Chemokine Expression Dynamics in Mycobacterial (Type-1) and Schistosomal (Type-2) Antigen-Elicited Pulmonary Granuloma Formation
2001; Elsevier BV; Volume: 158; Issue: 4 Linguagem: Inglês
10.1016/s0002-9440(10)64101-6
ISSN1525-2191
AutoresBoqin Qiu, Kirsten A. Frait, Filip Reich, Eric Komuniecki, Stephen W. Chensue,
Tópico(s)vaccines and immunoinformatics approaches
ResumoTranscript expression of 24 chemokines (CKs) was examined throughout 8 days in mouse lungs with type-1 (Th1) or type-2 (Th2) cytokine-mediated granulomas induced by bead-immobilized mycobacterial purified protein derivative or Schistosoma mansoni egg antigens. Where possible, CK protein levels were also measured. In addition, we examined effects of in vivo cytokine depletions. Findings were as follows: 1) bead challenge induced increases in 18 of 24 CK transcripts with type-1 and type-2 responses displaying different patterns. CKs fell into four categories: a) type-1-dominant (γ-interferon-inducible protein (IP-10), monokine induced by INF-γ (MIG), macrophage inflammatory protein-2 (MIP-2), lipopolysaccharide-induced chemokine (LIX), rodent growth-related oncogene homologue (KP), macrophage inflammatory protein-1α (MIP-1α) and -1β (MIP-1β), lymphotactin), b) type-2-dominant (eotaxin, monocyte chemotactic protein-2 (MCP-2) and -3 (MCP-3), liver and activation-regulated chemokine (LARC), T cell activation protein-3 (TCA-3), c) type-1 and type-2 co-dominant (MCP-1, MCP-5, monocyte-derived chemokine (MDC), thymus and activation-related chemokine (TARC), C10), and d) constitutive (lungkine, secondary lymphoid-tissue chemokine (SLC), EBI1-ligand chemokine (ELC), fractalkine, macrophage inflammatory protein-1γ (MIP1-γ), and stromal cell derived factor-1α (SDF1-α). 2) CKs displayed characteristic temporal patterns. CXC (IP-10, MIG, MIP-2, LIX, KC) and certain CC (MCP-1, MCP-5, MIP-1α, MIP-1β) CKs were produced maximally within 1 to 2 days. Others (MCP-2, MCP-3, eotaxin, lymphotactin, LARC, TCA-3) displayed peak expression later. 3) Interferon-γ neutralization profoundly abrogated MIG, but had little effect on other CKs. Tumor necrosis factor-α neutralization caused up to 50% reduction in a range of CKs. These findings indicate that type-1 and type-2 granulomas display characteristic CK profiles with coordinated expression that is under cytokine-mediated regulation. Transcript expression of 24 chemokines (CKs) was examined throughout 8 days in mouse lungs with type-1 (Th1) or type-2 (Th2) cytokine-mediated granulomas induced by bead-immobilized mycobacterial purified protein derivative or Schistosoma mansoni egg antigens. Where possible, CK protein levels were also measured. In addition, we examined effects of in vivo cytokine depletions. Findings were as follows: 1) bead challenge induced increases in 18 of 24 CK transcripts with type-1 and type-2 responses displaying different patterns. CKs fell into four categories: a) type-1-dominant (γ-interferon-inducible protein (IP-10), monokine induced by INF-γ (MIG), macrophage inflammatory protein-2 (MIP-2), lipopolysaccharide-induced chemokine (LIX), rodent growth-related oncogene homologue (KP), macrophage inflammatory protein-1α (MIP-1α) and -1β (MIP-1β), lymphotactin), b) type-2-dominant (eotaxin, monocyte chemotactic protein-2 (MCP-2) and -3 (MCP-3), liver and activation-regulated chemokine (LARC), T cell activation protein-3 (TCA-3), c) type-1 and type-2 co-dominant (MCP-1, MCP-5, monocyte-derived chemokine (MDC), thymus and activation-related chemokine (TARC), C10), and d) constitutive (lungkine, secondary lymphoid-tissue chemokine (SLC), EBI1-ligand chemokine (ELC), fractalkine, macrophage inflammatory protein-1γ (MIP1-γ), and stromal cell derived factor-1α (SDF1-α). 2) CKs displayed characteristic temporal patterns. CXC (IP-10, MIG, MIP-2, LIX, KC) and certain CC (MCP-1, MCP-5, MIP-1α, MIP-1β) CKs were produced maximally within 1 to 2 days. Others (MCP-2, MCP-3, eotaxin, lymphotactin, LARC, TCA-3) displayed peak expression later. 