrated that fibronectin-treated filters did not induce spontaneous chemotaxis in the absence of chemokines.

rhIP-10 (200 ng/ml) and rhMig (1,000 ng/ml) chemokines were used to evaluate the migratory properties of pulmonary T lymphocytes. The CXCR3 and CXCR3+ cell lines (300-19, kindly provided by Dr. B. Moser, Theodor-Kocher Institute, University of Bern, Switzerland) were used as negative and positive controls. Then 30 µl of chemokines or control medium were added to the bottom wells, and 50 µl of 5.0 × 10⁶ cells/ml T cells or CXCR3/+ cells resuspended in RPMI 1640 were added to the top wells. The chamber was incubated at 37° C with 5% CO₂ for 2 h. The membranes were then removed, washed with PBS on the upper side, fixed and stained with Diff-Quik (Dade AG, Düdingen, Switzerland). Cells were counted at magnification ×800 in three fields per well. All assays were performed in triplicate. In blocking experiments, cell suspensions were preincubated before chemotaxis assay for 30 min at 4° C with antihuman CXCR3 mAb at the concentration of 20 µg/ml.

Chemotactic Activity of AM Supernatants

The CXCR3 and CXCR3+ cell lines were also used to evaluate the chemotactic activities of AM supernatants. Supernatants from cell cultures obtained as reported above were used undiluted; different concentrations of IP-10 and Mig were utilized as positive control. Chemotactic assays were performed as reported above. In blocking experiments anti-IP-10 and anti-Mig mAbs were added to the cell supernatants before chemotaxis assay at the concentration of 20 µg/ml.

Statistical Analysis

Data were analyzed with the assistance of the Statistical Analysis System. Data are expressed as mean ± SD. Mean values were compared using the ANOVA test. To investigate the correlation coefficients (r) between IP-10 levels and BAL cell findings, Spearman's nonparametric Rank correlation test was used. A p value < 0.05 was considered as significant.

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Differential BAL cell counts in 11 HIV-infected patients and seven normal subjects are reported in Table 1. In all normal subjects the total cell recovery ranged from 95,000 to 190,000 cells/ml of BAL fluid; a percentage, ranging from 5 to 8% of BAL cells, were lymphocytes; both CD4 helper-related and CD8 cytotoxic/suppressor-related cells were present in approximately the same proportions as in peripheral blood (BAL CD4/CD8 ratio, 2.04 ± 0.3).

Cell recovery was significantly higher in six of the 11 patients with HIV infection (p < 0.001) who were defined as having an alveolitis. With regard to the differential count of BAL cells, these patients showed a T-cell alveolitis documented by the increased absolute number of CD8+ T cells (range, 66.0 to 106.6 × 10³ T cells/ml BAL fluid versus 8.6 ± 2.0 × 10³ T cells/ml BAL fluid in normal subjects); CD4+ T cells were less than 5% in all BAL specimens. As a consequence of the decrease in the CD4⁺ T-cell population and the increase of CD8⁺ T cells the BAL CD4/CD8 ratio was significantly decreased (CD4/CD8: 0.03 ± 0.02 versus 2.04 ± 0.3 of normal subjects, p < 0.001).

In five patients with advanced HIV infection and who were receiving antiretroviral therapy (Patients 7 to 11 in Table 1), cell recovery was below or within the normal range. The absolute number of BAL T cells was normal or decreased (range, 1.6 to 5.5 × 10³ T cells/ml BAL fluid versus 8.6 ± 2.0 × 10³ T cells/ml BAL fluid in normal subjects); the CD4/CD8 ratio was significantly decreased in all these patients since CD4+ T cells were virtually absent in BAL fluid (CD4/CD8: 0.01 ± 0.01 versus 2.04 ± 0.3 of normal subjects, p < 0.001).

