RANTES Potentiates Antigen-Specific Mucosal Immune Responses

RANTES Potentiates Antigen-Specific Mucosal Immune Responses1 James W. Lillard, Jr.,*† Prosper N. Boyaka,† Dennis D. Taub,‡ and Jerry R. McGhee2† RANTES is produced by lymphoid and epithelial cells of the mucosa in response to various external stimuli and is chemotactic for lymphocytes. The role of RANTES in adaptive mucosal immunity has not been studied. To better elucidate the role of this chemokine, we have characterized the effects of RANTES on mucosal and systemic immune responses to nasally coadministered OVA. RANTES enhanced Ag-specific serum Ab responses, inducing predominately anti-OVA IgG2a and IgG3 followed by IgG1 and IgG2b subclass Ab responses. RANTES also increased Ag-specific Ab titers in mucosal secretions and these Ab responses were associated with increased numbers of Ab-forming cells, derived from mucosal and systemic compartments. Splenic and mucosally derived CD4ⴙ T cells of RANTES-treated mice displayed higher Ag-specific proliferative responses and IFN-␥, IL-2, IL-5, and IL-6 production than control groups receiving OVA alone. In vitro, RANTES up-regulated the expression of CD28, CD40 ligand, and IL-12R by Ag-activated primary T cells from DO11.10 (OVA-specific TCR-transgenic) mice and by resting T cells in a dose-dependent fashion. These studies suggest that RANTES can enhance mucosal and systemic humoral Ab responses through help provided by Th1- and select Th2-type cytokines as well as through the induction of costimulatory molecule and cytokine receptor expression on T lymphocytes. These effects could serve as a link between the initial innate signals of the host and the adaptive immune system. The Journal of Immunology, 2001, 166: 162–169.

M

ammals are protected against pathogens through various natural immunity mechanisms. Although the epidermis and mucosal epithelium provide a physical barrier, early-acting innate effector molecules are also required to protect the host against potentially lethal agents. As a protective mechanism, a major function of mucosal epithelial cells is the transport of polymeric IgA into external secretions for mucosal immune responses. In addition, intestinal epithelial cells produce a number of cytokines including IL-1␣, IL-1␤, IL-6, IL-8, IL-10, and TNF-␣ (1, 2). Recently, human nasal- and adenoid-derived epithelial cells have been shown to secrete chemokines such as IL-8, monocyte chemoattractant protein-1 and RANTES in response to infection with respiratory syncytial virus (3, 4). RANTES is a CC chemokine that binds CCR1, CCR3, CCR4, and CCR5 and is produced by epithelial cells (3– 8), lymphocytes (9, 10), and platelets (11), and acts as a potent chemoattractant for monocytes (12, 13), NK cells (14), memory T cells (12, 13, 15), eosinophils (11), dendritic cells (16), and basophils (17). In addition, RANTES and other chemokines can selectively activate their corresponding lymphoid cell targets (14, 18 –21). *Department of Microbiology and Immunology, Morehouse School of Medicine, Atlanta, GA 30310; †Department of Microbiology, The Immunobiology Vaccine Center, University of Alabama at Birmingham, Birmingham, AL 35294; and ‡Laboratory of Immunology, Gerontology Research Center, National Institute on Aging, Baltimore, MD 21224 Received for publication February 16, 2000. Accepted for publication September 29, 2000. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This work was supported by the National Institutes of Health Grants AI 18958, AI 43197, GM 08248, RR 03034, DK 58967, and DK 44240; the United Negro College Fund-Merck Postdoctoral Science Research Fellowship; American Digestive Health Foundation Research Scholar Award; and Institutional Funds from the Morehouse School of Medicine. 2 Address correspondence and reprint requests to Dr. Jerry R. McGhee, University of Alabama at Birmingham, Department of Microbiology, The Immunobiology Vaccine Center, 761 BBRB, 845 19th Street South, Birmingham, AL 35294-2170. E-mail address: [email protected].