3) Interferon-γ neutralization profoundly abrogated MIG, but had little effect on other CKs. Tumor necrosis factor-α neutralization caused up to 50% reduction in a range of CKs. These findings indicate that type-1 and type-2 granulomas display characteristic CK profiles with coordinated expression that is under cytokine-mediated regulation. A boggling array of chemokines (CKs) has been described and detected at local inflammatory sites.1Zlotnik A Yoshie O Chemokines: a new classification system and their role in immunity.Immunity. 2000; 12: 121-127Abstract Full Text Full Text PDF PubMed Scopus (3232) Google Scholar In vitro studies indicate that they are produced to different degrees by a wide variety of cell types in response to injury, endogenous mediators, and exogenous stimulants. However, the in vivo organization and regulation of CKs is primarily unknown. It is a widely accepted hypothesis that tissue inflammation is the result of leukocytes interacting with cytokines and CKs. Each of these elements changes constantly with time. Therefore, a comprehensive, systematic approach is needed for analysis. In the present study we applied such an approach using defined, polarized models of T-cell-mediated type-1 and type-2 pulmonary granuloma formation to test the hypothesis that inflammatory responses can be defined based on profiles of CK expression. Granulomas represent a sequestration response elicited by a remarkably diverse group of agents. They are formed by an influx of inflammatory leukocytes that aggregate often in association with a poorly digestible nidus and include mononuclear phagocytes as a defining component. In immunologically active granulomas, additional cells, such as lymphocytes and in some instances plasma cells, myofibroblasts, B cells, and eosinophils may also be recruited. Granuloma macrophages and lymphocytes are heterogeneous and change constantly throughout time with each producing a myriad of secretory products.2Williams GT Williams WJ Granulomatous inflammation—a review.J Clin Pathol. 1983; 36: 723-733Crossref PubMed Scopus (244) Google Scholar, 3Sheffield EA The granulomatous inflammatory response.J Pathol. 1990; 160: 1-2Crossref PubMed Scopus (23) Google Scholar CKs are beginning to be recognized as one important group of mediators of granuloma formation.4Ben-Baruch A Michiel DF Oppenheim JJ Signals and receptors involved in recruitment of inflammatory cells.J Biol Chem. 1995; 270: 11703-11706Crossref PubMed Scopus (365) Google Scholar The present study monitored the expression of 24 CKs throughout an 8-day study period in lungs with granulomas induced by agarose bead-immobilized Mycobacteria bovis purified protein derivative (PPD) or Schistosoma mansoni egg antigens (SEA).5Chensue SW Warmington KS Ruth JH Lincoln P Kunkel SL Cytokine function during mycobacterial and schistosomal antigen-induced pulmonary granuloma formation. Local and regional participation of IFN-gamma, IL-10, and TNF.J Immunol. 1995; 154: 5969-5976PubMed Google Scholar Granulomas induced by PPD-coated beads are driven primarily by the Th1 (type-1)-associated cytokines interferon (IFN)-γ and tumor necrosis factor (TNF)-α, whereas those induced by SEA-coated beads are mediated by the Th2 (type-2)-associated cytokines, IL-4 and IL-13.6Chensue SW Warmington K Ruth JH Lukacs N Kunkel SL Mycobacterial and schistosomal antigen-elicited granuloma formation in IFN-gamma and IL-4 knockout mice: analysis of local and regional cytokine and chemokine networks.J Immunol. 