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Originally published online as doi:10.1189/jlb.0903444 on September 8, 2004

Published online before print September 8, 2004
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(Journal of Leukocyte Biology. 2004;76:1240-1247.)
© 2004 by Society for Leukocyte Biology

Granulocyte chemotactic protein-2 mediates adaptive immunity in part through IL-8Rß interactions

Udai P. Singh*, Shailesh Singh*, Prosper N. Boyaka{dagger}, Jerry R. McGhee{dagger} and James W. Lillard, Jr*,{dagger},1

* Department of Microbiology and Immunology, Morehouse School of Medicine, Atlanta, Georgia; and
{dagger} University of Alabama at Birmingham

1 Correspondence: Morehouse School of Medicine, Department of Microbiology, Biochemistry, and Immunology, 720 Westview Drive, Atlanta, GA 30310-1495. E-mail: Lillard{at}msm.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Chemokines constitute a large family of structurally related proteins that play a role in leukocyte migration and differentiation. Indeed, the early expression of human CXC chemokine receptor 1 (hCXCR1) and hCXCR2 [homologous to mouse interleukin (IL)-8Rß] ligands by the epithelium is a hallmark of the mucosal host defense. Mice lack IL-8; however, granulocyte chemotactic protein-2 (GCP-2)/lipopolysaccharide-induced CXC chemokine, a murine homologue of human GCP-2, has 32% and 61% sequence identity to human IL-8 and GCP-2, respectively, and binds hCXCR1, hCXCR2, and mouse IL-8Rß. To better understand the role of GCP-2 in adaptive immunity and as a nasal adjuvant, we characterized the exogenous effects of this CXC chemokine on cellular and humoral mucosal immune responses. GCP-2 significantly enhanced serum immunoglobulin G (IgG) and mucosal IgA antibodies through increased cytokine secretion by CD4+ T cells. These alterations in humoral and cellular responses were preceded by an increase in the number of B cells in the nasal tract, a decrease in the number of CD4+ T cells in the nasal tract as well as cervical lymph nodes, and an increase in the number of neutrophils in the nasal tract 12 h after GCP-2 immunization. This chemokine also modulated CD28 expression by CD4+ T cells during CD3{varepsilon} stimulation of wild-type mice. GCP-2 increased CD80 and CD86 expression on B cells during in vitro stimulation in a dose-dependent manner. In contrast, cytokine and costimulatory molecule enhancement by GCP-2 was not induced by lymphocytes from IL-8Rß–/– mice, suggesting that GCP-2 modulates cellular immunity in part through IL-8Rß interactions.

Key Words: adjuvant • Th1/Th2 • B7


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The mucosa serves as the first innate defense against mucosal pathogens and is a source of numerous effector molecules, namely, defensins, chemokines, and cytokines, which initiate mucosal adaptive immune responses [1 2 3 4 5 6 ]. Numerous studies have demonstrated the ability of chemokines to regulate the migration of lymphocytes to sites of disease, an important prerequisite for host defense. Previous studies from our laboratory suggest that chemokines such as lymphotactin [3 ], regulated on activation, normal T expressed and secreted (RANTES) [4 ], macrophage inflammatory protein-1{alpha} and -1ß (MIP-1{alpha} and MIP-1ß) [5 ], as well as interferon-{gamma} (IFN-{gamma})-inducible protein-10, monokine induced by IFN-{gamma}, and IFN-inducible T cell {alpha}-chemoattractant [6 ] can modulate mucosal adaptive immunity. Hence, chemokines may play important roles in bridging innate and early inflammatory responses with the adaptive immune system.

The inflammatory condition is composed of multiple mediators that regulate leukocyte functions (e.g., activation and migration). Interleukin (IL)-8 is secreted by epithelial and endothelial cells as well as by leukocytes and is chemotatic for neutrophils [7 ]. Indeed, this CXC chemokine plays a crucial role in neutrophil migration during many inflammatory conditions [8 , 9 ]. IL-8 binds with equal affinity to human CXC chemokine receptor 1 (hCXCR1) and hCXCR2. It is important to understand the cellular and molecular mechanism of hCXCR1 and hCXCR2 ligands in leukocyte activation and differentiation. Mice lack a clear-cut homologue of hIL-8, but mouse granulocyte chemotactic protein-2 (GCP-2) is 61% identical to hGCP-2 [10 ] and is a functional murine homologue for hIL-8, with 32% identity [11 12 13 ].

