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* Mercer University, Atlanta GA 30341;
Morehouse School of Medicine, Atlanta, GA 30310;
Centers for Disease Control and Prevention, Atlanta, GA 30333;
University of Alabama at Birmingham, Birmingham, AL 35294; and
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National Institute on Aging, National Institutes of Health, Baltimore, MD 21224
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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-secreting CD4+ T cells and increasing the number of Ag-specific IL-10-producing CD4+ T cells; these changes combined also corresponded with the transition from pneumococcal carriage to lethal pneumonia. These data suggest that CCL5 is an essential factor for the induction and maintenance of protective pneumococcal immunity. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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CCL5 is a CC chemokine that binds CCR1, CCR3, CCR4, and CCR5 and is produced by epithelial cells (5, 6, 7, 8, 9, 10), lymphocytes (11, 12), and platelets (13) and acts as a potent chemoattractant for monocytes (14, 15), NK cells (16), memory T cells (14, 15, 17), eosinophils (13), dendritic cells (18), and basophils (19). Importantly, CCL5 expression is increased by human nose- and adenoid-derived epithelial cells when infected with mucosal pathogens (5, 9) and has been shown to induce lymphocyte migration into the nasal mucosa of allergic patients (7). Its expression can also be induced by infecting human bronchial tissue- or nasal polyp-derived epithelial cells with the influenza virus (8).
In previous studies we demonstrated that, following nasal immunization, CCL5 initiates and enhances Ag-specific humoral and cellular immune responses in both mucosal and systemic compartments (20). We have also shown that CCL5 augments immune responses by activating host macrophages, B cells, and T cells and by enhancing Ag presentation as well as costimulatory molecule up-regulation. Others have shown that CCL5 induces T cell adhesion to ICAM-1, VCAM-1, fibronectin, collagen, and laminin of the extracellular matrix (21). In addition, CCL5 can selectively activate corresponding CCR5+ lymphoid cell targets (16, 22, 23, 24, 25).
Recently, contrasting results have been reported regarding the effect of this chemokine and its receptors on the outcome of Th1- and Th2-mediated immunity and disease. For example, anti-CCL5 Ab treatment was shown to decrease mycobacterium-inducible Th1-type lesions while increasing schistosome-inducible Th2-type granulomas in mice (26). It was also demonstrated that CCL5 inhibited IL-4 secretion through CCL5-CCR1 interactions, suggesting the potential role of CCL5 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 (27). Although these studies have addressed the importance of this CCR5 ligand in response to microbial infections, the role of CCL5 in the outcome of infectious disease and mucosal immunity remains illusive.
It has been shown that genetic variations in CCL5 contribute to differences in infectious disease progression. Indeed, polymorphisms in CCR5 and CCL5 modulate immune responses that play critical roles in susceptibility and progression to infectious diseases, namely HIV-1 and AIDS, respectively (28, 29, 30). However, it is not certain what effects these variations have on S. pneumoniae disease susceptibility, progression, and/or protective immunity. Moreover, it is also unknown whether mucosal or merely systemic immune responses are needed to prevent carriage or progressive/invasive S. pneumoniae infections.
Pneumococci in nasopharyngeal carriage are thought to be the main human reservoir for these potentially lethal bacteria. Moreover, nasopharyngeal carriage is thought to be an intermediate stage that precedes invasive disease. To this end, Pneumococcus has evolved to asymptomatically colonize the upper respiratory tract; yet, it is still recognized by the host as an invasive pathogen once it enters the lung (31, 32, 33, 34, 35, 36). Our study specifically addresses an important question: what relationship, if any, exists between CCL5 expression and pneumococcal infection that determines S. pneumoniae susceptibility and progression? This is a fundamental question for understanding how to better prevent, diagnose, and treat diseases caused by S. pneumoniae.
We have used a novel human isolate of capsular group 19 pneumococci that was passed in mice to yield S. pneumoniae strain EF3030, which has a greater propensity to cause nasal or pulmonary infections than to cause sepsis when given intranasally (37). The current study uses this unique mouse model to unravel the cellular and molecular mechanisms of pneumococcal carriage. Through Ab-mediated inhibition, we show that CCL5 is essential for optimal mucosal and systemic cellular and humoral pneumococcal immunity.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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Murine CCL5 was purchased from PeproTech. CCL5 and Freund’s or incomplete Freund’s adjuvants (Sigma-Aldrich) were used to generate anti-CCL5 Ab titers of 
1:106 such that 10 µl of rabbit anti-CCL5 antiserum neutralized 20 ng of CCL5. This antiserum was titrated by direct ELISA, and no cross-reactivity was detected when tested against other CCR5 ligands (CCL3 and CCL4), chemokines (CXCL1, CXCL9, CXCL11, CXCL12, CXCL13, XCL1, CCL2, CCL3, CCL4, CCL5, CCL7, CCL8, and CCL11), and cytokines (IL-2, IL-4, IL-5, IL-6, IL-10, IL-12, and TNF-
). Subsequently, normal or anti-CCL5 sera were heat-inactivated and purified using an IgG isotype-specific protein A column (Pierce). Anti-CCL5 Ab titers were adjusted to 1:4 x 105 (i.e., 50x dilution) in PBS (CCL5 Ab solution).
