Published online before print January 2, 2004
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Department of Immunology, Lerner Research Institute, Cleveland Clinic Foundation, Ohio
1Correspondence: Department of Immunology NB30, Cleveland Clinic Foundation, 9500 Euclid Avenue, Cleveland, OH 44195. E-mail: hamiltt{at}ccf.org
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express predominantly KC and very little MIP-2, and peritoneal exudate neutrophils produce as much or more MIP-2 as KC following stimulation in vitro. Although a collection of exogenous stimuli can induce expression of KC and MIP-2, the quantitative ratio for expression reflects the cell type and not the stimulus. The selective expression of KC over MIP-2 in endothelial cells results from markedly greater KC gene transcription and not from alterations in the rate of mRNA decay. These results demonstrate that distinct CXC chemokines show restricted expression in myeloid versus nonmyeloid cell types and that patterns of chemokine expression at sites of inflammation in vivo reflect the temporally ordered contribution of these distinct cell types.
Key Words: chemokines • endothelial cells • fibroblasts • macrophages
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Members of a subset of CXC chemokines containing a glycine-leucine-arginine (ELR) motif immediately preceding the CXC residues are known to selectively target neutrophils [3 , 4 , 6 , 8 , 9 ]. In the mouse, KC and macrophage-inflammatory protein-2 (MIP-2) appear to be the major ELR CXC chemokines expressed at sites of tissue inflammation following injury and/or infection [10 11 12 13 14 ]. The KC gene was originally discovered by differential hybridization screening of a cDNA library from platelet-derived growth factor (PDGF)-stimulated mouse fibroblasts [15 , 16 ], and MIP-2 was first isolated from the lipopolysaccharide (LPS)-stimulated Raw 264.7 macrophage-like cell line [17 , 18 ]. Like many chemokine genes, expression of the KC and MIP-2 genes is inducible in multiple, different cell types and in response to a variety of proinflammatory stimuli [13 , 17 , 19 , 20 ]. Furthermore, both chemokines are known to be expressed in a broad spectrum of acute and chronic inflammatory settings and are believed to be critical determinants of the nature and magnitude of the ensuing inflammatory reaction [10 , 11 , 13 , 14 , 21 22 23 ].
We have recently reported that the KC and MIP-2 genes each exhibit a distinct temporal pattern of expression following surgical injury in the skin. KC production occurs early and during the first 8 h of response, levels are two- to fourfold higher than MIP-2. In contrast, MIP-2 levels increase continuously and reach a peak between 16 and 24 h after injury, which equals or exceeds the level of KC. This biphasic pattern of expression suggests that the two genes are controlled by distinct mechanisms in vivo. Furthermore, the distinct kinetics for their expression could reflect the participation of multiple cell types and/or stimuli. In the present study, we have examined the cell types responsible for KC and MIP-2 expression in the injury model. The results demonstrate that different populations of cells in vivo produce KC and MIP-2 and that this differential expression preference is replicated in a set of primary and long-term, cultured cell lines in vitro. These findings support the hypothesis that temporal and spatial patterns of chemokine expression in vivo result from the differential contribution by different cell populations exhibiting distinct regulation of individual, functionally related chemokine genes.
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[32P]-deoxycytidine 5'-triphosphate (dCTP). Mouse interleukin (IL)-1, mouse tumor necrosis factor (TNF-), and enzyme-linked immunosorbent assay (ELISA) kits for mouse chemokines were obtained from R&D Systems (Minneapolis, MN). Epidermal growth factor (EGF), fibroblast growth factor (FGF), PDGF, and vascular endothelial growth factor (VEGF) were purchased from PeproTech (Rocky Hill, NJ).
Cell isolation and culture
Mouse primary dermal fibroblasts were isolated from adult murine epidermal/dermal tissue explants. Under sterile conditions, the dermal tissue was separated from the epidermis, minced into 1 mm2 fragments, and rinsed in Hanks’ balanced salt solution, and individual fragments were placed in 100 mm tissue-culture dishes in Dulbecco’s modified Eagle’s medium/F12 medium, supplemented with 5% fetal bovine serum (BioWhittaker, Walkersville, MD) and antibiotics (100 U/ml penicillin, 100 mg/ml streptomycin, 0.25 mg/ml fungizone). Cultures were maintained at 37°C in 5% CO2 for 7–10 days at which time residual tissue debris was removed, and the culture medium was replenished. When cultures were confluent, they were split at a 1:3 ratio. Peritoneal exudate cells were elicited by intraperitoneal (i.p.) injection of 1 ml Brewer’s thioglycollate broth (TG; Difco Laboratories, Detroit, MI) and were harvested after 6 h (neutrophils) or 72 h (macrophages) as described previously [24
]. H5V, a mouse endothelial cell line, was cultured as described previously [25
].