BAL T Cells from Patients with HIV Infection are CD8+ T Cells Expressing IFN- and an Upmodulation of CXCR3

Profiles shown in Figure 1 are representative of the six HIV-infected patients with T-cell alveolitis, the five HIV-infected patients without T-cell alveolitis, and the four uninfected, normal subjects. As shown in Table 1, CD8+ T cells were markedly increased in the lung of patients with alveolitis. Flow cytometry analysis showed that these cells were preactivated CXR3+ T cells bearing IFN- but not IL-4, a pattern that has been reported to be characteristic of Tc1 cells (16). In all patients with T-cell alveolitis, CXCR3 was found to be expressed at higher intensity than in patients without T-cell alveolitis or in normal subjects. In fact, as shown in Figure 1, normal BAL T cells and T cells from patients with normal cellular recovery expressed low or no levels of CXCR3 and did not express cytoplasmic cytokines.

View larger version (32K): [in this window] [in a new window]   Figure 1.   The flow cytometry profile of BAL T cells recovered from two representative patients with T-cell alveolitis (Patients 2 and 4 in Table 1), two representative patients with normal T-cell recovery (Patients 9 and 10 in Table 1) and two healthy control subjects (control subjects 1 and 2). The expression of CXCR3 and cytoplasmic cytokines was determined as reported in METHODS. BAL T cells isolated from the lung of HIV-infected patients with T-cell alveolitis are CD8+ T cells (Table 1) bearing CXCR3 and IFN-. This phenotype is characteristic of Tc1-type T cells.

CXCR3 Expressed by Pulmonary CD8+ T Cells from HIV-Infected Subjects Mediates Their Chemotaxis

To characterize the biologic properties of the CXCR3 chemokine receptor, highly purified T cells isolated from the BAL of patients with T-cell alveolitis were assessed for their migratory capabilities in response to different concentrations of IP-10 and Mig. The impossibility of recovering an adequate number of pulmonary CD8+ T cells from the BAL of HIV- infected subjects who did not have a CD8+ T-cell alveolitis and normal subjects prevented the evaluation of the migratory capabilities of lung T cells in these subjects. For this reason, the 300-19 T-cell line expressing high levels of CXCR3 or not expressing CXCR3 were used as positive and negative controls for the in vitro chemotaxis assay.

As shown in Figure 2, pulmonary T cells expressing CXCR3 exhibited a high migratory capability in response to CXCR3 ligands. The migratory capability was influenced by CXCR expression. In fact, to further verify the functional role of CXCR3 in signaling lung T-cell chemotaxis, in parallel experiments pulmonary T cells responsive to IP-10 and Mig were preincubated with anti-CXCR3 mAb (Figure 3). The blocking of the CXCR3 receptor with specific antibodies determined a marked inhibition of both IP-10- and Mig-induced chemotaxis. Taken together, these data suggest that pulmonary CD8+ T cells from HIV-infected patients with T-cell alveolitis express a functional CXCR3 receptor and actively migrate in response to CXCR3 ligands.

View larger version (19K): [in this window] [in a new window]   Figure 2.   Chemotactic activity of IP-10 (20 ng/ml) and Mig (100 ng/ml) on BAL Tc1 cells obtained from the BAL of three HIV-infected patients with T-cell alveolitis and on the CXCR3+ T-cell line (positive control). Flow cytometry reveals that both BAL Tc1 cells (A, B, and C ) and CXCR3+ T-cell line (D) expressed high levels of CXCR3. Panels E-H show chemotactic activity of IP-10 and Mig using a modified Boyden chamber. The assays were performed in triplicate, and data are given as mean ± SD. Both CXCR3 ligands exerted significant chemotactic activity on BAL Tc1 cells (E, F, and G) and the CXCR3+ T-cell clone (H ). CXCR3 negative T-cell clone did not migrate in response to IP-10 and Mig (negative control; data not shown).

View larger version (14K): [in this window] [in a new window]   Figure 3.   The effects of anti-CXCR3 monoclonal antibodies on the IP-10- and Mig-dependent migratory capabilities of pulmonary T cells isolated from a representative patient with T-cell alveolitis (Patient 6) and the CXCR3+ T-cell line. When added to the assays at the beginning of culture, the above-quoted antibody significantly inhibited the migration of both highly purified pulmonary Tc1 cells and CXCR3+ T-cell line. The assays were performed in triplicate, and data are given as mean ± SD.

Pulmonary Macrophages Express CXCR3 Chemokines

Because AMs represent a source of cytokine hyperproduction in the lungs of HIV-infected persons, in a second set of experiments we evaluated whether these cells express and release ligands capable of interacting with CXCR3 expressed by pulmonary CD8+ T cells.