Mice
Pneumococcal carriage-susceptible female BALB/c mice, ages 8–12 wk, were procured from The Jackson Laboratory. All mice were housed in horizontal laminar flow cabinets free of microbial pathogens. To ensure that mice were pathogen free, the mice were routinely screened for a large panel of pathogens, and histological analysis was performed on organs and tissues. Experimental groups consisted of 10 mice, and studies were repeated up to six times. The guidelines proposed by the Committee for the Care of Laboratory Animal Resources Commission of Life Sciences, National Research Council (Washington, DC), were followed to minimize animal pain and distress. All procedures involving mice were approved by the Medicine Institutional Review Board of the Morehouse School of Medicine (Atlanta, GA).
Pneumococcal strains and establishment of pneumococcal carriage
S. pneumoniae capsular group 19 strain EF3030 (37) was obtained from A. Parkinson of the Arctic Investigations Laboratory, Centers for Disease Control (Anchorage, AK). S. pneumoniae strain EF3030 was among the human isolates of capsular group 19 that were examined previously and found to be relatively noninvasive in mice (38). Pneumococci were grown in Todd-Hewitt broth and stored frozen in aliquots at –70°C in sterile lactated Ringer’s injection solution (Abbott Laboratories). To establish nasal carriage, groups of BALB/c mice were nasally administered 106.88 CFU of S. pneumoniae strain EF3030 in 15 µl of Ringer’s solution (39).
Pneumococcal surface Ag A (PsaA)3
Recombinant lipidated PsaA was prepared with the QIAexpress system (Qiagen). The expression host, Escherichia coli SG 13009, was transformed with pAB247, a recombinant plasmid that carries the psaA gene from the serotype 2 S. pneumoniae strain D39 cloned into pQE30. The His-tagged recombinant PsaA was purified by Ni-NTA chromatography (40). The potential level of endotoxin contamination was quantified by the chromogenic Limilus amebocyte lysate assay (Associates of Cape Cod) and shown to be <5 ELISA units/mg.
Sample and tissue collection
To obtain individual nasal wash samples from each mouse, the interior of the nasal tract was flushed by placing polyethylene tubing (1-mm diameter; BD Biosciences), attached to a 1-cc syringe through the trachea toward the nasal cavity, and rinsed with 150 µl of sterile PBS. Blood samples were collected by retro-orbital plexus puncture using heparinized capillary tubes for PsaA-specific Ab analysis by ELISA. Mice were sacrificed by CO2 inhalation to collect the spleen and mucosal lymphoid tissues (nasopharyngeal-associated lymphoid tissue (NALT), cervical lymph node (CLN), and lung) for single-cell isolation of lymphocytes.
Cell isolation
Single-cell suspensions of spleen, lung, CLNs, and NALT were prepared by aseptically removing tissues and passage through a sterile wire screen. The lower respiratory tract (lungs and mediastinal lymph nodes) was perfused with 1 ml of cold PBS to remove blood, dissected into small pieces, and subjected to collagenase digestion using 1 mg/ml collagenase type IV (Sigma-Aldrich) in RPMI 1640 (collagenase solution) (41). Nasal tract lymphocytes were isolated by gently washing nasal cavities with 200 µl of cold PBS to remove blood. Next, NALT was removed by scraping, and the resulting tissue was then passed through a sterile glass wool column (41). NALT and lung cells were further purified using a discontinuous Percoll (Amersham Biosciences) gradient, collecting large, low density cells at the 40–55% interface or small, high density cells (i.e., lymphocytes) at the 55–75% interface (41, 42).
T cell fractions were obtained by passing single cell suspensions over nylon wool column for 1 h at 37°C (> 98% purity). Subsequently, CD4+ T cells were enriched using mouse CD4 Cellect Plus columns according to the manufacturer’s protocol (Biotex Laboratories). Cell suspensions were washed twice in RPMI 1640, and lymphocytes were maintained in complete medium that consisted of RPMI 1640 supplemented with 10 ml/L nonessential amino acids (Mediatech), 1 mM sodium pyruvate (Sigma-Aldrich), 10 mM HEPES (Mediatech), 100 U/ml penicillin, 100 µg/ml streptomycin, 40 µg/ml gentamicin (Elkins-Sinn), and 10% FBS (Sigma-Aldrich).
RNA isolation, primer design, and RT reaction
Nucleotide sequences for mouse IL-4, IL-10, TNF-, IFN-, IL-12p40, CCL5, CCL3, CCL4, and CCR5 mRNAs and 18S rRNA were obtained from National Institutes of Health/National Center for Biotechnology Information GenBank database under accession numbers NM_021283, NM_010548, NM_013693, K00083, M86671, NM_013653, NM_011337, NM_013652, D83648, and X00686.1, respectively. These sequences were then used to design primers for RT-PCR analysis, which generated amplicons 90, 96, 100, 98, 102, 97, 102, 110, 100, and 149 bp in size for IL-4, IL-10, TNF-, IFN-, IL-12p40, CCL5, CCL3, CCL4, and CCR5 mRNAs and 18S rRNA, respectively. Primers were designed using the Primer 3 software program from Whitehead Institute at the Massachusetts Institute of Technology (Boston, MA). Thermodynamic analysis of the primers was conducted using the computer programs Primer Premier (Integrated DNA Technologies) and MIT Primer III. The resulting primer sets for IL-4, IL-10, TNF-, IFN-, IL-12p40, CCL5, CCL3, CCL4, and CCR5 were compared against the entire human genome to confirm specificity and to insure that the primers flanked mRNA splicing regions.
