1Divisions of Cancer Biology and Vascular Biology, La Jolla Institute for Molecular Medicine, San Diego 92121; and 2Department of Immunology, The Scripps Research Institute, La Jolla, California 92037
Submitted 25 September 2002 ; accepted in final form 23 April 2003
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ABSTRACT |
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novel source; novel target cell
Expression of the mRNA for PARC has been detected in human lung, particularly in alveolar macrophages, in follicular dendritic cells in the germinal centers of lymph nodes, in peripheral blood monocytes following stimulation with lipopolysaccharide (LPS), and in low concentrations in bone marrow (16). It has also been detected by in situ hybridization in atherosclerotic plaque (27). The known sources of PARC, primarily lung macrophages, give little information about its possible functions. Because PARC has been detected only in primates to this point, insights into its function from animal studies have been limited (13). However, in a recent gene array study in a monkey model of allergic asthma, PARC, along with eotaxin and monocyte chemotactic protein (MCP)-1, was one of the most highly induced genes in the lung tissue following allergen challenge (35), suggesting a role for PARC in allergic inflammation. Therefore, we systematically investigated which leukocytes, in particular those associated with allergic inflammation, were capable of producing PARC. Apart from monocytes, we found that eosinophils produce PARC, which was detected both at the mRNA level and at the protein level in the supernatants of peripheral blood eosinophils.
The cellular responses caused by PARC have not been investigated thoroughly. It has been shown that PARC is a chemotactic factor for various subclasses of lymphocytes in vitro (1) and in vivo (13). When PARC was injected into mice, only a moderate number of lymphocytes accumulated at the site of administration (13). However, since it is doubtful that mice express a PARC equivalent (31), and chemokine/chemokine receptor usage in rodents is quite different from that seen in humans, the lymphocyte-specific response observed in mice might be only a partial presentation of the human response. For this reason, we searched for additional cells that could respond to PARC and found that peripheral blood monocyte/macrophages in culture for 3-4 days respond to recombinantly expressed PARC. Freshly isolated monocytes did not show this activity (16). This distinction between freshly isolated monocytes and monocyte/macrophages may help in the identification of the PARC receptor.
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MATERIALS AND METHODS |
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For cDNA preparation RNA was purified with the RNeasy kit (Qiagen, Valencia, CA) followed by reverse transcription with the Promega RT-PCR kit (Promega, Madison, WI) according to the manufacturer's recommendations. Possible genomic DNA contamination could be excluded since the PARC PCR primers would have included an intron region in the genome.
MIP-1, an ELISA kit for MIP-1
, and neutralizing antibodies against the IL-8 receptor 1 (CXCR1) and CXCR2 were purchased from R&D Systems (Minneapolis, MN). IL-8 and growth-related protein (GRO)-
were expressed recombinantly and purified as previously described (29). LPS (Re595 Salmonella minnesota) was from Sigma Chemical (St. Louis, MO), and tumor necrosis factor-
(TNF-
) from Biosource (Sunnyvale, CA). Indo 1-AM and FITC-phalloidin were obtained from Molecular Probes (Eugene, OR).
Purification of PARC. Recombinant PARC expression in Escherichia coli strain HMS 174(DE3) (Novagen) was induced with 0.1 mM isopropylthiogalactoside for 3 h at 37°C. After centrifugation, the bacterial pellet was suspended in 20 mM sodium acetate, pH 5.0/0.15 M NaCl/5 mM EDTA/10 mM benzamidine/20 mM -amino caproic acid/0.2% Triton X-100, sonicated for 30 s, frozen, and thawed. After centrifugation, the pellet was dissolved in 8 M urea, 10 mM HEPES, pH 6.5, and 0.15 M NaCl, and the sample was absorbed to and eluted from Tris(carboxymethyl)ethylinediamine-Sepharose, which had been charged with NiCl2 (24). Expressed PARC was eluted with 40 mM sodium acetate, pH 4.5, 6 M guanidine HCl, and dialyzed against 10 mM HEPES/0.15 M NaCl, pH 7.0. The oligohistidine leash was excised from the expressed polypeptide by incubation with thrombin for 15 h at 23°C using an enzyme to substrate weight ratio of 1:4,000. After inactivation of the thrombin with 0.1 mM PMSF, PARC was separated from contaminating polypeptides by cation exchange chromatography on a dextran sulfate Sepharose column with a linear salt gradient from 0.15-1.5 M NaCl. Matrix-assisted laser desorption-time of flight mass spectroscopy of isolated PARC was performed as a service at The Scripps Research Institute.