Flow cytometry analysis of IP-10 and Mig expression by pulmonary macrophages is shown in Figure 4. AMs isolated from patients with CD8+ T cell alveolitis bore IP-10 protein, whereas Mig was expressed by AMs of three of the six patients. Specifically, in BAL samples expressing these chemokines, a percentage ranging from 51 to 78% of AMs bore IP-10 and a percentage ranging from 44 to 69% of AMs bore Mig. Nevertheless, as demonstrated by the Kolmogorov-Smirnov analysis, in all subjects the peak of positive IP-10 and Mig cells was significantly shifted with respect to the negative controls, indicating that the entire AM population bears these cytokines.

View larger version (35K): [in this window] [in a new window]   Figure 4.   Flow cytometry profile of AMs recovered from two representative patients with T-cell alveolitis (Patients 1 and 6 in Table 1), two representative patients with normal T-cell recovery (Patients 7 and 9 in Table 1) and two healthy subjects (control subjects 2 and 3). The expression of cytoplasmic cytokines was determined as reported in METHODS. Alveolar macrophages isolated from the lung of HIV-infected patients with T-cell alveolitis express the CXCR3 ligands IP-10 and Mig.

Profiles shown in Patients 7 and 9 are representative of IP-10 and Mig staining in patients with normal BAL cell recovery. Pulmonary macrophages from 1 of the 5 patients expressed low levels of cytoplasmic IP-10 but not Mig; other samples were negative. Pulmonary macrophages from control subjects expressed low levels or did not bear IP-10 and Mig. BAL T cells from both HIV patients and control subjects did not show CXCR3 ligand expression.

Because IL-15 is able to modulate chemokine and chemokine receptors (17) and IL-15 is actively released in the lungs of HIV-infected persons (18), we verified whether BAL cells bearing CXCR3 ligand coexpress IL-15. A percentage ranging from 51 to 67% of AMs from patients with T-cell alveolitis expressed IL-15 (mean, 70.1 ± 7.3%). Interestingly, BAL samples expressing IL-15 also expressed IP-10 or Mig (Figure 4). Less than 5% of AMs isolated from HIV-infected patients with normal cell recovery and AMs isolated from HIV-negative normal subjects bore IL-15. Taken together, these data indicate that AMs from patients with HIV infection are in state of activation with respect to normal AMs.

AM Supernatants Show Biologic Activity on the CXCR3+ T cell line and CXCR3 Ligands May Be Demonstrated in the BAL Fluid

We also evaluated whether AMs from patients with HIV infection and high intensity CD8+ T-cell alveolitis release functionally active CXCR3 ligands that mediate T-cell chemotaxis. Cell-free supernatants were obtained from AMs cultured in different experimental conditions and tested for their capabilities of inducing T-cell migration.

As shown in Figure 5, after 24 h of culture, supernatants obtained from unstimulated AMs isolated from six patients with T-cell alveolitis exerted significant chemotactic activity on the CXCR3+ cell line; the CXCR3 cell line did not migrate in presence of AM supernatants (data not shown). Interestingly, AMs obtained from patients with T-cell alveolitis and cultured for 24 h in the absence of stimulation exerted significantly higher chemotactic activity on the CXCR3+ cell line than did unstimulated AMs of patients with normal BAL (p > 0.001) or control subjects (p < 0.01). When AMs were cultured in the presence of IFN- and, to a lesser extent, LPS, the chemotactic activity of cell-free supernatants from HIV-infected patients and normal subjects was higher than the levels obtained from unstimulated AMs (data not shown).

View larger version (9K): [in this window] [in a new window]   Figure 5.   Chemotactic activity of AM supernatants on the CXCR3+ 300-19 T-cell line. The three columns show chemotactic activity of supernatants obtained from unstimulated AMs of six patients with T-cell alveolitis, five patients without T-cell alveolitis, and four normal subjects, respectively. Data are given as mean ± SD of pooled results obtained in different groups of patients. AMs obtained from patients with T-cell alveolitis and cultured for 24 h in absence of stimulation exerted significantly higher chemotactic activity on the CXCR3+ T cell clone than did unstimulated AMs of patients without T-cell alveolitis or normal subjects.