GCP-2 acts as a potent chemoattractant for neutrophils in the course of acute inflammation. Endothelial and bronchial epithelial cells produce GCP-2 after lipopolysaccharide (LPS) and/or IL-1ß exposure [14 15 16 ]. Moreover, GCP-2 is produced during peritonsillar abscess [17 ]. Similarly, GCP-2 is expressed in the synovial tissue during rheumatoid arthritis [18 ]. The recurring expression of GCP-2 coincides with the relapsing nature of these inflammatory diseases, which are also T cell-dependent. Hence, we have used a mouse model, which mucosally administers GCP-2 (multiple times) along with a T cell-dependent antigen, to study this chemokine’s role in mucosal adaptive immune and inflammatory responses.

The multiple biological activities related to the immunopathogenesis of GCP-2 are poorly understood. Effective leukocyte infiltration along with activation can initiate immunostimulating cascades that help to bridge innate and adaptive host responses. The present study has determined some of the cellular and molecular mechanisms that GCP-2 uses to modulate adaptive immunity.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Immunogens
GCP-2/LPS-induced CXC chemokine [19 ] was purchased from PeproTech (Rocky Hill, NJ). The potential level of endotoxin contamination was quantified by the chromogenic Limulus amebocyte lysate assay (Cape Cod Inc., East Falmouth, MA) to be <5 endotoxin units/mg. Chicken egg albumin (OVA) and bovine serum albumin (BSA) were purchased from Sigma Chemical Co. (St. Louis, MO).

Mice and immunizations
Female BALB/c and IL-8Rß–/– on BALB/c background mice, aged 6–8 weeks, were purchased from Jackson Laboratories (Bar Harbor, ME). All mice were housed in horizontal laminar flow barrier cabinets free of microbial pathogens. Routine antibody (Ab) screenings for a large panel of pathogens and histological analyses of organs and tissues were performed to ensure that mice were pathogen-free. Following anesthesia, mice were nasally immunized on days 0, 7, and 14 with 75 µg OVA alone or OVA plus 1 µg GCP-2 in 15 µl phosphate-buffered saline (PBS; 7.5 µl per nare). Experimental groups consisted of five mice, and studies were repeated three 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 1 ml PBS containing 0.1% sodium azide per 100 mg fecal pellet. Following suspension by vortexing for 10 min, fecal samples were centrifuged, and supernatants were collected for analysis. Blood samples were collected by supra-orbital capillary puncture, and serum was obtained following centrifugation. Serum and mucosal secretions were collected 1 week after the last immunization and analyzed for OVA-specific Ab responses by enzyme-linked immunosorbent assay (ELISA). Mice were killed by CO2 inhalation 1 week after the last immunization to quantify the OVA-specific CD4+ T cell responses present in immune compartments.

Cell isolation
After nasal immunization with PBS and/or 75 µg OVA alone or OVA plus 1 µg GCP-2, leukocytes were obtained from single-cell suspensions of spleen, lung, cervical lymph node (CLN), and nasal tract [3 4 5 ]. To isolate lower respiratory tract lymphocytes, lungs were injected with 10 ml cold PBS to remove blood, dissected into small pieces, and digested in collagenase type IV (Sigma Chemical Co.) in RPMI 1640 (collagenase solution) for 45 min with stirring at 37°C [2 3 4 5 ]. Nasal tract lymphocytes were isolated by gently washing nasal cavities with 200 µl cold PBS to remove blood. Next, the nasal tract mucosal tissue was removed by scrapping. Cell suspensions were washed twice in RPMI 1640. Lung and nasal tract lymphocytes were further purified using a discontinuous Percoll (Pharmacia, Uppsala, Sweden) gradient, collecting at the 40–75% interface.