1997; 159: 3565-3573PubMed Google Scholar Transcript analysis of 24 CKs in these models indicated that 18 were induced and expression showed close association with granuloma development. Moreover, among these, 13 showed type-specific and temporal differences in their expression patterns. In addition, the role of cytokine-mediated regulation was tested by intraperitoneal injection of antibodies against IFN-γ or TNF-α. MIG expression was dramatically reduced by the neutralization of IFN-γ, whereas other CKs showed no significant changes. In contrast, TNF-α neutralization reduced a broad range of CKs. This study emphasizes the importance of a comprehensive approach to CK analysis and indicates that CK profiling may be a feasible approach to define inflammatory responses for CK targeted therapies. Female CBA/J mice were purchased from The Jackson Laboratory (Bar Harbor, ME). All mice were maintained under specific pathogen-free conditions and used at 7 to 8 weeks of age. Types 1 and 2, secondary Ag-bead granulomas were generated as previously described.5Chensue SW Warmington KS Ruth JH Lincoln P Kunkel SL Cytokine function during mycobacterial and schistosomal antigen-induced pulmonary granuloma formation. Local and regional participation of IFN-gamma, IL-10, and TNF.J Immunol. 1995; 154: 5969-5976PubMed Google Scholar Briefly, mice were sensitized by a subcutaneous injection of 20 μg of Mycobacteria bovis purified protein derivative (PPD) (Department of Agriculture, Veterinary Division, Ames, IA) incorporated into 0.25 ml of complete Freund’s adjuvant (CFA) (Sigma, St. Louis, MO) or 3,000 Schistosoma mansoni eggs suspended in 0.5 ml of phosphate-buffered saline (PBS). Fourteen to 16 days later, PPD- and schistosome egg-sensitized mice were respectively challenged by tail vein injections with 6,000 Sepharose 4B beads (in 0.5 ml of PBS) covalently coupled to PPD or to soluble schistosome egg antigens (SEA) obtained from the World Health Organization (Geneva, Switzerland). Groups of mice were killed at 1, 2, 4, and 8 days of granuloma formation. After perfusion with cold RPMI, lungs excluding trachea and major bronchi were excised. The right upper lung of each mouse was used for mRNA isolation. The left lower lobe was postinflated and formalin-fixed for granuloma size determination. The remaining lung was placed in cold RPMI medium then granulomas were isolated and dispersed as previously described.6Chensue SW Warmington K Ruth JH Lukacs N Kunkel SL Mycobacterial and schistosomal antigen-elicited granuloma formation in IFN-gamma and IL-4 knockout mice: analysis of local and regional cytokine and chemokine networks.J Immunol. 1997; 159: 3565-3573PubMed Google Scholar For differential counting, duplicate cytospin preparations were prepared from the remaining dispersed granuloma cells and stained with Wright’s stain. Granulomas were measured blindly from formalin-inflated lungs that were paraffin-embedded, sectioned, then stained with hematoxylin and eosin. Granuloma area was measured by computerized morphometry. Only granulomas sectioned through the central bead nidus were measured. A minimum of 20 lesions was measured per lung. The total number of cells in a granuloma cross-section was determined morphometrically by directly counting cell number in an area of 2.5 × 103 μm2. This was determined to be 32 ± 7 with each cell occupying an average of 80 ± 18 μm2. The proportion of cell types was calculated by multiplying the total cell number by the percentages obtained by direct differential analysis of dispersed granulomas. At the time of bead challenge, mice were given an intraperitoneal injection of 5 mg of rabbit IgG with specificity for murine IFN-γ or TNF-α prepared by protein A column purification. Nonimmune rabbit IgG served as a control. The specificity and potency of these preparations was previously reported.5Chensue SW Warmington KS Ruth JH Lincoln P Kunkel SL Cytokine function during mycobacterial and schistosomal antigen-induced pulmonary granuloma formation. Local and regional participation of IFN-gamma, IL-10, and TNF.J Immunol. 