Total RNA was isolated from the spleen, NALT, CLNS, and lung using Tri-reagent (Molecular Research Center) according to the manufacturer’s protocol. Potential genomic DNA contamination was removed from these samples by treatment with RNase-free DNase (Invitrogen Life Technologies) for 15 min at 37°C. RNA was then precipitated and resuspended in RNAsecure (Ambion). Next, cDNA was generated by reverse transcribing 1.5 µg of total RNA using TaqMan reverse transcription reagent (Applied Biosystems) according to the manufacturer’s protocol and amplified with specific cDNA primers using SYBR Green PCR Master Mix reagents (Applied Biosystems). Levels of mRNA copies (>10) relative to 18S rRNA copies of these targets were evaluated by RT-PCR analysis using the Bio-Rad iCycler and software.
Ag-specific Ab detection by ELISA
PsaA-specific Abs in nasal secretion and serum samples were measured by ELISA (42). Briefly, 96-well Falcon 3912 flexible ELISA plates (Fisher Scientific) were coated with 50 µl of 5 µg/ml PsaA in coating buffer (sodium carbonate buffer) overnight at 4°C and blocked with 10% FBS in PBS (FBS-PBS) for 3 h at room temperature. Individual samples (100 µl) were added and serially diluted in FBS-PBS. After overnight incubation, plates were washed three times using PBS containing 0.05% Tween 20 (PBS-T), and concentrations of IgG or IgA were determined by the addition of a 0.33 µg/ml HRP-conjugated, goat anti-mouse ,, or µ H chain-specific antisera (Southern Biotechnology Associates) in FBS-PBS-T. Similarly, 100 µl of biotin-conjugated rat anti-mouse 1 (G1-7.3 at 12.5 ng/ml), 2a (R19-15 at 125 ng/ml), 2b (R12-3 at 12.5 ng/ml), and 3 (R40-82 at 50 ng/ml) (BD Pharmingen) H chain-specific mAbs were used to determine IgG subclass Abs (42). After incubation and washing steps, 100 µl of 0.5 µg/ml HRP-anti-biotin Ab (BD Pharmingen) in FBS-PBS-T were added to IgG subclass Ab detection wells 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 µl of 1.1 mM 2,2'-azino-bis (3)-ethylbenzthiazoline-6-sulfonic acid (Sigma) in 0.1 M citrate-phosphate buffer (pH 4.2) containing 0.01% H2O2 (ABTS solution).
Th cytokine analysis by ELISA
Purified CD4+ T cells and irradiated feeder cells were cultured at a density of 5 x 106 and 106 cells per ml, respectively, in complete medium containing 5 µg/ml PsaA at 37°C in 5% CO2. For the assessment of cytokine production, 1 ml of culture supernatants from 12-well flat-bottom plates (Corning Glass Works) were harvested after 3 days of ex vivo Ag-restimulation. IL-2, IL-4, IL-6, IL-10, TNF-, IFN- and GM-CSF levels in cell culture supernatants were determined by ELISA following the manufacturer’s instructions (e-Biosciences). Briefly, 96-well microtiter plates were coated with 100 µl (1/250 dilution) of rat anti-mouse IFN-, GM-CSF, TNF-, IL-2, IL-4, IL-6 and IL-10 in 0.1 M bicarbonate buffer (pH 8.2) overnight at 4°C and blocked with 10% FBS in PBS at room temperature for 3 h. Next, 100 µl of serially diluted recombinant murine cytokines, as standards or cultured supernatant samples, were added in duplicate and incubated overnight at 4°C. The plates were washed with PBS-T and incubated with (1/200 dilution) of biotinylated secondary murine cytokine detection Abs in FBS-PBS-T for 3 h at room temperature. After washing with PBS-T and PBS alone, wells were incubated for 2 h in 100 µl of avidin-HRP (1/250 dilution) and developed with tetramethylbenzidine solution. The cytokine ELISAs were capable of detecting 8 pg/ml IFN- and TNF-, 2 pg/ml IL-2, 4 pg/ml IL-4, IL-6, and GM-CSF, or 15 pg/ml IL-10.
CD4+ T cell proliferation
Lymphocyte proliferation was measured by BrdU absorption and detection (Roche Diagnostic Systems). In brief, purified CD4+ T cells were cultured at a density of 5 x 106 cells/ml with 106 irradiated feeder cells/ml in complete medium containing 5 µg/ml PsaA at 37°C in 5% CO2. After 2 days of ex vivo Ag restimulation, 1 x 106 cells/ml were transferred to polystyrene 96-well plates (Corning Glass Works). Ten microliters of BrdU labeling solution (10 µM/ml final concentration per well) was added, and the cells were incubated for 18 h at 37°C with 5% CO2. The cells were then fixed and incubated with 100 µl of nuclease in each well for 30 min at 37°C. Next, cells were washed with complete medium and incubated with BrdU-peroxidase solution for 30 min at 37°C. The incorporation of BrdU was correspondingly developed by adding 100 µl of tetramethyl-benzidine substrate. The substrate reaction was allowed to continue for 20 min. The OD was read at 405 nm with reference wavelength at 490 nm.