T cell fractions were obtained by passing single-cell suspensions over nylon wool for 1 h at 37°C (>98% purity). Subsequently, CD4+ T cells were enriched (>98% purity) using Mouse CD4 Cellect® plus columns, according to the manufacturer’s protocols (Biotex Laboratories, Edmonton, Alberta, Canada). Lymphocytes were maintained in complete medium, which consisted of RPMI 1640 supplemented with 10 ml/L nonessential amino acids (Mediatech, Washington, DC), 1 mM sodium pyruvate (Sigma Chemical Co.), 10 mM HEPES (Mediatech), 100 U/ml penicillin, 100 µg/ml streptomycin, 40 µg/ml gentamycin (Elkins-Sinn, Cherry Hill, NJ), 50 µM mercaptoethanol (Sigma Chemical Co.), and 10% of fetal calf serum (FCS; Atlanta Biologicals, Norcross, GA).

Cytokine and OVA-specific Ab detection by ELISA
For the assessment of cytokine production by the spleen, lungs, nasal tract, and CLNs, culture supernatants were harvested after 3 days of ex vivo restimulation. The presence of T helper cytokines, IL-2, IL-4, IL-5, IL-6, IL-10, IFN-{gamma}, and tumor necrosis factor {alpha} (TNF-{alpha}), in cell culture supernatants was determined by ELISA following the manufacturer’s instructions (E-Biosciences, San Diego, CA). Fecal and serum sample levels of OVA-specific Ab were measured by ELISA, as previously described [2 ]. Briefly, 96-well Falcon 3912 flexible ELISA plates (Fisher Scientific, Pittsburgh, PA) were coated with 100 µl 1 mg/ml OVA in PBS overnight (O/N) at 4°C and blocked with 10% FCS (Atlanta Biologicals) in PBS (B-PBS) for 3 h at room temperature. Individual samples (100 µl) 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, IgA, or IgG subclasses were determined by the addition of 100 µl biotinylated detection Ab (BD PharMingen, San Diego, CA). After incubation and wash steps, 100 µl 1:3000 dilution of antibiotin horseradish peroxidase Ab (Vector Laboratories, Burlingame, CA) in B-PBS-T was added to IgG subclass detection wells and incubated for 1 h at room temperature. Following incubation, all plates were washed six times, and the color reaction was developed by adding 100 µl 1.1 mM 2,2'-azino-bis(3)-ethylbenzthiazoline-6-sulfonic acid (ABTS; Sigma Chemical Co.) in 0.1 M citrate-phosphate buffer (pH 4.2) containing 0.01% H2O2 (ABTS solution). The plates were read at 415 nm after 10 min.

T cell proliferation assay
Antigen-specific lymphocyte proliferation was measured by a 5-bromo-2'-deoxy uridine (BrdU) absorption-detection kit, according to the manufacturer’s instructions (Roche Diagnostics, Dusseldörf, Germany). Subsequently, BrdU incorporation was detected using a scanning multiwell spectrophotometer (SpectraMax 250 ELISA reader, Molecular Devices, Sunnyvale, CA). In brief, after 2 days of culture with OVA (1 mg/ml), CD4+ T cells at the density of 5 x 106 cells/ml with 106 cells/ml {gamma}-irradiated feeder splenocytes were transferred to polystyrene 96-well plates (Corning Glass Work, Midland, MI). BrdU labeling solution (10 µl; 10 µM final concentration per well) was added, and cells were incubated for 18 h at 37°C with 5% CO2. The cells were then fixed and incubated with 100 µl nuclease in each well for 30 min at 37°C. The cells were washed with complete media and again incubated with BrdU-peroxidase solution for 30 min at 37°C. The incorporation was developed with a ABTS solution, and the change in optical density was read at 450 nm (OD450).