Immunological detection of PARC. Polyclonal antibodies to recombinant PARC were raised in rabbits by Strategic Biosolutions (Ramona, CA), and a sandwich ELISA was developed with this antibody preparation. The rabbit IgG was affinity purified on recombinant PARC covalently bound to Sepharose using the cyanogen bromide reaction, and the purified antibody was biotinylated using NHS-LC-biotin (succinimidyl-6-biotinamide-hexanoate; Pierce Chemical, Rockford, IL). Polyclonal antibody (10 µg/ml) was absorbed to microtiter wells (Nunc maxisorp; Nalge Nunc International, Rochester, NY) for 16 h in 150 mM sodium carbonate, pH 9.6. In later experiments monoclonal anti-PARC (2 µg/ml, R&D Research) was used, resulting in slightly increased signal-to-noise ratio. Each plate was washed 3x with 20 mM imidazole HCl, pH 7.3/0.15 M NaCl, blocked with 2% BSA/0.5% Tween 20 in 0.1 M sodium phosphate, pH 7.0/0.15 M NaCl for 1 h and washed. Cell supernatants or PARC standards in Ultraculture (Bio-Whittaker, Walkersville, MD) containing 1 mg/ml BSA and 1 mg/ml ovalbumin were added and incubated for 2 h at room temperature. After three further washes, 100 µl of biotinyl-anti-PARC-IgG were added, and the plate was incubated for 1 h at room temperature. The plate was again washed, incubated with avidin-horseradish peroxidase conjugate (DAKO, Carpinteria, CA), and finally developed with 2-2'-azino-di-3-ethylbenzazoline sulfonate (Roche Molecular Biochemicals, Indianapolis, IN), at 0.5 mg/ml in 50 mM sodium phosphate/20 mM sodium citrate, pH 5/0.03% H2O2, and the absorbance was read at 405 nm on a Molecular Devices Vmax reader (Molecular Devices, Sunnyvale, CA). All samples were analyzed at least in duplicate from cell preparations from 11 donors. The ELISA was specific for PARC and did not cross-react with human MIP-1 or MIP-1
, the two most closely related chemokines. Nor was there cross-reactivity with regulated on activation normal T-cell expressed and presumably secreted (RANTES), MCP-1, eotaxin, IL-8, or GRO-
.
Preparation of leukocytes. Mononuclear and neutrophil fractions were purified from fresh human acid citrate dextrose blood from healthy donors by Hetastarch sedimentation followed by Percoll gradient centrifugation (34) (Amersham Pharmacia Biotech, Piscataway, NJ). The neutrophil fraction was >98% pure. We further separated the mononuclear fraction into monocytes and lymphocytes by allowing the monocytes to adhere to tissue culture plastic in RPMI 1640 containing 10% FCS for 90 min at 37°C. Nonadherent lymphocytes in the supernatant were then removed, and the remaining monocytes were thoroughly washed and cultured for 1-4 days in RPMI 1640 containing 10% FCS (GIBCO-BRL). For calcium mobilization and actin polymerization assays, adherent monocytes were removed from the surface by a 10-min incubation with cell dissociation buffer (GIBCO-BRL), and the nonadherent fraction was added. The combined cells were washed and resuspended in Hanks' balanced salt solution (HBSS; 140 mM NaCl, 5 mM KCl, 3.8 mM KH2PO4, 2.2 mM Na2HPO4, and 5.5 mM glucose, pH 7.4). For chemotaxis, only the nonadherent cells were used to avoid possible inactivation of adhesion molecules due to cell processing. Overnight cultures of monocytes used in the ELISA were maintained in Ultraculture (Bio-Whittaker). Fluorescence-activated cell sorting (FACS) analysis showed that these cells were between 94 and 98% monocytes and 2 and 6% lymphocytes as assessed by size and granularity and by determining the percentage of CD14-positive cells using anti-CD14 (M5E2 monoclonal; Pharmingen, La Jolla, CA) followed by staining with FITC-anti-mouse IgG (Biosource, Camarillo, CA).