To evaluate whether the ability of AM supernatants to induce T-cell line migration was mediated by Mig and IP-10 and/ or by other proteins, specific neutralizing antibodies were added to AM supernatants. The addition of anti-IP-10 and anti-Mig antibodies (Figure 6), but not of a control antibody (data not shown), inhibited chemotactic activities of supernatants. Interestingly, in Patient 2 and, to a lesser extent, in Patients 3 and 6 the presence of both anti-IP-10 and anti-Mig antibodies did not completely abrogate the migratory capability induced by AM supernatants, indicating that other chemotactic proteins able to interact with the CXCR3+ T-cell clone are likely to be released by AMs of patients with T-cell alveolitis (e.g., I-TAC and other unknown CXCR3 ligand).

View larger version (17K): [in this window] [in a new window]   Figure 6.   The effects of anti-IP-10 and anti-Mig neutralizing antibodies on the chemotactic activity of supernatants obtained from unstimulated AMs of three representative patients with T-cell alveolitis. The above-quoted antibodies, when added to the assays at the beginning of culture, significantly inhibited the migration of CXCR3+ T-cell line. The assays were performed in triplicate, and data are given as mean ± SD.

In order to evaluate whether CXCR3 ligands are actively released in the pulmonary microenvironment, the fluid component of nine BAL samples was evaluated for their chemotactic activity on the CXCR3+ cell line (Figure 7); a definite biologic activity was demonstrated in four of the nine samples examined.

View larger version (21K): [in this window] [in a new window]   Figure 7.   Chemotactic activity exerted by the fluid components of the BAL on the CXCR3+ T-cell line (A and B from patients with and without T-cell alveolitis, respectively), and, as a comparison, migratory response of the same cell line in response to IP-10 and Mig (B). The assays were performed in triplicate, and data are given as mean ± SD.

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Pulmonary infection with HIV may result in a massive lymphocyte infiltration characterized by the accumulation of activated CD8+ T cells. The findings that these cells express high levels of IFN- and CXCR3 and migrate in response to IP-10 and Mig suggest a role for CXCR3 ligands in augmenting T-cell recruitment to the lung of HIV-infected persons. Furthermore, the fact that AMs strongly express IP-10 and Mig and release CXCR3 ligands that exert chemotactic activity on CXCR3+ T-cell lines further points out the involvement of macrophages in the migration of CTL into peripheral tissues of HIV infected patients.

Recent data have suggested the role of IFN- and IFN--dependent chemokine IP-10 in the development of CD8+ T-cell immune responses taking place in the lung of the cynomolgus macaques inoculated intravenously with a pathogenic isolate of simian immunodeficiency virus (19, 20). In the current study we have provided the first evidence for a connection between the expression of IFN--dependent lymphotactic chemokines and the development of a local CTL immune response during a human retroviral infection: pulmonary CD8+ T cells from patients with HIV infection express IFN- and CXCR3 at high density, a phenotype characterizing Tc1 cells, and lung immunocompetent cells of HIV-infected patients appear to be a major source of CXCR3 ligands.

The high expression of CXCR3 by T cells retrieved from the BAL of patients with HIV infection might be considered a by-product of the in vivo cell hyperactivity of the pulmonary T-cell compartment in this disease. In fact, recent data clearly indicate that CXCR3 and its ligands become functional on recently activated T cells, including CD8 T cells (12). After antigenic challenge or in response to stimulation through the T-cell receptor (TCR), T cells express CXCR3, respond with chemotaxis to CXCR3 ligands, and produce IFN-. Furthermore, in the presence of persistent antigenic stimulations, CXCR3 expression is maintained and poised for rapid upregulation with reactivation (12). A similar sequence of events could take place in the lungs of HIV-infected persons. In fact, as previously reported (21), the study of the molecular organization of the TCR has made it possible to determine that T cells proliferating in the lungs of HIV-infected persons show a preferential usage of definite TCR gene regions, thus indicating an ordered immune response in which the TCR has been triggered. In addition, Twigg and colleagues (11) recently showed that pulmonary CD8+ T cells of HIV-infected patients are preactivated T cells expressing IFN- (Tc1).