Flow cytometry analysis of costimulatory molecules
Lymphocytes were isolated from the spleens of normal and IL-8Rß–/– mice and added at a density of 106 cells/ml in complete medium containing 0, 1, 10, 100, or 1000 ng/ml GCP-2. Anti-CD3{varepsilon} Ab-coated plates were used to activate primary CD4+ T cells from normal or IL-8Rß–/– mice. After incubation for 3 days, the cells were stained with phycoerythrin (PE)-conjugated rat anti-mouse CD28, CD80, or CD86 plus fluorescein isothiocyanate (FITC)-conjugated rat anti-mouse CD4 or B220 monoclonal Ab (mAb; BD PharMingen) for 30 min with shaking. Lymphocytes were then washed with fluorescein-activated cell sorter (FACS) buffer (PBS with 1% BSA), fixed in 2% paraformaldehyde in PBS, and analyzed by flow cytometry (Becton Dickinson, San Diego, CA). The percent increase (or decrease) of the costimulatory molecule expression by resting or CD3{varepsilon}-activated splenocytes from normal and IL-8Rß–/– mice in cultures with supernatant containing GCP-2 was calculated as CD28 or CD80 and CD86 expression on CD3+ CD4+ or CD3– B220+ cells, respectively, cultured with GCP-2 ligands minus the percent gated of double-positive cells in cultures without GCP-2, divided by the latter.

Flow cytometry analysis of leukocyte migration
Mice were immunized with OVA alone or OVA plus GCP-2, as before. After 12 h, leukocytes from the spleen or mucosal tissues were stained with CY5-conjugated CD4, FITC-conjugated CD8, and/or B220 mAb along with PE-conjugated CD11b, CD11c, NK1.1, and LY6.G (BD PharMingen) for 30 min with occasional shaking. Labeled cells were washed with FACS staining buffer (PBS with 1% BSA), fixed in 2% paraformaldehyde in PBS, and 104 cells were analyzed using a FACScanTM flow cytometer and CellquestTM software (BD PharMingen).

Statistics
The data are expressed as the mean ± SEM and compared using a two-tailed Student’s t-test or an unpaired Mann Whitney U-test. The results were analyzed using the Microsoft Excel program (Seattle, WA) and were considered statistically significant if Pvalues were <0.05. When cytokine levels were below the detection limit (BD), they were recorded as one-half the lower detection limit (e.g., 10 pg/ml for IL-10) for statistical analysis.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
GCP-2 stimulates OVA-specific, systemic Ab responses
The optimal doses required for chemokines to induce chemotaxis have been well-documented [20 21 23 24 ]; however, previous studies from our laboratory yield consistent and significant increases when 1 µg chemokine(s) is given as mucosal adjuvants [4 5 6 ]. To determine the optimal dose of GCP-2 as adjuvant, which would affect antigen-specific serum Ab responses, mice were nasally administered (three times at weekly intervals) with 75 µg OVA alone or in the presence of increasing concentrations of GCP-2 (e.g., 0.0, 0.5, 1.0, 2.5, and 5.0 µg). Accordingly, we analyzed OVA-specific IgA, IgG, and IgM Ab isotypes in sera. Significant titers of OVA-specific Ab responses were elicited when mice received 1 µg GCP-2 as adjuvant (Fig. 1 ). Although higher doses of GCP-2 also enhanced humoral responses, there was no significant increase in host responses when >1 µg GCP-2 was used as adjuvant. Therefore, subsequent studies used 75 µg OVA plus 1 µg GCP-2 as the immunization regimen.



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  		Figure 1. Dose response of OVA-specific serum Ab responses following immunization using GCP-2 as adjuvant. Groups of five BALB/c mice were nasally immunized on days 0, 7, and 14 with 75 µg OVA alone or 0.5, 1.0, 2.5, and 5.0 µg GCP-2 in 15 µl PBS. OVA-specific IgA (•), IgG ({blacksquare}), and IgM ({blacktriangleup}) Ab titers in the serum on day 21 were determined by ELISA, and data presented are the mean Ab titers ± SEM of four separate experiments. (*) Statistically significant differences (i.e., P<0.05) from OVA-specific IgG Ab titers of mice immunized with OVA alone.

 
Mice nasally immunized three times with 75 µg OVA plus 1 µg GCP-2 displayed significant increases in antigen-specific serum IgG responses when compared with mice receiving OVA alone (Figs. 1 and 2A ). The humoral adjuvant activity of GCP-2 induced a significant increase in serum IgG1 responses followed by IgG2b responses (Fig. 2B) . We next asked whether the adjuvant activity of nasally coadministered GCP-2 could promote mucosal secretory IgA (S-IgA) Ab responses. Analysis of OVA-specific S-IgA responses in mucosal secretions revealed significant S-IgA Ab titers in fecal extracts (Fig. 2C) .