1995; 154: 5969-5976PubMed Google Scholar Two days after challenge, lungs were excised for mRNA measurement and morphometric analysis. Mice were anesthetized and bled before sacrifice for lung harvest, which was done at 1, 2, 4, and 8 days after the bead embolization. Unchallenged mice at 14 days after sensitization served as time 0 controls, whereas lungs of normal mice provided baseline determinations. After perfusion with cold RPMI, lungs, excluding the trachea and major bronchi, were excised. The left lower lung lobe of each mouse was postinflated and formalin-fixed. The right lobe was snap-frozen in liquid nitrogen for mRNA isolation, and the remaining lobes were uses for protein extraction. mRNA was isolated from the frozen tissues using Poly(A)Pure mRNA isolation kits (Ambion, Austin, TX). For protein extraction, frozen lungs were placed into Dulbecco’s modified Eagle’s medium and were ground using a homogenizer. Samples were centrifuged and supernatant fluids collected. Primers and probes were designed with primer design software (Primer Premier; Biosoft International, Palo Alto, CA) using murine sequences obtained from the National Center for Biotechnology Information (NCBI) GenBank. Table 1 shows the sequences and NCBI reference numbers. Oligonucleotides were obtained from Operon Technologies, Inc., Alameda, CA.Table 1Chemokine Probe and Primer SequencesChemokineNCBI accession No.Probe (5′-3′)Sense primer (5′-3′)Antisense primer (5′-3′)LIXMMU27267TGA GGA CTC TGA CCC CAG TGA AAAT GCA CTC GCA GTG GAA AGACT TGT GAG ATG AGC AGG AAG CMIGM34815GGC CTG TCT GTT TGC TGG TGA GGGG CAA GTG TCC CTT TCC TTCGGG CTC TAG GCT GAC CCA AATIP-10MUSIP10CTC ATC CTG CTG GGT CTG AGT GGCA TCA GCA CCA TGA ACC CAA GCT ATG GCC CTC ATT CTC ACT GMIP-2MMMIP2GCT GTC CCT CAA CGG AAG AAC CCAA GGC TAA CTG ACC TGG AAA GCAT AAC AAC ATC TGG GCA ATGKCJ04596AGC GCT GCT GCT GCT GGCCCA GCC ACC CGC TCG CTTGGG CCC TGA GGG CAA CACMIP-1αMUSMIP1ATTG AGC CGA ACA TTC CTG CCA CCATC ACT GAC CTG GAA CTG AAT GCAA GTG AAG AGT CCC TCG ATGMIP-1βMUSMIP1XGGC TCT GAC CCT CCC ACT TCC TGAC CAT GAA GCT CTG CGT GTCAA GCT GCC GGG AGG TGT AAGMCP-1MUSGFJECTG CAT CTG CCC TAA GGT CTT CATGC TGA CCC CAA GAA GGA ATGC TTG AGG TGG TTG TGG AAA AGGMCP-2AB023418TAC ATG GAG ATC CTT GAC CAG ACTT CGG GTG CTG AAA AGC TACTG CCT GGA GAA GAT TAG GGG AGMCP-3S71251TTC CTC ACC GCT GTT CTT TCT GGTG CCT GAA CAG AAA CCA ACC TCAT TCC TTA GGC GTG ACC ATTMCP-5MMU50712CAC AAG CAG CCA GTG TCC CCGAGG TAT TGG CTG GAC CAG ATGTTC TCC TTG GGG TCA GCA CAGLymphotactinMMU15607AGG AGC CCA GAG GTC CAC CAG CACAG GGC CAG TAC CAG AAA GAA CATG GGT TTG GGA ACT GAG ATG AGCEotaxinU26426CTC CAT CCC AAC TTC CTG CTGTTC TAT TCC TGC TGC TCA CGGAGG GTG CAT CTG TTG TTG GTGTCA-3MMTCA3GCCC TGC TAA CCG GGG TGA AGA TGAAA GAT GGG CTC CTC CTG TCCTGG AGG ACT GAG GGA AAC TGCLARCAB015136AAG CAG AAC TGG GTG AAA AGGGCA GCA AGC AAC TAC GAC TGCTC TTA GGC TGA GGA GGT TCMDCAF052505GGA GGA CCT GAT GAC CAT GGG TCAGG CAG GTC TGG GTG AAG AAG CTGG ATG GAG GTG AGT AAA GGT GGCC10MUSC10TTT AGA GCA GTC AAC AGT ATT CACA CCC ACT TTC TTC TGT CTT CCTA CAA CTC CAG ATG GCT CTA ACCTARCMMU242587AGG CCG TGA CCT TCC CGC TGACAG GGA TGC CAT CGT GTT TCTGGT CAC AGG CCG CTT TAT GTTLungkineAF082859CCC CTT TAT TGA CTG ACA AAC TATAT TCC CGC GTT AGT CTG GTGG CCC ATA GTG GAG TGG GAT AAGSLCAF001980TCT AAG CCT GAG CTA TGT GCA AATC CCG GCA ATC CTG TTC TCA CCGC CCT TGG AGC CCT TTC CTT TCELCAF059208CCC CTG TGA ACC CGT CGG AGC CTAGG ACA TCT GAG CGA TTC CAGAG TCT TCC GCA TCA TTA GCA CFractalkineAF071549TGT GCT GAC CCG AAG GAG AAA TGCT CAT CCG CTA TCA GCT AAA CTTG TCC ACC CGC TTC TCA AACMIP-1γMMU49513CTC CTT CCT CAT TCT TAC AAC TGTGC CCA CTA AGA AGA TGA AGCCAA TTT CAA GCC CTT GCT GTGSDF-1αMUSSDF1ATCG ACA GAT GCC TTG TCC TGA GTGAG AAA CCT TCC ACC AGA GCA GGG CAC TGA ACT GGA TAA AGG AGCCyclophilinMUSCYCLOACAT CGT GTC ATC AAG GAC TTC AGTG GGC TCC GTC GTC TTC CTT TC TTT CCT CCT GTG CCA TCT CCC Open table in a new tab Approximately, 1 μg of mRNA was reverse-transcribed in a 20-μl reaction in a PCR reaction tube using Reverse Transcription System kits (Promega, Madison, WI). Five to 10 reactions were conducted on the same mRNA preparation in parallel to minimize variability. The cDNA product from each tube was pooled for analysis. The concentration of each cDNA sample was adjusted so that the signal obtained with the housekeeping gene, cyclophilin, when amplified by a 22-cycle PCR reaction and detected by a PCR-ELISA, would give an absorbance value at ∼0.5. After adjusting the cDNA concentration, a sample of cDNA from normal lungs was amplified by a PCR reaction using