Flow cytometry
After nasal challenge with either PBS or EF3030, spleen, lung, CLN, and NALT cells were isolated, and changes in small, high density leukocytes and large, low density cell subpopulations were determined by FACS analysis. Specifically, after selective single-cell isolation, fluorescently tagged Abs (BD Pharmingen) were used to determine the changes in leukocyte subpopulations by flow cytometry. Lymphocytes were washed three times in PBS (supplemented with 1% BSA) and treated with 1 µg of PE-, FITC-, allophycocyanin-, or CyChrome 5-conjugated hamster anti-mouse CD3
(145-2C11) or rat anti-mouse CD4 (RM4-5), CD8 (53-6.7), CD11b (M1/70), or CD19 (1D3) IgG Abs (BD Pharmingen) per 105 cells at 4°C for 30 min in FACS staining buffer that consisted of pH 7.4–7.6 PBS containing 1 µg of Fc Block (BD Pharmingen) per 106 cells in 1% BSA. Similarly, control cells were stained with the appropriate rat IgG1, IgG2a, and IgG2b and/or hamster IgG1 isotype controls (BD Pharmingen) in FACS staining buffer. Subsequently, cells were washed with 1.0 ml of FACS buffer to remove unbound Abs. Next, labeled cells were fixed in 500 µl of 2% paraformaldehyde in PBS, and 104 cells were analyzed by flow cytometry using a FACScanTM flow cytometer and CellQuest software (BD Pharmingen).
For intracellular CCL5, IFN-, and IL-10 or CCR5 cell surface staining, PE-, FITC-, APC-, or CyChrome 5-conjugated hamster anti-mouse CD3 or CD11c (HL3; BD Pharmingen) and rat anti-mouse CCR5 (C34-3448; BD Pharmingen), CD4, CD8, CD11b, or CD11c were used to stain the surface of cells as before. Rat anti-mouse CCL5 (MAB478; R&D Systems), chicken anti-mouse cytokeratin 8 (Abcam catalog code ab14053), and rabbit anti-mouse Toll/IL-1R (TIR8)/single Ig IL-1-related receptor (SIGIRR) (Abcam catalog code ab22053) along with isotype control (Abcam) Abs were conjugated to allophycocyanin, CyChrome 5, or FITC, respectively, using custom fluorochrome conjugation kits (Prozyme or Pierce). Cells were resuspended in Cytofix/Cytoper solution and washed in 1x Perm/Wash solution (BD Pharmingen). Next, cell pellets were stained using FACS staining buffer along with 1 µg per 106 cells of fluorochrome-conjugated anti-mouse CCL5, IFN- (XMG1.2; BD Pharmingen), IL-10 (JES5-16E3; BD Pharmingen), TIR8, and/or cytokeratin 8 Abs.
Pneumococci Quantification
Single-cell suspensions of spleen, lung, NALT, and CLN tissues were prepared by passage through a sterile wire screen as described above. Cells from each tissue were suspended in 2 ml of sterile Ringer’s solution. Five serial 10-fold dilutions were made and plated (in quadruplicate) on blood agar plates containing 4 µg/ml gentamicin sulfate. CFUs per organ were enumerated 24 h after plating and incubation in a candle jar at 37°C.
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 Statview II statistical program (Abacus Concepts) for Macintosh computers and were considered statistically significant if p values were <0.05. When cytokine levels were below the detection limit, they were recorded as one-half of the lower detection limit (e.g., 2 pg/ml for IL-6) for statistical analysis.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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To study the role of innate or early acting host factors in protection against pneumococcal carriage as well as pneumonia, a challenge model of carriage or focal pneumonia in the absence of generalized sepsis was used. It has been reported that S. pneumoniae capsular group 19 strain EF3030 could cause carriage or focal pulmonary infections in mice when 5 x 106–107 CFUs are given intranasally to anesthetized mice in a 15- or 50-µl volume of Ringer’s solution, respectively (37). To determine the initial modulation of early acting chemokine and adaptive immune factors induced during pneumococcal carriage, changes in IL-4, IL-10, IFN-, and IL-12p40 as well as CCL5, CCL4, CCL3, and CCR5 mRNA were evaluated in both mucosal and systemic immune compartments (Fig. 1). IL-10, but not IL-4, mRNA levels were dramatically increased by CLN lymphocytes 24 h after the establishment of pneumococcal carriage. Similar expression of IL-4 and IL-10 was observed by NALT 1 day after infection. However, only modest and time-attenuated increases in IL-10 and IL-4 mRNA were expressed by lung tissues. Both IFN- and IL-12p40 mRNA were significantly increased in nasal tracts 1 day after carriage challenge. Similarly, IL-12p40 > IFN- was elevated in the CLNs during the same time period. IFN- and IL-12p40 mRNA levels were also transiently expressed by the lung from days 1 to 7 postinfection.