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  		Figure 2. OVA-specific serum and fecal Ab responses following nasal immunization. Groups of five BALB/c mice were nasally immunized on days 0, 7, and 14 with 75 µg OVA alone or 1.0 µg GCP-2 in 15 µl PBS. (A and B) Ig isotype and IgG subclass Ab titers, respectively, in the serum. (C) IgA and IgG Ab titers in fecal secretions. OVA-specific serum and fecal Ab titers on day 21 were determined by ELISA, and data presented are the mean Ab titers ± SEM or those BD of four separate experiments. (*) Statistically significant difference (i.e., P<0.05) from Ab titers of mice immunized with OVA alone.

 
Proliferation and cytokine responses induced by GCP-2
As GCP-2 enhanced mucosal and systemic Ab responses, we next examined the pattern of the T helper cytokine responses it promoted. CD4+ T cells isolated from the spleen, nasal tract, CLNs, or lungs of mice immunized with OVA plus GCP-2 exhibited marked increases in OVA-specific, proliferative responses as compared with CD4+ T cells from mice immunized with OVA alone (Fig. 3 ). CD4+ T cells from the spleen, nasal tract, CLNs, and lungs of mice immunized with OVA plus GCP-2 also showed significant increases in IL-2, TNF-{alpha}, and IFN-{gamma} secretions by OVA-restimulated T cells compared with controls (Fig. 3) . As an adjuvant, GCP-2 also increased Th2 responses; most notably, IL-5 and IL-6 secretions by ex vivo-restimulated T lymphocytes were dramatically elevated in immunized mice (Fig. 3) . GCP-2 promoted relatively low IL-4 or IL-10 responses. Taken together, these results show that GCP-2 enhanced IL-2, IL-5, and IL-6 as well as TNF-{alpha} and IFN-{gamma} T cell responses.



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  		Figure 3. Proliferation and T helper cell type 1 (Th1)/Th2-type cytokine secretion by OVA-restimulated CD4+ T cells from previously immunized mice. Groups of five BALB/c mice were nasally immunized on days 0, 7, and 14 with 75 µg OVA alone or with 1.0 µg GCP-2 in 15 µl PBS. One week after the last immunization with OVA alone (open bar) or OVA plus GCP-2 (solid bar), lung-, spleen-, nasal tract-, and CLN-derived CD4+ T cells were purified and cultured at a density of 5 x 106 cells/ml with 1 mg/ml OVA for 3 days. Cytokine ELISA was determined in culture supernatant productions. Proliferation was measured by BrdU incorporation. The data presented are the mean – OD450 for proliferative responses or IL-2, TNF-{alpha}, IL-4, IL-5, IL-6, IL-10, and IFN-{gamma} secretion (pg/ml) ± SEM of quadruplicate cultures. (*) Statistically significant difference (P<0.05) between OVA alone and OVA plus GCP-2-immunized mice.

 
Primary Th cell responses induced by GCP-2
When GCP-2 was used as an adjuvant, it up-regulated systemic and mucosal Ab responses as well as the responses of CD4+ T cells from the spleen, nasal tract, CLNs, and lungs. We next examined whether these effects were mediated through IL-8Rß interactions. CD3{varepsilon} stimulation was required to increase Th1 and Th2 cytokine secretion by wild-type or IL-8Rß–/– T cells. IL-2 secretion patterns of CD3{varepsilon}-stimulated CD4+ T cells from normal and IL-8Rß–/– mice were not affected by GCP-2, which increased TNF-{alpha} and IFN-{gamma} primary Th1 responses after CD3{varepsilon} stimulation in normal mice but not by IL-8Rß–/– CD4+ T cells, treated in a similar manner (Fig. 4 ). GCP-2 leads to a robust increase in the secretion of IL-5 and IL-6 cytokines by wild-type, primary, CD3{varepsilon}-stimulated T cells but not by similarly treated IL-8Rß–/– T cells. It is interesting that GCP-2 induced IL-4 and IL-10 secretion by CD4+ T cells from IL-8Rß–/– mice after CD3 stimulation when compared with resting CD4+ T cells or CD3{varepsilon}-stimulated, wild-type Th cells. Taken together, these results show that GCP-2 increases IL-4 and IL-10 secretion patterns by CD3{varepsilon}-stimulated, IL-8Rß–/– naïve CD4+ T cells but enhances IL-5, IL-6, TNF-{alpha}, and IFN-{gamma} production by CD3{varepsilon}-stimulated, wild-type, primary CD4+ T cells.