primer pairs specific for a given CK. A series of PCR reactions was performed at different cycle numbers, then the PCR products were detected by using PCR-ELISA detection kits (Roche Molecular Biochemicals, Mannheim, Germany). The cycle number giving an absorbance value nearest 0.2 was used for that particular CK primer-probe set. This was done for each of the CKs examined. Each CK cDNA was amplified in a 40-μl PCR reaction, then 20 μl of the product was assayed using a biotinylated probe and PCR-ELISA (DIG Detection) kits (Roche Molecular Biochemicals). In each PCR-ELISA detection assay, PCR products were detected in duplicates. Water was used as negative control. Cyclophilin mRNA of each sample, amplified in a separate tube, was included for normalization and confirmation of cDNA equivalency. To validate the calibration curve (see below) for a particular assay, a selected preparation was assayed at both 10- and 40-μl volume, which should result in a one to four relationship in relative concentration. The basis for the quantification of mRNA expression is that the absorbance [optical density (OD450)] reading increases directly with the copy number of the CK mRNA being measured at the optimal PCR cycle number. However, the relationship between the OD reading and the copy number may not be linear at high OD450 readings. The system was therefore calibrated to correct for possible nonlinear relationships because of high copy number expression. Standard curves were generated using serially diluted cDNA samples from normal lung amplified with cyclophilin primers. The resulting curve was best fitted to obtain the following correction formulas: 1) for OD450 > 1.0: log (dilution factor) = log (OD450 /0.2)/0.7 and 2) for OD450 0.05 were considered to indicate lack of significance. The kinetics of type-1 (PPD) and type-2 (SEA) bead granuloma formation are shown in Figure 1 along with leukocyte differential analyses indicating the numbers of each major cell type present in an average granuloma cross-section. It can be seen that the period of rapid cellular accumulation occurred between 0 and 4 days. Day 4 represented a time of lesion sustenance whereas day 8 marked the period of lesion involution. Although both type-1 and type-2 lesions contained significant components of lymphocytes by day 2, the type-1 lesion was characterized by recruitment of greater numbers of neutrophils in this period. Thereafter, on days 4 and 8, the type-2 lesion was distinguished by a significant component of eosinophils. In both types of lesions mononuclear phagocytes reached a maximum on day 4. This analysis provided a base of histopathological observations to which CK expression dynamics could be related. The expression profiles of 24 CK transcripts were determined for type-1 and type-2 granulomas throughout an 8-day study period. Samples from 5-day points for each of the two models were measured in a single PCR-ELISA experiment and the relative abundance of each chemokine RNA was compared between the samples. Three repetitions were performed for each chemokine in three separate experiments, and only one experiment is presented. As is required for all relative RNA quantification methods, compared samples were measured together in each experiment. Although results could not be pooled among experiments, the relationships were consistent within the three studies. Of the 24, 18 CKs showed significant up-regulation (greater than twofold) during granuloma development. These are presented in Figure 2, Figure 3. Figure 2 shows the profiles of three ELR+ CXC CKs (MIP-2, KC, and LIX), two ELR- CXC CKs (IP-10 and MIG), and one known C class CK (lymphotactin). The differences between the type-1 and type-2 granulomas were easily seen. Of these, all but KC showed a predominant expression in the type-1 response. Moreover, in the complete Freund’s adjuvant-PPD sensitized mice, transcript levels for several of these (KC, LIX, IP-10, and lymphotactin) were significantly elevated before bead challenge (time 0) as compared to lungs of