Eosinophils were isolated from blood of healthy, mildly allergic donors. After the preparation of the polymorphonuclear cell fraction as described above, eosinophils were purified by negative selection using anti-CD16 with magnetic bead separation (14). The eosinophil preparations were >99% pure with neutrophils as the only trace contaminating cells. Mononuclear cells were never observed in eosinophil preparations as assessed by hematoxylin/eosin Y staining. When determining the release of PARC from eosinophils, cells were incubated overnight in Ultraculture with the addition of 5 ng/ml of IL-5 and granulocyte-macrophage colony-stimulating factor (GM-CSF) (both from R&D Systems).
All leukocytic cell lines were obtained from ATCC and grown in RPMI 1640 containing 10% FCS.
Calcium mobilization. To detect changes in intracellular calcium, we labeled leukocytes for 30 min with indo 1-AM (Molecular Probes) as previously described (29). Fluorescently labeled cells (2 x 105) were warmed to 37°C for 2 min in a stirred cuvette containing 200 µl of modified Gey's buffer (MGB; 140 mM NaCl, 5 mM KCl, 1.9 mM KH2PO4, 0.5 mM MgCl2, 1.1 mM Na2HPO4, 1.5 mM Ca2Cl, 10 mM HEPES, and 5.5 mM glucose, pH 7.4), the stimulus was added, and the emission ratio at 400/480 nm was followed kinetically on an SLM 8000 fluorometer (SLM Aminco, Rochester, NY) as described previously (32). Calcium concentrations were calculated as described in Ref. 12.
Actin polymerization. For the determination of actin polymerization, 4-day-old monocyte/macrophages or lymphocytes were stimulated with PARC for the indicated time at 37°C and pipetted into a mixture of formaldehyde, lysophosphatidic acid, and FITC-phalloidin (Sigma Chemical) as previously described (7). The mean fluorescence of the cell population was detected by FACSCAN analysis (Becton Dickinson, Research Triangle Park, NC) of the monocyte or lymphocyte population that was selected by its characteristic size and granularity.
Chemotaxis assay. Monocytes (2 x 105) in HBSS containing 0.5% BSA were applied to the top compartment of 5-µm pore-size Transwell filters (Costar; Corning, Acton, MA) and allowed to transmigrate into the bottom compartment containing varying concentrations of PARC for 90 min at 37°C. At this time, 5 mM EDTA was added to the lower compartment to release monocytes that adhered to the underside of the filters, and transmigrated cells were counted manually in 25 low-power magnification fields. In some experiments the filters were coated with 200 µl/ml Matrigel (Becton Dickinson, San Diego, CA) overnight at 4°C and washed three times with 150 mM NaCl and 10 mM HEPES, pH 7.3, to mimic basement membrane-like conditions before the addition of monocytes.
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RESULTS |
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Expression of PARC mRNA by various leukocytes. Screening for expression of PARC in various tissues indicates that PARC mRNA was not limited to the lung but was produced by various cells of leukopoietic lineage. RT-PCR indicates that it was expressed in bone marrow, by peripheral monocytes following 90 min of adhesion to plastic, and by freshly isolated eosinophils (Fig. 2), but not by neutrophils. The trace of a PCR product observed in lymphocytes may have been caused by monocyte contamination. A PCR product for PARC was obtained from the eosinophil mRNA of three consecutive donors (Fig. 2), and DNA sequencing confirmed that the eosinophil-derived PARC PCR product was identical to the recombinant PARC derived from lung cDNA.
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Detection of PARC by ELISA. A sandwich ELISA was established to determine PARC protein concentrations in cellular supernatants. Consistent with the PCR results, both eosinophils and monocytes expressed PARC protein constitutively. Three out of the 11 eosinophil donors presented with peripheral eosinophil counts in the normal range (<3% of the leukocyte population). In contrast to the eight eosinophilic donors, PARC levels in these three donors were below the detection limit of the ELISA. The large variation in PARC concentrations observed with different donors (290+/-200 pg/106 eosinophils and 760+/-420 pg/106 monocytes; Fig. 3, A and B) is not unusual for the expression of a chemokine. Although eosinophils produced only a third of the amount of PARC obtained from monocytes, this may represent a sizable contribution under conditions of allergic inflammation, where tissue eosinophils can be the dominating cell population.