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  		Figure 4. Cytokine secretion by CD3{varepsilon}-stimulated, naïve T cells from IL-8Rß–/– and normal mice. CD4+ T cells from IL-8Rß–/– (open and checkered bars) and normal mice (solid and dotted bars) were cultured at a density of 5 x 106 cells/ml with 0, 1, 10, 100, or 1000 ng/ml GCP-2 on uncoated (checkered and dotted bars) or anti-mouse CD3{varepsilon} Ab-coated plates (solid and open bars). IL-2, TNF-{alpha}, and IFN-{gamma} as well as IL-4, IL-5, IL-6, and IL-10 production was determined by ELISA of cultured supernatants. ({cjs3576}) Statistically significant differences (P<0.05) between cultured, resting, naïve lymphocytes from IL-8Rß–/– or normal mice. (*) Statistically significant differences (P<0.05) between cultured, CD3{varepsilon}-stimulated, naïve lymphocytes from IL-8 Rß–/– or normal mice.

 
GCP-2 modulates CD28 and B7 expression
Earlier studies have shown that chemokines can differentially modulate the expression of costimulatory molecules by lymphocytes [4 5 6 , 20 21 22 23 24 ]. To better elucidate the effects of GCP-2 on adaptive immune responses, we assessed its potential to modulate the expression of costimulatory molecules by lymphocytes from normal and IL-8Rß–/– mice. GCP-2 had minimal or no effects on resting wild-type or IL-8Rß–/– lymphocytes (Fig. 4 and data not shown). However, GCP-2 significantly increased the expression of CD28 by CD3{varepsilon}-stimulated lymphocytes from wild-type mice but not from IL-8Rß–/– mice (Fig. 5 ). Similar to the induction of CD28 expression, GCP-2 also modestly increased the expression of CD80 and CD86 by B220+ B cells from normal mice but not from IL-8Rß–/– mice costimulated by anti-CD3{varepsilon}, mAb-treated T cells in culture. As described previously by others [20 21 22 23 24 ] and our laboratory [4 5 6 ], chemokines optimally induce chemotaxis at ~10 ng/ml and affect costimulatory molecule expression and leukocyte activation at 10–50 ng/ml. In confirmation, we also show that GCP-2 behaves in a similar manner to increase CD28 and B7 expression in part through IL-8Rß interactions.



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  		Figure 5. Modulation of CD28 CD80, and CD86 expression by GCP-2. CD3{varepsilon}-stimulated, naïve lymphocytes from normal (•) or IL-8Rß–/– ({circ}) mice were incubated with 0, 1, 10, 100, and 1000 ng/ml GCP-2 in 96-well culture plates. The percent increase (or decrease) in the expression of the costimulatory molecules by normal or IL-8Rß–/– lymphocytes was calculated as the percent of double-positive (CD4+ CD28+, B220+ CD80+, or B220+ CD86+) cells in cultures containing GCP-2 minus the percent gated of double-positive cells in cultures without GCP-2, divided by the latter. Studies were repeated four times, and the data presented are the mean percent change ± SEM of these experiments.

 
GCP-2-mediated in vivo migration of leukocyte subpopulations
To further establish the effect of GCP-2 on the modulation of cellular and humoral immunity, mice were nasally immunized as before with OVA plus GCP-2 or with PBS and/or with OVA alone. Nasal immunization (with OVA alone) did not significantly alter the number of leukocytes in the spleen or lungs (Table 1 ). When compared with OVA alone, GCP-2 plus OVA significantly decreased the number of CD4+ T cells in the nasal tract and CLNs but caused a modest increase in the number of B cells in the CLNs and a significant increase in the number of nasal tract B220+ B cells. A modest yet statistically significant increase in the number of CD11c+ dendritic cells in CLNs was observed 12 h after OVA plus GCP-2 immunization.