unsensitized, unchallenged mice (dashed line). In addition, other than lymphotactin, which reached maximum expression on day 4, the others displayed maximal expression during the rapid recruitment stage, days 1 and 2.Figure 3CC chemokine mRNA expression profiles of lungs during type-1 (PPD) and type-2 (SEA) Ag-bead lung granuloma formation. The mRNA expression levels were determined by PCR-ELISA analysis and the results are presented in arbitrary units (AU) as described in Materials and Methods. The profiles are representative of at least three independent experiments. In each experiment mRNA was prepared from three to four mice for each time point. Dashed linesindicate levels in normal lung controls.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Figure 3 shows the profiles obtained for 12 selected CC CKs, MIP-1α, MIP-1β, MCP-1, MCP-2, MCP-3, MCP-5, TCA-3, eotaxin, LARC, TARC, C10, and MDC. Among these, MIP-1α and MIP-1β appeared to be more strongly expressed in the type-1 lesions especially on day 2. In contrast, MCP-2, MCP-3, TCA-3, eotaxin, and LARC tended to greater expression or were sustained longer in the type-2 response, especially during the later stages of granuloma formation, days 4 and 8. It was also noted that baseline levels of eotaxin transcripts were increased in lungs after sensitization (compare day 0 to dashed line). Others such as MCP-1, MCP-5, MDC, and TARC were significantly induced in both responses, but despite trends, definite patterns of dominance could not be established. However, MCP-5 transcripts did show a unique pattern appearing to dominate early in the type-2 but later in the type-1 response. Among the 24 CK transcripts analyzed, six were found to be expressed constitutively by lung tissue and showed no change, minimal induction, or partial inhibition after sensitization and challenge. These included ELC, fractal-kine, lungkine, SLC, SDF-1α, and MIP-1γ (Figure 4). It is noteworthy that among these, SDF-1α, ELC and SLC have been implicated in the physiological recirculation of lymphocytes to lymphoid tissues.7Forster R Schubel A Breitfeld D Kremmer E Renner-Muller I Wolf E Lipp M CCR7 coordinates the primary immune response by establishing functional microenvironments in secondary lymphoid organs.Cell. 1999; 99: 23-33Abstract Full Text Full Text PDF PubMed Scopus (1877) Google Scholar, 8Hedrick JA Zlotnik A Identification and characterization of a novel beta chemokine containing six conserved cysteines.J Immunol. 1997; 159: 1589-1593PubMed Google Scholar, 9Hromas R Kim CH Klemsz M Krathwohl M Fife K Cooper S Schnizlein-Bick C Broxmeyer HE Isolation and characterization of Exodus-2, a novel C-C chemokine with a unique 37-amino acid carboxyl-terminal extension.J Immunol. 1997; 159: 2554-2558PubMed Google Scholar, 10Nagira M Imai T Hieshima K Kusuda J Ridanpaa M Takagi S Nishimura M Kakizaki M Nomiyama H Yoshie O Molecular cloning of a novel human CC chemokine secondary lymphoid-tissue chemokine that is a potent chemoattractant for lymphocytes and mapped to chromosome 9p13.J Biol Chem. 1997; 272 (1951): 8-24Crossref Scopus (232) Google Scholar, 11Randolph DA Huang G Carruthers CJ Bromley LE Chaplin DD The role of CCR7 in TH1 and TH2 cell localization and delivery of B cell help in vivo.Science. 1999; 286: 2159-2162Crossref PubMed Scopus (161) Google Scholar, 12Sallusto F Lenig D Forster R Lipp M Lanzavecchia A Two subsets of memory T lymphocytes with distinct homing potentials and effector functions [see comments].Nature. 