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Peak levels of CCL5, CCR5, IFN-, and IL-10 mRNA coincided with significant protein expression from these transcripts by high density leukocytes and large, low density cytokeratin 8+ and TIR8/SIGIRR+ cells after challenge. To better elucidate the contribution of the mucosal cells in the host response against pneumococcal carriage, the change in the number of CD4+ and CD8+ T cells, CD11b+ monocytes, CD11b+CD11cHi cells, and low density TIR8/SIGIRR+cytokeratin 8+ cells that expressed CCL5, CCR5, IL-10,and/or IFN- were determined by flow cytometry. Analysis of the fold increase of these mucosal subsets revealed that the number of CCL5-producing NALT and lung CD4+ and CD8+ T cells, monocytes, and dendritic cells significantly increased following strain EF3030 challenge as compared with those in control (uninfected) mice (Table I). Although the majority of CCL5 was provided by leukocytes (high density), the percentage of lung-derived large, low density TIR8+cytokeratin 8+ cells (i.e., epithelial cells) expressing CCL5 nearly doubled following strain EF3030 challenge (Fig. 2). To this end, a dramatic increase in the number of CCR5+ leukocytes were also detected, with the highest increases occurring in the lung and NALT. In particular, the number of CCR5+ CD11b+ cells increased by 6-fold as compared with controls.
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- and/or IL-10-producing mucosal leukocytes from infected mice as compared with controls (Table I). The numbers of CLN-derived CD8+ T cells that produced IFN- and/or IL-10 were significantly increased following bacterial challenge as compared with controls. The numbers of IFN-+ and/or IL-10+CD11b+CD11cHi (dendritic) cells from the lung, but not from NALT, were greatly increased compared with controls or other cells making these Th1 and/or Th2 cytokines, respectively. Although examination of these leukocyte populations revealed their significant contribution to the Th cytokine milieu in the mucosa during pneumococcal carriage, we next examined the contribution of TIR8+ cytokeratin 8+ cells to this cytokine mix. IL-10 expression patterns of nasal tract-derived TIR8+cytokeratin 8+ cells was largely unchanged; however, the percentage of lung-derived, large, low density IL-10+TIR8+cytokeratin 8+ cells nearly doubled following strain EF3030 challenge. It is widely held that epithelial cells do not secrete IFN-; nonetheless, a percentage of the large, low density cells from the NALT (3 to 4%) and lung (5.7 to 16.6%) produced IFN-. Although the percentage of nasal tract-derived IFN-+TIR8+cytokeratin 8+ cells did not change, the percentage of similar cells from the lung nearly tripled following bacterial challenge.
Together, these data show that leukocytes contribute significantly to CCL5, IFN-, and IL-10 during pneumococcal carriage. The lung contained more CCL5+ leukocytes than other compartments studied; yet, the number of NALT-derived monocytes and CD8+ T cells that produced this chemokine were also significantly increased following strain EF3030 challenge. Similarly, the lung compartment contained a higher percentage of CCL5+TIR8+cytokeratin 8+ cells than NALT (compared with controls) following bacterial challenge. Although the lung held a greater increase in IL-10+ APCs, CLN-derived CD8+ T cells produced IL-10 and/or IFN- following strain EF3030 challenge as compared with controls. Coinciding with these CCL5, IFN-, and IL-10 expression patterns by leukocytes, large, low density lung TIR8+ cytokeratin 8+ cells produced significant levels of these factors following pneumococcal carriage as compared with controls, all of which supported the associated increase in the numbers and possible differentiation of CCR5+ leukocytes in the mucosa.
CCL5 modulates Th cytokine, CCL5/CCR5 expression, and pneumococcal disease
CCL5 was largely expressed by NALT, CLN, and lung lymphocytes during pneumococcal carriage. To determine the effect(s) of CCL5 on pneumococcal carriage, S. pneumoniae strain EF3030-challenged mice were treated with anti-CCL5 or control Abs. CCL5 blockade dramatically decreased the IL-4 mRNA levels expressed by NALT and lung cells, which peaked 1 day after pneumococcal challenge (Fig. 1). Similar decreases in IFN- and IL-12p40 as well as CCR5 mRNA levels were observed in NALT, CLN, and lung cells. CCL5 blockade did not decrease CCL5 mRNA expression by cells isolated from NALT, CLN, or lung. In fact, the CCL5 mRNA expression induced by pneumococcal carriage challenge peaked at 1 day after challenge (similar to infected controls); however, high transcript levels of this chemokine continued through day 14 in anti-CCL5 Ab-treated mice. Similarly, anti-CCL5 Ab treatment during pneumococcal carriage did not dramatically affect levels of IL-10 mRNA but extended the expression of this transcript by NALT, CLN, and lung cells.
CCL5 blockade also corresponded with pneumococcal carriage progression to invasive and potentially lethal pneumonia (Fig. 3). The majority of the lethal effects of simultaneous CCL5 inhibition and pneumococcal carriage occurred 6 days after S. pneumoniae strain EF3030 challenge. In the absence of CCL5, pneumococcal carriage developed into pneumonia without bacteremia/sepsis. To explain, no detectable S. pneumoniae strain EF3030 CFUs were detected in the spleen, and <2 CFUs were detected in CLNs of infected mice (data not shown). Bacterial counts in NALT and lungs peaked 2–4 days after pneumococcal challenge. Although 102–103 S. pneumoniae strain EF3030 CFUs were routinely cultured from NALT after challenge, the bacterial load in the lung receded to <10 CFUs 7 days after challenge. In contrast, CCL5 blockade resulted in a dramatic (104-fold) increase in strain EF3030 CFUs from both NALT and lung. These heightened bacterial loads, relative to infected controls, continued 28 days after challenge.