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  		Table 1. GCP-2 Effects on In Vivo Leukocyte Migration

 
We also noted changes in the number of LY-6G+ neutrophils after nasal administration of OVA alone when compared with OVA plus GCP-2; a significant increase in neutrophils was noticed in the nasal tract 12 h after immunization. Taken together, these data suggest that GCP-2 (+OVA) mediates leukocyte recruitment to and from the nasal tract and CLNs, 12 h after nasal immunization when compared with groups given OVA alone.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
GCP-2 acts as a functional murine homologue of hIL-8 and GCP-2 [11 12 13 ], and its ability to interact with CXCR1 and CXCR2 provided the rationale(s) to test our hypothesis that GCP-2 enhances adaptive immunity. Previous studies from our laboratory showed that RANTES, when used as an adjuvant, induces predominantly Th1-driven, antigen-specific IgG2a, followed by IgG2b, IgG3, and IgG1 Ab [4 ], and that MIP-1{alpha} functions as a Th1 inducer to propagate cytotoxic T cell responses and serum IgG2a and mucosal IgA Ab responses [5 ]. Lymphotactin also acts as an innate mucosal adjuvant to induce Th2 > Th1 responses and dramatically increased serum IgG subclasses with robust mucosal IgA Ab responses [3 ]. In the present study, we have shown that GCP-2 fosters Th2 and Th1 responses as well as CD28-B7 expression, in part through IL-8Rß ligation.

The increases in IgG1 and the subsequent expression of IgG2b and IgG2a OVA-specific Ab titers were most likely a result of the mixed Th2/Th1 cytokine help provided by CD4+ T cells as well as by use of a soluble protein antigen [25 , 26 ]. In this regard, Th2 cytokines support IgG1 [26 27 28 ] and IgG2b [29 ] Ab generation, and the levels of anti-OVA IgG1 and IgG2b Ab induced by GCP-2 plus OVA were consistent with the cytokine secretion patterns of IL-5 and IL-6 expression by CD4+ T cells from mice immunized with GCP-2 as compared with mice immunized with OVA alone. Indeed, IL-5 has been shown to increase the Ig secretion of IgA-, IgG1-, and IgE-committed B cells [30 , 31 ].

GCP-2, as an adjuvant, also increased antigen-specific IL-2, TNF-{alpha}, and IFN-{gamma} CD4+ T cell responses compared with mice immunized with OVA alone. IFN-{gamma} production is often associated with IgG2a and IgG3 Ab production [32 ]. The low doses of IFN-{gamma} (1500 units) have been shown to increase IgG2a production in vivo, and considerably higher doses of IFN-{gamma} (12,500 units) are required to induce decreases in IgG1 and IgE responses [33 ]. Taken together, the analysis of the OVA-specific, humoral responses was supported by mixed Th2/Th1 cytokine help.

The precise cytokine signals required for S-IgA production and for mucosal immunity in general are not completely understood. It has been shown that mucosal IgA responses require Th2-type, cell-derived cytokines (e.g., IL-5, IL-6, and IL-10) [34 ]. Studies have supported that Th1- and Th2-type, cell-derived cytokines are important for S-IgA responses [34 35 36 ]. We have previously shown that lymphotactin, MIP-1{alpha}, MIP-1ß, and RANTES induce mucosal IgA [3 4 5 ]. The level of the particular cytokines(s) required for B cells to express the IgA isotype was also provided in our experimental model. Although it has been reported that IL-4, IL-5, and IL-6 do not induce IgA switching, IL-5 and IL-6 induce surface IgA+ B cells to secrete IgA [34 ]. The cytokine produced by CD4+ T cells in systemic and mucosal compartments after GCP-2 immunization explains why an increase in OVA-specific IgA occurred in mucosal secretions. The heightened mucosal Ab responses generated by GCP-2 also correlated with the Th2 and Th1 responses displayed by OVA-restimulated CD4+ T cells from immunized mice.