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PARC concentrations were comparable to MIP-1 concentrations of the same monocyte supernatants, both in terms of concentration and variability (620+/-390 pg/106 cells, Fig. 3B). It should also be noted that our method of monocyte purification by means of their adhesion to plastic has been found to activate the release of a number of cytokines and chemokines without the need for inflammatory mediators (17, 30). In this sense these cells are not strictly unstimulated but stimulated in a fashion that mimics monocyte extravasation in vivo.
No PARC was detected in the supernatants of neutrophils or lymphocytes or in monocyte/macrophages kept in culture for 4 days. LPS increased protein expression of PARC by monocytes (Fig. 3C) as previously reported for PARC mRNA levels (16). Again, the increase of PARC in the supernatants of LPS-stimulated cells was in the same concentration range as that observed for MIP-1 (Fig. 3D). IL-8 also increased expression of PARC to a statistically significant degree (Fig. 3C; Student's t-test, P < 0.05). In contrast, GRO-
failed to induce increased PARC expression, suggesting that the response to IL-8 was mediated by the CXCR1, which is specific for IL-8 (Fig. 3), and not by the CXCR2, which has similar affinities for IL-8 and GRO-
(18, 29). To test this, monocytes were incubated with anti-CXCR1 or anti-CXCR2 antibody (10 µg/ml, R&D Systems) in the presence or absence of IL-8 (10 µg/ml, R&D Systems). Anti-CXCR1, but not anti-CXCR2, antibody significantly decreased the concentration of PARC in the supernatant of IL-8-stimulated cells (P < 0.01, Fig. 3C). Interestingly, anti-CXCR1 antibody by itself reduced PARC concentrations in unstimulated cells to levels below those seen in the absence of antibody (Fig. 3C), suggesting that IL-8 produced by the monocytes, which are known to produce IL-8 under our culturing conditions, contributed to the basal PARC production in these cells.
Screening for cell lines responding to PARC. Because calcium mobilization is a facile assay that allows fast screening of many different cell types and uses small cell numbers (2 x 105 cells/sample), we used this assay to assess cell types that express receptors for PARC. Numerous leukocytic cell lines failed to respond to PARC. These included THP1, HL60, U937, Ramos, Raji, Jurkat, and RBL2H3 cells and included differentiation with PMA for HL60 and THP1 cells, with dibutyryl-cAMP for HL60 cells and U937 cells and interferon- and G-CSF for U937 cells. Among primary leukocytes, only lymphocytes and 3- to 4-day-old monocytes (Fig. 4) responded to PARC with calcium mobilization. Eosinophils and neutrophils showed no response.
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Calcium mobilization in monocytes/macrophages. Because lymphocyte stimulation by PARC had been described previously (1, 16), we primarily investigated the effect of PARC on monocytes. In 4-day-old monocytes/macrophages, PARC induced a calcium flux with a threshold dose of 5 x 10-9 M, and the maximal response, corresponding to a rise of intracellular Ca2+ from 140 nM to 1 µM, was reached with 10-7 M PARC (Fig. 4). To assure that the Ca2+ flux could not be attributed to contaminating lymphocytes, 2 x 104 lymphocytes, a cell number that is greater than the lymphocyte contamination in any of our monocyte preparations, were stimulated with PARC. Under these conditions no change in calcium fluorescence was observed, indicating that monocytes were the cells that responded to PARC.
Monocytes purified as the adherent cell fraction in the mononuclear cell mixture failed to respond to PARC, when used on the day of cell preparation. This refractory behavior of freshly isolated, adherent monocytes was not surprising, since these cells expressed mRNA for PARC and secreted PARC protein as shown above, which is expected to desensitize and/or downregulate any PARC receptors these cells may possess. The same behavior was observed for several other chemokines (IL-8 and GRO-), which like PARC failed to activate monocytes on days 0-2 but caused a calcium flux on days 3-4 (Fig. 4), which decreased again over the following days and became negative by 1 wk. The potency of PARC was slightly lower than that of MIP-1
, IL-8, or GRO-
(Fig. 4).