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To distinguish the potential cellular mechanism(s) that CCL5 has on bridging the innate or early response to pneumococcal challenge to adaptive immunity, leukocyte counts were determined 5 days after strain EF3030 challenge. Pneumococcal carriage resulted in a dramatic increase in the number of leukocytes in both inductive and effector sites (Fig. 4). However, CCL5 blockade led to a significant decrease in leukocyte cell counts in the CLNs, NALT, and lung 5 days post S. pneumoniae strain EF3030 challenge. There were modest decreases in the number of CD8+ T cells, neutrophils, B cells, and macrophages, and slight increases in the number of CD4+ T cells in the spleens of anti-CCL5 Ab-treated mice. However, there were statistically significant decreases in the number of CLN and lung CD4+ and CD8+ T cells, B cells, macrophages, neutrophils, and NK cells from mice treated with anti-CCL5 Abs, compared with infected controls.
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CCL5 modulation of pneumococcal humoral immunity
CCL5 was largely expressed by NALT, CLN, and lung lymphocytes during the early or innate (i.e., up to 4 days) response to pneumococcal carriage. This chemokine was also necessary for optimal protection against pneumococcal pneumonia. To determine the effects of CCL5 on pneumococcal T-dependent Ag responses, PsaA-specific humoral and cellular immunity was characterized 28 days after S. pneumoniae strain EF3030 challenge during anti-CCL5 or control Ab treatments. CCL5 blockade significantly reduced PsaA-specific IgG2a and IgG1 Ab levels in serum after 28 days (Fig. 5). In addition, CCL5 inhibition greatly reduced PsaA-specific IgA Ab levels in nasal washes compared with control Ab-treated mice challenged in a similar fashion (Fig. 6). Anti-CCL5 Ab treatment also reduced PsaA-specific nasal IgG Abs, although, this decrease was not statistically significant. Together, these data show that CCL5 is essential for optimal mucosal and peripheral pneumococcal humoral immunity.
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To determine whether the changes in PsaA-specific CD4+ T cell responses contributed to the changes noted in pneumococcal humoral responses, the effects of CCL5 inhibition on Th cytokine secretion patterns and proliferation were also observed. In general, pneumococcal carriage led to substantial increases in PsaA-specific proliferation and the corresponding IL-2 secretion responses in pneumococcal carriage controls (Fig. 7). However, anti-CCL5 Ab treatment resulted in significant decreases in ex vivo PsaA-restimulated splenic, but not CLN-, NALT-, or lung-derived CD4+ T cell proliferation. Similar reductions in PsaA-specific CD4+ T cell IL-2-secretion were also noted following CCL5 blockade.
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and TNF- are known to play a profound role in pneumococcal immunity (43, 44, 45). CCL5 inhibition during pneumococcal carriage resulted in the generation of less IFN- and TNF- PsaA-reactive mucosal and systemic CD4+ T cells as compared with that in control Ab-treated mice. The most dramatic reductions were observed in IFN- production by CLN CD4+ T cells and TNF- patterns by Th lymphocytes from the NALT.
In contrast to the negative modulation of PsaA-specific T cell proliferation and IL-2, IL-4, IL-6, GM-CSF, IFN- and TNF- secretion, CCL5 blockade led to an overall increase in IL-10-secreting PsaA-specific CD4+ T cells. CLN- and NALT-derived CD4+ T cells from anti-CCL5 Ab-treated mice secreted more IL-10 following PsaA-restimulation than controls. Indeed, splenic PsaA-specific Th2 cells secreted significantly higher levels of IL-10 as compared with control Ab-treated and naive controls. Together, our data suggest that CCL5 blockade increased IL-10 but decreased IL-2, IL-4, IFN-, TNF-, GM-CSF, and IL-6 secretion patterns by PsaA-specific CD4+ T cells from S. pneumoniae strain EF3030-challenged mice.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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In our model, CCL5-mediated adaptive immune responses played a partial yet substantial role in protective pneumococcal immunity. The potential lethal effects of pneumococcal challenge (and CCL5 blockade) occurred 6 days post infection during the end of the innate response and the beginning of the recognition phase of the adaptive immune response. The changes due to CCL5 blockade were illustrated by decreases in CD4+ and CD8+ T cell as well as B cell and monocyte counts per lymphoid organ at day 5. Notably, these lymphocytes were important sources of IFN-, IL-10, and CCL5 4 days following the challenge. It has been shown previously that CCL5 plays an important role in the motility of lymphocytes that express CCR5, CCR4, CCR3, and CCR1 (13, 15, 16). We have also shown that CCL5 can increase the proliferation and activation of Ag-stimulated T lymphocytes (20). Hence, the hypercellular mucosal response to pneumococcal challenge is partially mediated by the ability of CCL5 to enhance recruitment and proliferation of leukocytes. These findings coincided with marked increases in CCL5 and IL-10 production by leukocytes as well as mucosal (NALT and lung) large/low density TIR8+cytokeratin 8+ cells.