The end result of the immunization strategy we used was increased humoral and cell-mediated, adaptive immune responses. However, the mechanism of GCP-2 adjuvantcy remained uncertain. We have previously shown that chemokines can modulate cytokine and costimulatory molecule expression by activated lymphocytes [3 4 5 ]. Now, we show that GCP-2 modulates cytokine and costimulatory molecule expression by activated T cells (Figs. 4 and 5) . However, cytokine secretion alone does not completely explain the adjuvant effects of GCP-2.

CD28 is equally expressed by CD4+ and CD8+ T cells, which cooperatively regulate T cell activation through B7 and T cell receptor stimulation [37 ]. CD28 supplies a coactivation signal for T cell activation [38 , 39 ] and is required for mucosal and T cell-mediated immunity [40 , 41 ]. We have previously shown that RANTES and MIP-1{alpha} act as mucosal adjuvants, partly through CD28 up-regulation [4 , 5 ]. Similarly, CXCR3 ligands may enhance adaptive immune responses through CD28 modulation [6 ]. GCP-2 significantly increased CD28 expression by CD3{varepsilon}-stimulated wild-type CD4+ T cells in a dose-dependent manner but not by IL-8Rß–/– Th cells treated in a similar manner.

To address another potential mechanism of adjuvant activity, we investigated how GCP-2 affects the expression of B7 molecules on B cells, which also express IL-8Rs [42 ]. Previous studies first showed that the CD28 binds B7-1, B7-2, and B7-H1 on antigen-presenting cells [43 , 44 ]. It has been reported that the mucosal adjuvanticity of CT involves the selective up-regulation of CD86 expression [45 ] and that MIP-1{alpha} significantly up-regulates the expression of CD80 [5 ], like RANTES [4 ], but also increases the CD86 surface level on resting B cells. Similarly, our data suggest that GCP-2 modestly enhances B7-1 and B7-2 surface expression on B cells in a dose- and IL-8Rß-dependent manner to presumably support primary adaptive immune responses. Hence, local GCP-2 expression during the innate host response could act on adjacent or nearby lymphocytes to enhance the expression of IL-5, IL-6, TNF-{alpha}, and IFN-{gamma} after initial antigen stimulation. Together, this would help initiate and direct adaptive immune response.

It is generally accepted that neutrophils are among the first leukocytes recruited to sites of injury or infection. Indeed, increases in IL-8R ligands are an indication of neutrophil recruitment to inflammatory loci. In the present study, we have shown that GCP-2 provides signals to bridge innate and adaptive immunity. Potentially, these characteristics would allow GCP-2-activated leukocytes to contain and engulf mucosal pathogens and subsequently, present foreign antigens to naïve lymphocytes to support protective and adaptive immune responses. Correspondingly, these mechanisms would also permit GCP-2 to markedly enhance antigen-specific host responses to microbes that enter the sterile, peripheral environment.

GCP-2 may use several mechanisms to facilitate the induction of adaptive mucosal immune responses. Our results show that GCP-2 enhances the recognition phase of the adaptive host response by modestly increasing the number of B cells, decreasing the number of CD4+ T cells from CLNs and the nasal tract, and most importantly, increasing neutrophils in the nasal tract 12 h after nasal immunization. The variances observed in humoral and cellular immunity induced by GCP-2 coincide with its ability to modulate leukocyte, especially neutrophil, migration to and from mucosal effector sites. This innate ability of GCP-2 to induce chemotactic and costimulatory signals has implications in mechanisms of immunity and the priming and maintenance of chronic inflammatory responses. IL-8R internalization may represent another mechanism that regulates the migration and retention of neutrophils to inflammatory sites as well as controlling the immune or inflammation-enhancing function of GCP-2 [46 ]. Our results suggest that GCP-2 dictates immune responses on concomitant exposure with leukocytes. Although further studies will be needed to elucidate the precise cellular and molecular contributions that GCP-2 makes toward host immune responses, our results have helped to clarify some of the cellular and molecular mechanisms that this chemokine uses to affect mucosal and systemic adaptive immunity and chronic inflammation.


   ACKNOWLEDGEMENTS
 
U. P. S. and S. S. contributed equally to this manuscript.

Received September 26, 2003; revised August 4, 2004; accepted August 17, 2004.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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