1999; 401: 708-712Crossref PubMed Scopus (4519) Google Scholar Because these CKs were not induced with inflammation, it suggested that CKs are functionally segregated. To determine the contribution of the background non-T-cell innate foreign body CK response, we also assessed CK mRNA produced after embolization of uncoated agarose beads. In general, Ag-free beads elicit minimal transient mononuclear cell inflammation and the CK response was correspondingly blunted. Most transcripts were not significantly induced, reaching only twofold or less than that in normal lungs (data not shown). However, LIX mRNA showed a fivefold induction similar to the type-2 response (as compared to 30-fold for the type-1 response), suggesting that the modest LIX response to SEA beads was essentially Ag-independent. Transient but significant induction was noted on day 1 for MIP-1α, MCP-1, MCP-5, and LARC, with fold increases greater than normal of 4.9, 2.7, 2.7, and 3.5, respectively. Although modest compared to the Ag-elicited responses, these inductions suggest participation of CKs in the foreign-body response. Because relative CK transcript expression may not necessarily reflect levels of translated protein, we assessed levels of CK in lungs with type-1 and type-2 granulomas by specific protein ELISA. At present, reagents are available to detect only a limited number of murine CKs but where possible such assays were performed. Figure 5 shows levels of CKs detected in lung aqueous extracts during the course of granuloma formation expressed as pg per mg lung protein. In general, CK protein and mRNA expression profiles matched well. As with mRNA, the CXC CKs, IP-10 and MIG dominated in the type-1 response during the early recruitment period. Similarly, MIP-1α and to a lesser extent MIP-1β tended to higher levels in the type-1 response. Likewise reflecting transcript levels, eotaxin dominated in the type-2 response after day 2. As in the transcript analysis, MCP-1/JE and MDC did not display definite polarization and both appeared early in granuloma formation. The KC protein profile differed most from the transcript analysis by showing a clear dominance in the type-1 response. Also, protein analysis showed high levels of C10 that were expressed in ng/mg amounts, which tended to be greater in the type-2 response mainly at the day 8 stage of granuloma formation. This was not apparent from transcript levels. Such discrepancies may be related to local tissue accumulation. Despite this, the findings suggested that semiquantitative mRNA analysis was a reasonably accurate reflection of local CK synthesis. Table 2 summarizes the expression profiles for the 24 CKs analyzed during type-1 and type-2 lung granuloma formation based on transcript and protein analyses.Table 2Summary of Chemokine mRNA Expression during Type-1 (PPD) and Type-2 (SEA) Bead Granuloma FormationType dominanceChemokineEarly/late-Fold increase over normalReceptors1LIXE>10CXCR1,21MIP-2E>10CXCR1,21KCE2 to 10CXCR1,21MIGE>10CXCR31IP-10E>10CXCR31MIP-1αE>10CCR1,51MIP-1βE2 to 10CCR51LymphotactinL2 to 10XCR12EotaxinL2 to 10CCR32MCP-2L>10CCR2,32TCA-3L>10CCR82LARCL>10CCR62MCP-3L2 to 10CCR21 & 2MDCE2 to 10CCR41 & 2MCP-1E>10CCR21 & 2MCP-5E and L>10CCR1,2,31 & 2C10L2 to 10CCR?1 & 2TARCE and L2 to 10CCR7,8CLungkinen/a1 to 2CXC?CSLCn/a1 to 2CXCR3,CCR7CELCn/a1 to 2CCR7CFractalkinen/a1 to 2CX3CR1CMIP-1γn/a1 to 2CCR1,3CSDF-1αn/a1 to 2CXCR4C, Constitutive expression; n/a, does not apply. Open table in a new tab C, Constitutive expression; n/a, does not apply. It is known that Th1-associated cytokines (IFN-γ, TNF-α) promote tissue responses induced by mycobacterial Ags, whereas the Th2-associated cytokines (IL-4, IL-5, and IL-13) mediate those induced by schistosomal egg Ags.5Chensue SW Warmington KS Ruth JH Lincoln P Kunkel SL Cytokine function during mycobacterial and schistosomal antigen-induced pulmonary granuloma formation. Local and regional participation of IFN-gamma, IL-10, and TNF.J Immunol. 1995; 154: 5969-5976PubMed Google Scholar Such cytokines may orchestrate inflammatory responses by regulating local CK expression. We previously provided evidence that Th2 cytokine-chemokine networks operate during type-2 lung granuloma formation in vivo.13Ruth JH Warmington KS Shang X Lincoln P Evanoff H Kunkel SL Chensue SW Interleukin 4 and 13 participation in mycobacterial (type-1) and schistosomal (type-2) antigen-elicited pulmonary granuloma formation: multiparameter analysis of cellularrecruitment, chemokine expression and cytokine networks.C
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