Copyright © 2001 by The American Association of Immunologists

RANTES has been shown to induce lymphocyte migration into the nasal mucosa of allergic patients (6) and its expression can be induced by infecting human bronchial tissue- or nasal polyp– derived epithelial cells with influenza virus (7). Freshly isolated human colon epithelial cells and HT-29 and Caco-2 epithelial tumor cell lines were shown to secrete RANTES and other chemokines in response to Salmonella typhimurium infection or cytokine stimulation (5). Recently, contrasting results were reported regarding the effect of this chemokine and its receptors on the outcome of Th1and Th2-mediated diseases. For example, anti-RANTES Ab treatment was shown to decrease mycobacterial-inducible Th1-type lesions, while increasing schistosomal-inducible Th2-type granulomas in mice (22). It was also shown that RANTES inhibited IL-4 secretion through RANTES-CCR1 interactions, suggesting the potential role of RANTES in Th1-mediated granuloma formation. In contrast, CCR5 gene knockout mice challenged with Leishmania donovani displayed augmented Th1 responses to L. donovani Ag, when compared with wild-type mice (23). Although these studies have addressed the expression of chemokines in response to microbial infection and the important role of RANTES in the outcome of inflammatory and infectious disease models, the mechanisms that RANTES uses to influence host mucosal immune responses remains elusive. We have previously shown that lymphotactin, a C chemokine, can enhance mucosal and systemic immunity, suggesting that chemokines could be major regulatory molecules for the induction of mucosal immunity (24). The current study seeks to determine whether a CC chemokine, RANTES, provides signals to immune cells to activate the acquired immune system. To determine the immunological contribution that RANTES makes toward mucosal and systemic immunity, we investigated its effects on primary and secondary Ag-specific immune responses. Our results demonstrate that, following nasal immunization, RANTES initiates and enhances Ag-specific humoral and cellular immune responses in both mucosal and systemic compartments. These studies suggest that this CC chemokine may play an important role in the induction and development of adaptive mucosal immunity. 0022-1767/01/$02.00

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Materials and Methods

Cell isolation

RANTES and immunogen

Single-cell suspensions of spleen, Peyer’s patches, mesenteric lymph nodes, and cervical lymph nodes were prepared by aseptically removing tissues and then passing them through a sterile wire screen. After the removal of Peyer’s patches, the small intestine was cut into 1-cm strips and stirred in PBS containing 1 mM EDTA at 37°C for 30 min. Next, intestinal lamina propria lymphocytes were isolated by digesting intestinal tissue in collagenase type IV (Sigma) in RPMI 1640 (collagenase solution) for 45 min with stirring at 37°C (25). The lower respiratory tract (lungs and mediastinal lymph nodes) was injected with 10 ml cold PBS to remove blood, dissected into small pieces, and subjected to collagenase digestion, as described for the isolation of lamina propria lymphocytes (24). Lymphocytes were further purified using a discontinuous Percoll (Pharmacia, Uppsala, Sweden) gradient, collecting at the 40 –75% interface (26). Nasal tract lymphocytes were isolated by gently washing nasal cavities with 200 ␮l cold PBS to remove blood. Next, the nasal tract mucosal tissue was removed by scraping; the resulting tissue was then passed through a sterile wire mesh (24). Cell suspensions were washed twice in RPMI 1640. Lymphocytes were maintained in complete medium, which consisted of RPMI 1640 supplemented with 10 ml/L of nonessential amino acids (Mediatech, Washington, DC), 1 mM sodium pyruvate (Sigma), 10 mM HEPES (Mediatech), 100 U/ml penicillin, 100 ␮g/ml streptomycin, 40 ␮g/ml gentamicin (Elkins-Sinn, Cherry Hill, NJ), 50 ␮M mercaptoethanol (Sigma), and 10% FCS (Atlanta Biologicals, Norcross, GA). T cell fractions were obtained by passing single-cell suspensions over nylon wool for 1 h at 37°C. CD4⫹ T cells were enriched (⬎ 98% purity) using Mouse CD4 Cellect plus columns according to the manufacturer’s protocols (Biotecx Laboratories, Edmonton, Alberta, Canada).

Murine RANTES was purchased from PeproTech (Rocky Hill, NJ). The potential level of endotoxin contamination was quantified by the chromogenic Limulus amebocyte lysate assay (Associates of Cape Cod, Falmouth, MS) and was shown to be ⬍5 EU/mg. Chicken egg albumin (OVA) and BSA were purchased from Sigma (St. Louis, MO).

Mice and immunizations Female C57BL/6 and BALB/c mice, aged 5– 6 wk, were procured from The Jackson Laboratory (Bar Harbor, MA). DO11.10 mice were generously provided by Dr. Casey Weaver (University of Alabama at Birmingham, Birmingham, AL). All mice were housed in horizontal laminar flow cabinets free of microbial pathogens. Routine Ab screening for a large panel of pathogens and histological analysis of organs and tissues were performed to ensure that mice were pathogen-free. C57BL/6 and BALB/c mice used in immunization studies were 8 –12 wk of age. Following anesthesia, mice were nasally immunized on days 0, 7, and 14 with 75 ␮g OVA in the presence or absence of 0.01–5 ␮g of RANTES in 10 ␮l PBS (5 ␮l/nare). Mice that received 75 ␮g OVA and 1 ␮g cholera toxin (CT) in 10 ␮l PBS served as positive controls (25), while mice given 75 ␮g hen egg lysozyme, OVA, or PBS alone were negative controls (24) BALB/c mice of the same age were also immunized to confirm the results obtained using C57BL/6 mice. Experimental groups consisted of five mice and studies were repeated three to five times. The guidelines proposed by the committee for the Care of Laboratory Animal Resources Commission of Life Sciences National Research Council were followed to minimize animal pain and distress.