TIR8, also known as SIGIRR, is a member of the IL-1R family with unique properties. It has been reported that TIR8 is expressed by epithelial cells and sentinel dendritic cells (47). Similarly, cytokeratin 8 has been shown to be predominantly expressed by epithelial cells; however, it is important to note that distinct dendritic cell subsets can endocytose and transport apoptotic epithelial cells (48). In the present study, we show that NALT- and lung-derived large/low density TIR8+ cytokeratin 8+ cells (i.e., epithelial and/or dendritic cells) from strain EF3030-challenged mice express CCL5, IL-10, and IFN-. These data suggest that leukocytes (and epithelial cells) participate in the recognition and subsequent production of inflammatory cytokines during pneumococcal carriage. However, additional studies are required to dissect the precise cellular contribution of Th cytokines and determine whether nasal tract and/or lung-derived live or apoptotic epithelial cells also contribute to pneumococcal responses.
CCL5 inhibition did not reduce the mRNA expression of this chemokine. However, CCL5 blockade reduced the expression of IL-4, IFN-, and IL-12p40 and extended the highest increased levels of IL-10 expression by the mucosal lymphocytes following pneumococcal challenge. Indeed, CCL5 inhibition resulted in an IL-10-biased host response as noted by the cytokine secretion patterns exhibited by PsaA-stimulated CD4+ T cells. No doubt the early shift in IL-10 secretion by mucosal lymphocytes following CCL5 blockade affected subsequent adaptive humoral and cellular immunity to Pneumococci. Studies are currently underway to better understand the changes in Th cytokine production during pneumococcal carriage, in the presence or absence of CCL5, that contribute to Th cell differentiation and Ag recognition.
As part of the many S. pneumoniae immune evasion mechanisms, pneumococcal lipoteichoic acids fail to induce IL-1
and TNF- secretion by human monocytes (49). TNF- levels are predictive of S. pneumoniae bacteremia, even in nontoxic appearing febrile children (50). IL-6 enhances the differentiation of pneumococcal specific B cells to plasma cells (51) and is essential for protection against pneumococcal pneumonia (52). We show that pneumococcal carriage leads to the generation of PsaA-specific TNF- and IL-6 responses that are dependent on the presence of CCL5.
Although little is known regarding Ag-specific IL-4 and GM-CSF responses during S. pneumoniae infection, we show that these Th cytokines are increased during pneumococcal carriage. CCL5 blockade also diminished the cellular responses that increased host susceptibility to lethal pneumococcal disease. IFN- has been shown to be required for protective host immunity against pneumococcal disease(s) (43). In confirmation of these findings, we show that IL-12p40 expression coincides with this IFN- production early on, which also corresponds with the heightened PsaA-specific IgG2a adaptive responses during carriage. Both Ag-specific IFN- CD4+ T cell secretion and IgG2a Ab responses were dramatically reduced in the absence of CCL5. 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 cell-derived cytokines (53, 54, 55). Clearly, Th1 and Th2 cytokines as well as IgA Ab responses were enhanced during pneumococcal carriage and largely reduced by CCL5 blockade.
CCR5 is preferentially expressed by Th1 cells (56). In contrast, Th2 cells preferentially express CCR4 and, more controversially, CCR3 (57). Hence, coordinate expression of CCR4 and CCR5 ligands (CCL5, CCL4, and CCL3) often follow subsequent recruitment of CCR5+ cells for Th1 responses as well as CCR4+ and/or CCR3+ cells for Th2-mediated immunity. We also show that the number of CCR5+ T cells and APCs significantly increased following pneumococcal challenge. Leukocyte subpopulations from the NALT, CLN, and lung tissues in CCL5-deficient mice contained fewer CD4+ and CD8+ T cells as well as CD11b+ macrophages relative to control Ab-treated mice. Taken together with the reduced capacity of the infiltrating T cells and APCs to mount substantial cytokine responses, these alterations in these leukocyte populations also had a profound effect on the transition of pneumococcal carriage to pneumonia. Intranasal infection of normal mice with strain EF3030 of capsular group 19 S. pneumoniae resulted in carriage without pneumonia; however, in the absence of CCL5, pneumococcal carriage developed to pneumonia as indicated by significantly higher EF3030 CFUs in the lungs of anti-CCL5 Ab-treated mice.
The biological determinants that influence the probability of S. pneumoniae transmission and progression can include, but are not limited to, the characteristics of the infecting strain (carriage vs invasive) and the susceptibility of uninfected hosts as well as infected individuals. Polymorphisms in CCL5 that negatively affect its expression are associated with enhanced susceptibility and progression to HIV-1/AIDS (28, 29, 30). This association accounts for an important health disparity; up to 67% of all African Americans carry at least one of these polymorphisms. Until now, it was not certain what effect CCL5 expression would have on S. pneumoniae disease susceptibility, progression, or protective immunity. It is tempting to speculate that some of the health disparities associated with pneumococcal disease may be partially mediated by this CCL5 genetic variance, which would result in diminished expression of CCL5 by individuals of African origin as compared with others. Although pneumococcal pneumonia has been shown to lead to an increase in CCL5, CCL4, and CCL3 mRNA expression, administration of neutralizing Abs against CCL5 or CCL3 did not prevent macrophage infiltration into infected alveoli (58). However, in the model used by Fillion et al. (58), pneumococcal infection resulted in 100% mortality in 4 days, which provided a relatively narrow window for observing the cellular and molecular mechanisms that CCR5 ligands could use to control pneumococcal disease through adaptive immunity.