Actin polymerization in monocytes and lymphocytes. To confirm that monocytes responded to PARC, filamentous actin (F-actin) was determined in monocyte/macrophage and lymphocyte populations after 4 days in culture. As shown in Fig. 5, PARC caused an increase in F-actin content in both cell types. These experiments clearly indicate that 4-day-old monocytes/macrophages respond to PARC. Lymphocytes similarly responded with actin polymerization. Their unusually high F-actin content (the resting cell level of fluorescence intensity seen in FITC phalloidin-stained lymphocytes was sixfold higher than that in the monocyte population) further guaranteed that all lymphocytes were excluded from the monocyte population. This further showed that the actin polymerization in monocytes could not be attributed to contaminating lymphocytes.
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Chemotaxis of monocytes. To test whether PARC plays a role in monocyte trafficking, we determined chemotaxis of monocyte/macrophages toward PARC. As already suggested by the F-actin response, a prerequisite for chemotaxis, PARC induced chemotaxis of 4-day-old monocytes (Fig. 6) with a threshold dose of 2 nM and a maximal response with 20-30 nM PARC. Checkerboard analysis indicates that the cells responded with chemotaxis rather than chemokinesis. Because leukocytes have to be able to migrate through the basement membrane during the inflammatory response, PARC-dependent cell migration through Matrigel-coated filters was determined. Under these conditions PARC was a more potent chemoattractant than IL-8 in terms of both sensitivity and numbers of attracted cells (Fig. 6B). As reported previously (1), freshly isolated monocytes did not migrate toward PARC.
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As a further functional assay in monocytes/macrophages, we determined the release of H2O2 (9) following stimulation with PARC but found no evidence for an oxidative burst (results not shown). Under the same experimental conditions, 100 ng/ml PMA caused the production of H2O2 (1.1 nmol·2 x 105 monocytes-1·60 min-1).
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DISCUSSION |
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The physiological role for eosinophil-derived PARC deserves further investigation, since it may initiate both pro- and anti-inflammatory reactions. The chemotactic effect of PARC on lymphocytes (1, 13, 16) and, as shown here, on monocyte/macrophages, is likely to cause mononuclear cell attraction to an area in which eosinophils have accumulated. Thus PARC has the potential to be an important factor in pulmonary leukocyte accumulation in allergic disease. The notion that PARC may play a role in allergic lung disease is strengthened by the recent observation that PARC was one of the most strongly upregulated genes in a monkey model of allergic asthma (35).
On the other hand, PARC has an antagonistic effect on the CCR3 (21). This property of PARC may limit further eosinophil influx caused by eotaxin, RANTES, MCP-3, and MCP-4, which all stimulate CCR3 (8, 10, 23, 33). In this respect PARC may limit eosinophil accumulation by competing with the ligands that activate CCR3. Future investigation will show which of these two mechanisms, the pro- or the anti-inflammatory side, prevails and whether PARC should be inhibited or whether on the contrary PARC-derived proteins may be useful as CCR3 antagonists in asthmatics as suggested by Nibbs et al. (21).
Until now the determination of PARC expression has relied largely on its detection on the mRNA level. The development of an ELISA for PARC greatly simplifies detection of PARC and allows quantification of the expressed protein. Our results establish that monocytes express PARC also on the protein level, which increases following stimulation with LPS (Fig. 3), as suggested by the earlier detection of increased PARC mRNA levels under this condition (16, 27). In addition, IL-8 was also found to induce increased PARC expression by monocytes. In contrast, GRO- failed to influence PARC production, suggesting that PARC induction was mediated by the CXCR1, which is specific for IL-8, and not by the CXCR2, which reacts equally well with IL-8 and GRO-
(18, 29). This was confirmed using specific antibodies. In fact baseline PARC concentrations were decreased by 50% in the presence of anti-CXCR1 antibody, suggesting that constitutive expression of IL-8 contributed to the expression of PARC by adherent monocytes. The same CXCR1-specific upregulation of monocytic chemokine production has recently been described for IL-8 itself, where IL-8, but not GRO-
, induces its own expression in an autocrine fashion (6). Eosinophils had to be cultured in the presence of IL-5 and GM-CSF to detect PARC in the supernatants. Because both these factors are, however, necessary for eosinophil survival in vitro (15), it is problematic to omit them. In addition, both IL-5 and GM-CSF are expressed by eosinophils at sites of allergic inflammation in asthmatics (5). In this respect their addition only mimics the situation seen in allergic inflammation. The concentrations of PARC found in the supernatants of eosinophils are comparable to those previously reported for IL-8 (20).