Sample and tissue collection Fecal samples were weighed and dissolved in PBS containing 0.1% sodium azide (e.g., 1 ml/100 mg of fecal pellet). Following suspension by vortexing for 10 min, fecal samples were centrifuged and supernatants were collected for analysis. Nasal and vaginal cavities were rinsed three times with 50 ␮l PBS. Blood samples were collected by tail vein bleeding and serum was obtained following centrifugation. Serum and mucosal secretions were collected on days 0, 7, 14, and 21 for OVA-specific Ab analysis by ELISA. Mice were sacrificed by CO2 inhalation 1 wk after the last immunization to quantify the OVA-specific Ab-forming cells (AFCs)3 and T cell responses present in immune compartments.

OVA-specific Ab detection by ELISA Fecal and serum levels of OVA-specific Abs were measured by ELISA, as previously described (26). Briefly, 96-well Falcon 3912 flexible ELISA plates (Fisher Scientific, Pittsburgh, PA) were coated with 100 ␮l of 1 mg/ml OVA in PBS overnight (O/N) at 4°C and blocked with 1% BSA (Sigma) in PBS (B-PBS) for 3 h at room temperature. Individual samples (100 ␮l) were added and serially diluted in B-PBS. After O/N incubation at 4°C and three washes using PBS containing 0.05% Tween 20 (PBS-T), titers of IgM, IgG or IgA were determined by the addition of a 0.33 ␮g/ml HRP-conjugated, goat anti-mouse ␣, ␥, or ␮ heavy chain-specific antisera (Southern Biotechnology Associates, Birmingham, AL) in B-PBS-T. Similarly, 100 ␮l of biotin-conjugated rat anti-mouse ␥1 (12.5 ng/ml G1-7.3), ␥2a (125 ng/ml R19-15), ␥2b (12.5 ng/ml R12-3), ␥3 (50 ng/ml R40-82), and ⑀ (1.25 ␮g/ml G1-7.3) (PharMingen, San Diego, CA) heavy chainspecific mAbs were used to determine IgG subclass and IgE isotype titers (26). After incubation and washing steps, 100 ␮l of 0.5 ␮g/ml HRP-antibiotin Ab (Vector Laboratories, Burlingame, CA) in B-PBS-T or 500 ng/ml polyHRP80 streptavidin (Research Diagnostics, Flanders, NJ) in PolyHRP Diluent (Research Diagnostics) were added to IgG subclass or IgE detection wells, respectively, and incubated for 3 h at room temperature. Following incubation, the plates were washed six times and the color reaction for ELISA was developed by adding 100 ␮l of 1.1 mM 2,2⬘-azino-bis-3ethylbenzthiazoline-6-sulfonic acid (Sigma) in 0.1 M citrate-phosphate buffer (pH 4.2) containing 0.01% H2O2 (ABTS solution). Endpoint titers were expressed as the reciprocal Log2 of the highest dilution, which indicated an OD that was 415 nm (OD415) of ⱖ 0.1 OD unit above the OD415 of negative controls after a 20-min incubation (27).

3 Abbreviations used in this paper: AFC, Ab-forming cells; ELISPOT, enzyme-linked immunospot; CD40L, CD40 ligand; O/N, overnight; PBS-T, PBS containing 0.05% Tween 20; B-PBS, 1% BSA in PBS; BD, below detection; CT, cholera toxin.

IgA, IgM and IgG enzyme-linked immunospot (ELISPOT) analysis An ELISPOT assay was employed to detect total or OVA-specific AFCs (27). In brief, 96-well Millititer HA nitrocellulose-based plates (Millipore, Bedford, MS) were coated with 100 ␮l of 1 mg/ml OVA in PBS, PBS only (negative control), or 0.5 ␮g/ml goat anti-mouse Ig (heavy and light) human-adsorbed polyclonal Ab (Southern Biotechnology Associates) and incubated O/N (12 h) at 4°C. Wells were subsequently blocked with B-PBS for 2 h and washed. Whole cells were added to wells in duplicate at 106, 5 ⫻ 105, and 105 cells/ml concentrations in complete medium and incubated for 6 h at 37°C in 5% CO2. After washing with PBS-T, individual AFCs were detected with HRP-labeled goat anti-mouse ␣-, ␮-, or ␥-chainspecific Abs (1 ␮g/ml; Southern Biotechnology Associates), visualized by adding 3-amino-9-ethylcarbazole buffer (Moss, Pasadena, MD) and counted using a dissecting microscope (Stereo Zoom H Series Microscope System; Olympus, Lake Success, NY).

Ag-specific CD4⫹ Th cell responses Purified CD4⫹ T cells were cultured at a density of 5 ⫻ 106 cells/ml with 1 ⫻ 106 cells/ml of T cell-depleted and -irradiated (3000 rads) splenic feeder cells in complete medium containing 1 mg/ml OVA at 37°C in 5% CO2. To ascertain Ag-specific proliferative responses, purified CD4⫹ T cells were cultured in 96-well round-bottom plates (Corning Glass Works, Corning, NY). After 3 days of culture, cells were pulsed with 0.5 ␮Ci methyl-[3H]thymidine (Amersham Life Sciences, Arlington Heights, IL) per well for 18 h. Cells were harvested on glass microfiber filter paper (Whatman, Clifton, NJ) and radioactivity levels were obtained by liquid scintillation counting.