The lung is a site of viral replication since the early phases of HIV disease. It is expected that, after the establishment of viral infection, upmodulation of functional CXCR3 directs CD8 precursors from secondary lymphoid tissues to the alveolar spaces where the CXCR3 ligands (Mig and IP-10) are being expressed. As shown in Figure 6, anti-IP-10 and anti-Mig antibodies only partially inhibited the migration of the CXCR3+ 300-19 T-cell line in response to AM supernatants. This indirectly suggests that other CXCR3 ligands are actively produced by AMs during HIV infection. Further studies are in progress in our laboratories to evaluate the functional importance of the chemokine repertoire in the regulation of T-cell traffic within the lung. In particular, given the ability of I-TAC to favor T-cell recruitment (14), we are investigating whether this newly identified non-ERL chemokine may influence entry of T cells into the lower respiratory tract of patients with different interstitial lung disorder.

Concerning the molecules that favor the expression of CXCR3 ligands in the lungs of HIV-infected persons, IFN- and IL-15 are ideal candidates to regulate the chemokine- induced recruitment of HIV-specific CTL. Although IL-15 per se may have chemotactic activity on T cells, there are data suggesting that the effects of IL-15 on T-cell motility are more similar to chemokinesis than chemotaxis because of its ability to stimulate motility in the absence of an established chemotactic gradient (22). Besides influencing Tc1-cell proliferation, IL-15 is a potent inducer of CC-, CXC-, and C-type chemokines (17). Furthermore, there are data indicating that IL-15 favors the expression of CXCR3 in other inflammatory diseases, including rheumatoid arthritis, multiple sclerosis, and sarcoidosis (13, 23, 24). Alveolar macrophages from HIV- infected patients constitutively express high levels of IL-15 (18). Because IFN- increases the expression of CXCR ligands (17) and favors IL-15 expression on cells belonging to macrophagic lineage (25), it is conceivable that IFN-, IL-15, and lymphotactic chemokines act in concert to sustain the inflammatory response against HIV. In line with this interpretation, it has been recently reported that IP-10 selectively upregulates human T-cell cytokine synthesis, with enhancement selectively targeted toward the promotion of IFN- expression (Th1-like) (26). This suggests a potential role for T-cell-focused chemokines in maintenance of the default Th1/Tc1 responses usually seen in response to environmental antigens. It remains to be established whether bronchial and endothelial cells, which may express IL-15 and CXCR ligand (14), efficiently cooperate in inducing the HIV-associated alveolitis, as demonstrated in other ILD in which IFN- is upregulated, including pulmonary tuberculosis (14).

It is important to mention that the effectiveness of the pulmonary immune system in controlling HIV infection varies during the different phases of HIV disease. In particular, a progressive decline in CTL activity and number can be documented during the course of HIV disease (1). In theory the complex chemokine-driven regulation of CTL trafficking might help the spread of the virus to tertiary lymphoid tissues. We clearly demonstrated that CD8+ T cells are HIV- infected in the lung of most patients with AIDS (27), the viral load of pulmonary CD8+ T cells being consistently higher than in the corresponding samples of peripheral blood CD8+ T cells (28). To explain how HIV gains entry into CD8+ T lymphocytes, it can be hypothesized that infected CD8 T cells derive from chemokine-recruited precursors originating from HIV-infected secondary lymphoid tissues and transiently coexpress CD4 antigen during their in vivo differentiation. In line with this interpretation, Kitchen and colleagues (29) recently reported that differentiation from a CD4⁺ precursor may allow productive infection of CD4-negative T cells. If this were the case, the network between IFN-, IL-15, and lymphotactic chemokines might paradoxically favor HIV infection of organs characterized by a massive CD8+ T-cell infiltration, including lung, lymph nodes, liver, salivary glands, kidney, and bone marrow of patients with diffuse infiltrative CD8+ lymphocytosis syndrome (30). Furthermore, an alteration of the pattern of cytokine production by lung immunocompetent cells could also explain the progressive decline in CTL activity. Because several CC-chemokines compete with IP-10 to bind CXCR3 with moderate affinity and can block IP-10-mediated receptor activation (31), efforts should be made to determine whether the local release of CC-chemokines may favor the progressive decline in CTL number and activity that is observed in patients with late-stage disease.