This may indicate that PARC expression was already induced in vivo in these eosinophilic subjects, but it is also possible that different stimulating agents are necessary to upregulate PARC expression in these cells.
The PARC ELISA will allow detection of this chemokine in patient samples, which is important, since the in vivo function of PARC is still unknown. Apart from its possible association with allergic disease, it may also play a role in atherogenesis, since PARC mRNA has been detected in atherosclerotic plaque (27). The detection of PARC in human fluids is essential, since its role is difficult to assess in animal models. PARC is supposed to have evolved quite recently by gene duplication from MIP-1 (13), and so far no animal equivalent of PARC has been reported. It does not appear to exist in mice (16), and its existence in rabbits is questionable. Using human PARC cDNA as a probe, we isolated five clones of rabbit MIP-1
from a rabbit lung library (Clontech) but no PARC equivalent (results not shown).
Furthermore, chemokine usage often differs between humans and rodents; for instance, mice have no IL-8 and CXCR1 equivalent but use MIP-1 for certain functions such as neutrophil chemotaxis that are mediated by IL-8 and the CXCR1 in humans. In mice, injected PARC attracted only lymphocytes and no monocytes (13). Because of the differing chemokine usage between rodents and humans, this observation does not preclude monocyte chemoattraction by PARC in humans in vivo, as would be suggested by our in vitro experiments (Fig. 6). Monocytes cultured for 3-4 days were activated by PARC as shown in calcium mobilization, actin polymerization, and chemotaxis assays. The activating concentration range was similar to that previously described for lymphocytes (16). It will be interesting to determine under which conditions monocyte/macrophages can be chemoattracted by PARC in vivo, a process that could play a role in various chronic inflammatory diseases. Because pulmonary alveolar macrophages are the most prominent source of PARC (16), PARC may contribute to the homing of monocytes to the lung.
Previous reports, which failed to show activation of monocytes by PARC (1, 16), analyzed freshly isolated monocytes, which did not respond to PARC in our hands either. This could be due either to the lack of PARC receptors at this point or to desensitization of PARC receptors from previous stimulation with cell-derived PARC. Previously, a similar refractory behavior has been observed toward IL-8 and GRO-, which failed to stimulate monocytes that were freshly purified by means of their adhesive property, which induces IL-8 production (17). Although both classes of IL-8 receptors, the CXCR1 and the CXCR2, were shown to be present on these cells (19), no cellular response to IL-8 could be evoked in freshly prepared adhesion-isolated monocytes, and the IL-8-mediated chemotactic response of monocytes was long overlooked in vitro (11) despite evidence for it in vivo (4). A similar condition may exist for the PARC receptor on monocytes, where PARC produced by the adherent monocytes might make PARC receptors refractory to the further addition of PARC. The testing of this hypothesis will have to await the description of the PARC receptor, which is still unknown. Alternatively, PARC receptor expression may be upregulated after 3-4 days in culture.
The PARC receptor is G protein coupled (1) and sensitive to inhibition by pertussis toxin (16). This coupling to Gi is a common feature of chemokine receptors (3, 28). Although chemokine receptors can activate cellular signaling through other G proteins, coupling to Gi
is necessary to induce a chemotactic response, which is mediated by the
,
-chains of Gi
(2). As already mentioned, the specific receptor activated by PARC is still unknown, but CCR1-10 have been excluded (16, 21). Our search for a cell line that responds to PARC has been similarly unsuccessful. None of the tested hematopoietic cell lines (THP1, HL60, U937, Ramos, Raji, Jurkat, and RBL2H3) was activated by PARC, which suggests that cloning of the PARC receptor has to rely on primary lymphocyte (1) or cultured monocyte/macrophage libraries, unless PARC can be shown to activate one of the existing orphan receptors.
In summary, we identified a novel source of PARC, eosinophils, and a novel function for it, the stimulation of monocyte/macrophages that were kept in culture for 3-4 days. Both these observations are of potential interest in chronic inflammatory processes.
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ACKNOWLEDGMENTS |
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Current address for H. Takamori: Dept. of Surgery, Kumamoto Univ., Kumamoto, Japan.
GRANTS
This work was supported by National Institutes of Health Grants HL-55657 (to I. Schraufstatter) and AI-35796 (to P. Sriramarao).
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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