Cytokine analysis by ELISA For the assessment of cytokine production, 2 ml of culture supernatants from 12-well flat-bottom plates (Corning Glass Works) were harvested after 5 days of incubation. Control wells consisted of cells only or cells cultured with BSA or 1 ␮g/ml Con A (Sigma). Cytokines in cell culture supernatants were determined by ELISA as described previously (26). Briefly, Falcon 3912 Microtest plates (Fisher Scientific) were coated with 100 ␮l of 2.5 ␮g/ml rat anti-mouse IFN-␥, IL-2, IL-4, IL-5, IL-6, and IL-10 (PharMingen) in 0.1 M bicarbonate buffer (pH 8.2) O/N at 4°C and blocked with 3% BSA in PBS at room temperature for 3 h. Next, 100 ␮l of serially diluted recombinant murine cytokines as standards (PharMingen) or cultured supernatant samples were added in duplicate and incubated O/N at 4°C. The plates were washed with PBS-T and incubated with 0.2 ␮g/ml of biotinylated-secondary-murine cytokine detection Abs (PharMingen) in BPBS-T for 3 h at room temperature. After washing with PBS-T and PBS alone, wells were incubated for 2 h in 100 ␮l of 0.5 ␮g/ml peroxidaseconjugated anti-biotin Ab (Vector Laboratories, Burlingame, CA) and developed with ABTS solution, as described above. The cytokine ELISA

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were capable of detecting 15 pg/ml IFN-␥; 5 pg/ml IL-2, IL-4, and IL-5; 100 pg/ml IL-6; and 200 pg/ml IL-10.

Effects of RANTES on naı¨ve or DO11.10 primary lymphocytes Ninety-six-well, round-bottom plates were coated with 0, 0.5, or 10 ␮g/ml of anti-mouse CD3⑀ mAb in carbonate-bicarbonate buffer (pH 9.4) for 12 h at 4°C. Splenocytes from naı¨ve C57BL/6 or DO11.10 mice were isolated at the 40 –75% interface of a discontinuous Percoll (Pharmacia) gradient and added at a density of 2 ⫻ 106 cells/ml in complete medium containing 0, 1, 10, 100, or 1000 ng/ml of RANTES. A class II-restricted OVA peptide containing 5 ng/ml amino acids 323–339, was used to activate primary OVA-specific TCR-transgenic CD4⫹ T cells from DO11.10 mice (28). Lymphocytes were also cultured with optimal doses of Con A (5 ␮g/ml) or anti-mouse CD3⑀ mAb (10 ␮g/ml-coated plates) as positive controls or alone as negative controls. After incubation for 2 days, cells were stained with rat anti-mouse CD28, CD40, CD40 ligand (CD40L), CD80, CD86, CD30, 4-1-BB, CD3, CD4, and/or B220 mAbs conjugated to either PE or FITC (PharMingen) or with rabbit anti-IL-4R␣, IFN-␥R␤, and IL-12R␣␤ plus goat anti-rabbit-FITC labeled polyclonal Abs (Santa Cruz Biotechnology, Santa Cruz, CA) and analyzed by flow cytometry.

Statistics The data are expressed as the mean ⫾ SEM and compared using a twotailed student’s t test or an unpaired Mann Whitney U test. The results were analyzed using the Statview II statistical program (Abacus Concepts, Berkeley, CA) for Macintosh computers and were considered statistically significant if p values were less than 0.05. When cytokine levels were below the detection limit (BD), they were recorded as one-half the lower detection limit (e.g., 50 pg/ml for IL-6) for statistical analysis.