IL-10 leads to macrophage/monocyte deactivation as well as suppression of the release of reactive oxygen species and nitrogen intermediates, which are known to be involved in the pathophysiology of pneumococcal meningitis (59). This Th2 cytokine also reduces pulmonary vascular leakage and the appearance of RBCs in the alveoli in a mouse model of pneumococcal pneumonia (60). IL-10 has also been shown to enhance susceptibility to pneumococcal infections (61). CCL5 blockade resulted in a shift to predominant IL-10 pneumococcus-specific Th cell responses, which was exemplified by increased levels of IL-10 production by PsaA-specific CD4+ T cells. Interestingly, this selective increase in IL-10 secretion is also representative of unique Th2 cell subsets that are often prevalent at the early stages of Th2 differentiation that diminish over time (62). Alternatively, IL-10+ CD4+ T cells also exist as T regulatory (Tr1) cells; indeed, IL-10 has been reported to be important in the generation of anergic T cells that fail to produce IL-2Th1/Th2, GM-CSFTh1/Th2, IFN-Th1, TNF-Th1, IL-4 Th2, and IL-5Th2 in response to Ag stimulation (63). Although additional studies are necessary to characterize the potential of CCL5 deficiencies to support an increase in CD4+CD25+ Tr1 cells or merely the lack of appropriate Th cell development, our data suggest that the development of these Ag-specific IL-10-secreting CD4+ T cells in the presence of diminished Th1 (e.g., IFN- and TNF-) and Th2 (IL-4 and IL-6) response do not support the protective immunity needed to contain pneumococcal carriage.
Although the precise role of CCL5 interactions that determine innate and adaptive pneumococcal immunity are not known, this study addresses important questions that are relevant to many individuals that display polymorphisms in either the CCL5 gene or its promoter that diminish its expression and have deleterious consequences in infectious disease outcomes. Understanding the cellular and molecular mechanisms that CCL5 uses to modulate Th1 and Th2 adaptive immune responses to Pneumococci is essential to develop vaccines, therapies, and diagnostic and prognostic tools to control pneumococcal disease.
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1 This work was supported in part by National Institutes of Health Grants AI057808 and RR03034. 
2 Address correspondence and reprint requests to Dr. James W. Lillard, Jr., Department of Microbiology, Biochemistry, and Immunology, Morehouse School of Medicine 720 Westview Drive, Atlanta GA 30310-1495. E-mail address: lillard{at}msm.edu
3 Abbreviations used in this paper: PsaA, pneumococcal surface antigen A; CLN, cervical lymph node; NALT, nasopharyngeal-associated lymphoid tissue; SIGIRR, single Ig IL-1-related receptor; TIR8, Toll/IL-1R.
Received for publication June 8, 2004. Accepted for publication December 5, 2005.
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by lower airway epithelial cells and eosinophils infected with respiratory syncytial virus. J. Virol. 72: 4756-4764. and chemokines induce NK cell migration and enhance NK-mediated cytolysis. J. Immunol. 155: 3877-3888. [Abstract], MIP-1, and RANTES on the chemotaxis and adhesion of T cell subsets. Adv. Exp. Med. Biol. 351: 139-146. [Medline] chemokines costimulate lymphocyte cytolysis, proliferation, and lymphokine production. J. Leukocyte Biol. 59: 81-89. [Abstract] chemokines costimulate human T lymphocyte activation in vitro. J. Immunol. 156: 2095-2103. [Abstract] are overcome to control infection with Leishmania donovani in CC chemokine receptor (CCR) 5-, macrophage inflammatory protein-1 -, or CCR2-deficient mice. J. Immunol. 163: 5519-5525. 
) Abs were administered by an i.p. route every 3 days, starting 2 days before challenge (A). Mice were intranasally challenged with 106.88 CFUs of S. pneumoniae strain EF3030 in a 15-µl volume of Ringer’s solution, and survival curves of both groups were measured over a 28-day period. Experimental groups consisted of 10 BALB/c mice, and studies were repeated 3 times. NALT- (B) and lung-associated (C) pneumococci were enumerated before and 1, 2, 4, 7, 14, 21, and 28 days post strain EF3030 challenge and are expressed as CFUs (± SEM) per mouse; data are representative of three different experiments using groups containing 10 mice each. Spleens as well as CLNs from infected or control mice were absent of detectable S. pneumoniae strain EF3030 CFUs (i.e., <2) and are not shown. Asterisk(s) indicate significant differences (*, p < 0.05) between anti-CCL5 and control Ab-treated groups.
) or control Abs (
) were administered by an i.p. route every 3 days, starting 2 days before challenge. NALT-, lung-, CLN-, and spleen-derived lymphocytes were counted by hemocytometry and flow cytometry. Experimental (
) consisted of 10 mice each, and studies were repeated three times. Asterisk(s) indicate significant differences (*, p < 0.05) between anti-CCL5 and control Ab-treated groups.