Results Nasal delivery of RANTES stimulates Ag-specific mucosal and systemic Ab responses Intranasal administration of Ag coadministered with lymphotactin has been previously shown to enhance Ag-specific mucosal and systemic Ab responses (24). In the current study, we first administered OVA (75 ␮g) with increasing concentrations (e.g., 0.0, 0.01, 0.1, 1.0, and 5.0 ␮g) of RANTES by the nasal route at weekly intervals to determine the optimal dose of RANTES that would effect Ag-specific serum Ab responses. Accordingly, we analyzed the OVA-specific Ab isotypes and IgG subclasses in sera and fecal samples. Administration of OVA alone elicited low Agspecific serum Ab responses; however, mice nasally immunized with OVA and ⱖ0.10 ␮g RANTES displayed significantly higher serum titers of anti-OVA IgM and IgG Abs (data not shown). Since doses of RANTES (ⱖ1.00 ␮g) were comparable and elicited significant titers of OVA-specific Ab responses, we used the 1.0-␮g dose of RANTES for subsequent experiments. After three immunizations, mice receiving RANTES plus OVA displayed significant ( p ⬍ 0.05) increases in Ag-specific IgM and IgG serum Ab levels, with IgG levels showing the steepest rise (Fig. 1). Following our immunization schedule, RANTES induced significant increases in anti-OVA IgG2a ⬎ IgG2b ⫽ IgG3 ⬎ IgG1 serum responses (Fig. 1). Increases of IgA Ab titers in fecal extracts were reached by day 14 and continued through day 21 (Fig. 1). Similarly, statistically significant increases in OVA-specific IgA Ab titers were also observed in vaginal and nasal wash samples by day 21 (Fig. 1). These findings demonstrate that OVAspecific Abs in the serum and external secretions were enhanced by the nasal coadministration of RANTES. Effects of RANTES on Ag-specific AFCs ELISPOT assays were performed on mucosal and systemic tissues to confirm that the enhanced mucosal OVA-specific Ab responses observed actually arose from mucosal effector sites and were not transudated from serum. The number of total Ig AFCs per 106 lymphocytes remained constant in both experimental and control groups of mice. Mice that received OVA alone did not display

FIGURE 1. OVA-specific serum, vaginal and nasal wash, and fecal Ab responses following nasal immunization with RANTES. Groups of five C57BL/6 mice were intranasally immunized on days 0, 7, and 14 with 75 ␮g OVA and 0.0 (䡺) or 1.0 ␮g (f) RANTES in 15 ␮l PBS. A and B, The Ig isotype and IgG subclass Ab titers, respectively, in sera. C, Mucosal IgA Ab titers in external secretions. The data presented are the mean Ab titers ⫾ SEM of three separate experiments. The distribution of OVA-specific serum and fecal Ab titers on day 21 was determined by ELISA. The asterisks indicate statistically significant differences (i.e., p ⬍ 0.05) from Ab titers of mice immunized with OVA alone.

substantial Ag-specific AFCs in any of the tissues analyzed. Coadministration of OVA and RANTES significantly increased Agspecific IgA AFCs in the upper (nasal cavity) and lower (lung) respiratory tracts as well as in the Peyer’s patches and intestinal lamina propria (Fig. 2). RANTES also substantially increased the OVA-specific IgM and IgG AFCs among splenic and respiratory tract lymphocytes, but not in intestinal lamina propria-derived cells (Fig. 2). Taken together, these results suggest that the nasal delivery of RANTES enhanced Ag-specific mucosal IgA as well as peripheral IgM, IgG, and IgA AFCs. Cytokine profiles and proliferative responses of OVA-specific CD4⫹ T cells Since our study showed that RANTES induced systemic and mucosal Ab responses with reduced or unchanged OVA-specific IgG1 and IgE responses, we next examined the ability of this CC chemokine to promote OVA-specific Th cytokine and proliferative responses. Marked increases in OVA-specific proliferative responses were observed by CD4⫹ T cells isolated from the lower respiratory tract, Peyer’s patches, cervical lymph nodes, and spleens of mice immunized with OVA and RANTES (Fig. 3). In addition, RANTES also enhanced the spleen-, mesenteric lymph node-, Peyer’s patch-, lower respiratory tract-, and cervical lymph node-derived CD4⫹ T cell cytokine responses to OVA. Th cells from these inductive tissues exhibited increased production of IFN-␥, IL-2, IL-5, and IL-6 but no statistically significant changes in IL-4 or IL-10 levels in OVA-restimulated cultures (Fig. 3). The Th cell subpopulations from mice that did not receive RANTES showed low or undetectable cytokine levels (Fig. 3). These results suggest that mucosal immunization with OVA and RANTES induced proliferative and Th1- (IL-2 and IFN-␥) type responses with

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FIGURE 2. OVA-specific AFCs in spleen, nasal tract, cervical lymph nodes, lower respiratory tissue, mesenteric lymph nodes, Peyer’s patches, and intestinal lamina propria. Groups of five C57BL/6 mice were nasally immunized on days 0, 7, and 14 with 75 ␮g OVA and 0.0 (䡺) or 1.0 ␮g (f) RANTES in 15 ␮l PBS. Levels of OVA-specific IgM (A), IgG (B), and IgA (C) AFCs present in these associated lymphoid tissues were determined by ELISPOT analysis 7 days after the last immunization. AFCs below detectable levels are designated BD. The data presented are the mean AFCs ⫾ SEM, in duplicate cultures, of three separate experiments. An asterisk indicates statistically significant differences (p ⬍ 0.05) from AFCs of mice immunized with OVA alone.

significant levels of IL-5 and IL-6 from all of the lymphoid tissues analyzed. Effects of RANTES on costimulatory molecule expression by primary resting or Ag-activated naı¨ve T and B cells The adjuvant effects of CT have been attributed to its ability to up-regulate CD86 expression (29), while RANTES has been shown to enhance CD80, but not CD86 (20). To better elucidate the adjuvant effects of RANTES on T cell responses, we assessed its potential to modulate costimulatory molecule expression by resting and OVA-stimulated T cells from the spleen or Peyer’s patch of DO11.10 mice that contain an OVA323–339-specific transgenic TCR. There was no statistically significant difference between the responses observed by splenic- and Peyer’s patch-derived T lymphocytes. RANTES modestly increased the expression of CD28, but not CD40L, by resting CD4⫹ T cells in a dosedependent fashion (Fig. 4 and Fig. 5). However, RANTES significantly enhanced ( p ⬍ 0.05) CD28, CD40L, and CD30, but not 4-1-BB, expression by DO11.10 T cells in a dose-dependent fashion, following OVA323–339 peptide stimulation (Fig. 4 and Fig. 5) with optimal increases at 50 ng/ml of RANTES. Our findings reveal for the first time that RANTES is effective at regulating CD28, CD40L, and CD30 expression on both resting and activated T lymphocytes. Modulation by RANTES of IL-12R expression by Ag-stimulated CD4⫹ T cells IL-12R expression marks Th1 cells (30). Since RANTES induced predominant Ag-specific Th1 cell and IgG2a ⬎ IgG1 Ab responses after immunization, we next determined the potential of RANTES to regulate IL-12R␣␤. RANTES was unable to affect IL-12R␣␤ expression by resting T cells from either the spleen or Peyer’s patch; however, Table I illustrates the regulatory effects of RANTES on OVA323–339-stimulated CD4⫹ T cells from DO11.10 mice. RANTES enhanced IL-12R␣␤ expression by OVA-stimulated splenic and Peyer’s patch CD4⫹ T cells from OVA-specific TCR transgenics in a dose-dependent fashion.

Discussion Local production or delivery of RANTES has been shown to cause chemotaxis of immune cells in vivo (6, 31). The mucosa contain large numbers of ␥␦ TCR⫹ intraepithelial lymphocytes (32), which secrete RANTES and other chemokines in response to stimulation (33). Interestingly, RANTES and its ligands, CCR5 and CCR1, are associated with Th1-type T cells and immune responses (20, 22, 34). Thus, the spectrum of mucosal cells expressing RANTES-specific receptors as well as the ability of this CC chemokine to attract lymphocytes and affect the outcome of Th1- and Th2-mediated disease pathologies, provided the rationale to test the effects of RANTES on acquired mucosal immunity. The results reported here support our hypothesis that RANTES is able to enhance the development of both humoral and cellular mucosal and systemic immunity. We have also shown that RANTES-mediated immunity involves largely Th1-type responses with synthesis of IL-5 and IL-6. The nasal immunization of mice with OVA and RANTES enhanced OVA-specific serum and mucosal Ab and CD4⫹ T cell proliferative and cytokine responses in both systemic and mucosal compartments. Although previous studies in our laboratory have shown that the classical mucosal adjuvant CT induces Ag-specific IgG1 and IgE Ab responses (25) following RANTES plus OVA administration, the most prevalent OVA-specific serum IgG subclass Ab response was IgG2a, followed by IgG2b, IgG3, and IgG1 (Fig. 1). These results were surprising since nasal immunization regimen that use soluble protein Ag often result in higher IgG1 Ag-specific Ab titers (35, 36). IL-4 supports IgG1 and IgE Ab production and generation; hence, the low levels of anti-OVA IgG1 and IgE Abs were consistent with the observed cytokine profiles (i.e., IL-2, IFN-␥, IL-5, and IL-6) of the OVA-restimulated CD4⫹ T cells of RANTES-treated mice (37–39). IFN-␥ production is often associated with IgG2a and IgG3 Ab production (40) and may account for the RANTES-mediated anti-OVA IgG2a and IgG3 Ab responses. Even low doses of IFN-␥ (1,500 U) have been shown to increase IgG2a production in vivo, while considerably

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FIGURE 3. Proliferation and Th cytokine secretion by OVA-stimulated CD4⫹ T cells. Groups of five C57BL/6 mice were nasally immunized on days 0, 7, and 14 with 75 ␮g OVA and 0.0 (䡺) or 1.0 ␮g (f) RANTES in 15 ␮l PBS. One week after the last immunization, lower respiratory tissue (lung and mediastinal lymph nodes)-, Peyer’s patch-, mesenteric lymph nodes-, spleen-, and cervical lymph node-derived CD4⫹ T cells were purified and cultured at a density of 5 ⫻ 106 cells/ml with 500 ␮g/ml OVA for 3 days with T cell-depleted, irradiated splenic feeder cells (1 ⫻ 106 cells/ml) in complete medium. Experimental groups consisted of five mice and studies were repeated three times. Proliferation was measured by [3H]thymidine incorporation. Unstimulated Peyer’s patch lymphocytes incorporated ⬃1000 cpm of [3H]thymidine, while the other unstimulated lymphoid tissues incorporated ⬃600 cpm of [3H]thymidine. The data presented are the mean stimulation index ⫾ SEM of quadruplicate cultures. The stimulation index corresponds to cpm of cell cultures containing OVA divided by the cpm of cultures without OVA. Cytokine production of cultured supernatants was determined by ELISA. Th1- and Th2-type cytokine profiles are presented as the mean cytokine levels (pg/ml) ⫾ SEM of duplicate cultures from each group. An asterisk indicates statistically significant differences (p ⬍ 0.05) from cytokine levels of mice immunized with OVA alone, while cytokines below detectable levels are designated BD.

higher doses of IFN-␥ (12,500 U) are required to induce decreases in IgG1 and IgE responses (41). Although the precise cytokine signals required for the induction of secretory-IgA are not completely understood, it has been shown that mucosal IgA responses are supported by both Th1- and Th2-type cell-derived cytokines (26, 42, 43). Clearly, both serum and mucosal Ag-specific Ab responses were enhanced as a result of the effects of RANTES. Further, anti-OVA AFCs observed in the intestinal lamina propria as

well as the upper and lower respiratory tract confirmed the Agspecific IgA Abs detected in mucosal secretions. The cytokines produced by CD4⫹ T cells after mucosal administration of RANTES and OVA only partially explained the increases in OVA-specific Ab and T cell responses ex vivo. RANTES may augment immune responses by activating host macrophages, dendritic cells, B and T cells, or enhancing Ag presentation. In this regard, RANTES has been shown to increase Ag

FIGURE 4. Flow cytometry analysis of CD28 and CD40L T cell expression induced by RANTES. A representative of three separate experiments are shown where splenic T cells from DO11.10 mice were incubated with 0 or 100 ng/ml RANTES in plates containing 5 ng/ml OVA peptide containing amino acids 323–339 (OVA323–339). The percent gated of doublepositive CD4⫹ and CD28⫹ or CD40L⫹ cells is illustrated in the upper right quadrant of the representative density plots of the FACS data analyzed using CellQuest version 3.3 software (Becton Dickinson, Mountain View, CA). Correspondingly, the relative cell count (x-axis) of CD4⫹ T cells expressing CD28 or CD40L (y-axis) of these density plots are also shown of the FACS data analyzed using CellQuest.

The Journal of Immunology

FIGURE 5. Regulation of CD28, CD40L, CD30, and 4-1-BB expression on T cells by RANTES. Splenic T cells from DO11.10 mice were incubated with 0, 10, 50, 100, 500, or 1000 ng/ml of RANTES in plates containing 0 or 5 ng/ml of OVA peptide containing amino acids 323–339 (OVA323–339). The percent increase (or decrease) of the costimulatory molecule expression by resting (open symbols) and OVA323–339-activated (filled symbols) T cells was calculated as the percent of double-positive CD4⫹ and CD28⫹ (E, F), CD40L⫹ (‚, Œ), CD30⫹ (䡺, f) as well as 4-1-BB⫹ (〫, ⽧) cells in cultures containing RANTES minus the percent gated of double-positive cells in cultures without RANTES, divided by the latter. Studies were repeated three times and the data presented are the mean percent change ⫾ SEM of these experiments. Asterisk(s) indicate statistically significant differences (i.e., p ⬍ 0.05) from lymphocytes incubated without RANTES.

uptake and activate human macrophages to kill Trypanosoma cruzi (44). Although these innate and chemotactic molecules directly aid in the accumulation of lymphocytes at infection or immunization sites; lymphocyte recruitment alone does not insure the initiation of an adaptive immune response, particularly not a mucosal immune response (45). Our results and those of others have shown that RANTES incubated with Ag-stimulated lymphocytes significantly enhances Th1- and modestly increases Th2-type cytokine secretion (20). Our results would suggest that RANTES also induced this T cell response pattern to nasally coadministered protein. CD28 is expressed by naı¨ve T cells and supplies a coactivation signal for cell growth (46, 47). This receptor binds B7-1 (CD80) and B7-2 (CD86) on APCs (48), and as more recently shown, B7-H1 (49). Indeed, the mucosal adjuvanticity of CT involves the up-regulation of CD86 expression (29, 50) Stimulation and signal transduction through CD28 acts in concert with the signals provided by Ag and TCR/CD3 interactions, which result in IL-2 production by precursor Th cells and subsequent cell division (51). Interestingly, RANTES enhanced CD28 expression by Ag-activated OVA323–339-specific TCR-transgenic CD4⫹ T cells (Fig. 4). Correspondingly, RANTES has also been shown to enhance CD80 expression by accessory cells (20).

167 In addition to B7 ligands, CD40 is another receptor important in B cell activation and differentiation (52). The T cell ligand for CD40 is gp39 or CD40L and is considered a major determinant in the outcome of T-B cell interactions (52). CD40L stimulation can drive B cell activation and IgA production (53, 54). Although we did not observe any significant changes in CD40 expression by B cells following stimulation with LPS and/or RANTES, dramatic increases in CD40L expression were obtained by RANTES- plus Ag-stimulated DO11.10 CD4⫹ T cells (Fig. 4). Taken together, these results suggest that the increased expression of CD28 and CD40L, as well as CD80 (20), represent major mechanisms in the mucosal adjuvanticity of RANTES. CD30 and 4-1-BB (CD137) interactions have been shown to control Th1 and Th2 differentiation and lymphocyte proliferation. CD30 is predominately expressed by T cells that secrete Th2-type cytokines and its expression is down-regulated by IFN-␥ (55). In contrast, 4-1-BB and IL-12R␣␤ are preferentially expressed on Th1-type T cells and naive T cells can be led to differentiate to Th1-type T cells after 4-1-BB and CD28 stimulation (30, 56). In our study, RANTES up-regulated CD30 expression on both resting and Ag-triggered CD4⫹ T cells from OVA-TCR-transgenic D011.10 mice, but failed to significantly enhance 4-1-BB expression (Fig. 5). Our results also show that RANTES up-regulates IL-12R expression by T cells in vitro (Table I). This observation supports the predominant Th1-type pattern of immune responses measured in mice that received RANTES. It is tempting to conclude that both Th1- and Th2-type cells are activated by RANTES, because our findings demonstrate that RANTES potentiates the development, in vitro and in vivo, of both Th1- (IFN-␥) and Th2(IL-5 and IL-6) type cytokines. Contradictory data have been reported regarding the stimulatory effect of RANTES on both Th1- and Th2-type responses. Recently, it has been shown that RANTES and MIP-1␣ can enhance IgE and IgG4 production by IL-4 and anti-CD40 or anti-CD58 mAb-stimulated human sIgE⫹ and sIgG4⫹ B cells (57). One could suggest that these effects observed in vitro with human B cell IgG4 and IgE responses do not reflect how RANTES affects host immune responses in vivo. In this regard, in vivo models of Mycobacterium and Schistosoma infection as examples of Th1 and Th2 inducers, respectively, were used to show that RANTES mainly promoted the development of Th1 cell-mediated pathology (22). Our results show that both Th1- and Th2-type pathways can be induced by mucosally administered RANTES, even though this molecule primarily induced Th1-type responses. Numerous studies have been published that demonstrate the ability of chemokines to regulate the migration of lymphocytes to

Table I. Regulation of IL-12R␣␤ expression on Ag-stimulated OVAspecific CD4⫹ T cells by RANTESa Dose of RANTES (ng/ml) Lymphoid Tissue

Spleen Peyer’s patches

10

(2%) 3%

100

1,000

10,000

29%ⴱ 35%ⴱ

19% 21%

6% 10%

a OVA-activated splenic or Peyer’s patch lymphocytes from DO11.10 mice were incubated with 0, 10, 100, 1,000, or 10,000 ng/ml of RANTES for 48 hours and prepared for FACS analysis. The percent increase (or decrease) of the IL-12R␣␤ expression by OVA-activated T cells was calculated as the percent of double-positive CD4⫹ and IL-12R␣␤⫹ cells in cultures containing RANTES minus the percent of gated double-positive cells in cultures without RANTES, divided by the latter. Studies were repeated three times and the data presented are the mean percent change of these experiments. SEM did not exceed 10% and are not disclosed. ⴱ, Statistically significant differences ( p ⬍ 0.05) relative to cultures without RANTES.

168 sites of disease. These same effects can also lead to severe inflammation and chronic disease if left unchecked. We have shown that, under certain conditions, RANTES can enhance or reduce immune cell function. Perhaps due to the importance of chemokines in host immunity, a number of pathogens have evolved endogenous chemokine homologues and binding proteins that presumably interfere with the chemotaxis of immune cells to sites of infection so that these microbes can evade immune detection (12, 58 – 67). Chemokines may use a variety of mechanisms to support their adjuvant activity. For example, lymphotactin is unable to affect the expression of IL-12R␣␤, CD28, CD80, CD86, CD40, or CD40L, while RANTES differentially affects these costimulatory molecules. Aside from chemotaxis, our data suggest that chemokines, such as lymphotactin (24) and now RANTES can serve as a potent and effective mucosal adjuvant for adaptive mucosal immunity. Further studies will be needed to elucidate the precise contributions that chemokines and their receptors make to the generation and demarcation of acquired immunity as well as effector and recognition immune cell interactions. However, our results have clarified the role chemokines play in acquired mucosal and systemic immunity. RANTES not only plays a role in acute immune cell functions (i.e., inflammation), but our current findings implicate that this CC chemokine affects adaptive immunity (i.e., cytokine secretion, Ab formation, and costimulatory molecule expression).

Acknowledgments We thank Dr. Kimberly McGhee for preparation of the written text of this manuscript. The content of this manuscript benefited from many fruitful conversations with members of the Morehouse School of Medicine and the University of Alabama at Birmingham Immunobiology Vaccine Center.

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