Surgical wound model
Female C57BL/6 mice were anesthetized by peritoneal injection of sodium pentobarbital (5 mg/ml) and subjected to a 20-mm full-thickness abdominal incision. The incisions were closed with an Ethibond 5/0 braided nylon suture. At specific times after wounding, mice were killed by CO2 asphyxiation, and the tissue surrounding the wound site (2 mm border on both sides) was harvested and used for protein extraction (see below) or fixed in formalin for histology and in situ hybridization. All tissue preparations were as described previously [26
]. The Animal Review Committee of the Cleveland Clinic Foundation (Ohio) approved animal use.
Preparation of tissue extracts and ELISA
Tissue sections for measurement of cytokine protein levels were homogenized by mechanical disruption with a PowerGen tissue homogenizer (Fisher Scientific, Pittsburgh, PA) in 150 mM NaCl, 50 mm Tris-HCl, pH 7.4, with Mini-mix protease inhibitors (Roche Pharmaceuticals, Indianapolis, IN). Tissue homogenates were centrifuged at 18,000 g for 10 min, and the supernatants were collected, aliquoted, and stored at –20°C. Protein concentrations were measured by Bio-Rad protein assay (Bio-Rad Laboratories, Hercules, CA). Quantification of KC and MIP-2 in tissue extracts and in cell culture supernatants was done using quantitative colorimetric sandwich ELISA according to the manufacturer’s instructions.
In situ hybridization
In situ hybridization was performed using formalin-fixed tissue as described previously [26
27
28
]. Radiolabeled cRNA probes for KC and MIP-2 were prepared by in vitro transcription from T3 and T7 promoters using KC and MIP-2 plasmids [24
] digested with Xba1 (sense) or Sal1 (antisense). The reaction was performed in a final volume of 25 µl containing 100 µCi [32P]-uridine 5'-triphosphate (UTP; 3000 Ci/mmol, 250 µM final concentration), 40 mM Tris-HCl, pH 7.5, 6 mM MgCl2, 2 mM spermidine, 10 mM NaCl, 10 mM dithiothreitol, 1 U/µl RNase inhibitor, 0.25 mM each adenosine 5'-triphosphate, CTP, and guanosine 5'-triphosphate, linearized template equivalent to 0.5 µg whole plasmid, and 20 units T3 or T7 polymerase. After phenol chloroform extraction and ethanol precipitation, probe length was reduced by alkaline hydrolysis in 0.2 M carbonate buffer, pH 8.2, for 10 min followed by neutralization and ethanol precipitation.
Tissue to be hybridized was fixed in 10% buffered formalin for 18 h, embedded in paraffin, sectioned, and placed on polylysine-coated slides. Hybridization was performed using 1.1 x 107 dpm radiolabled cRNA for 18 h at 50°C. After washing, slides were dehydrated and air-dried before autoradiography using NTB-3 autoradiographic emulsion (Eastman Kodak, Rochester, NY) at 4°C for 2 weeks. After developing, the slides were counter-stained with hematoxylin.
Preparation of RNA and Northern hybridization
Total RNA from cultured cells was prepared and used in Northern hybridization as described previously [26
]. Blots were exposed using XAR-5 X-ray film (Eastman Kodak).
Nuclear transcription assay
Cultures of 5 x 107 macrophages were treated as indicated in the text, and nuclei were isolated as described previously [29
, 30
]. Transcription initiated in intact cells was allowed to complete in the presence of [32P]-UTP, and the RNA was isolated and hybridized to slot-blotted plasmids containing specific cDNA (7 µg DNA/slot), essentially as described elsewhere [29
, 30
]. Blots were hybridized for 72 h and exposed to X-ray film for 2–4 days. The -tubulin gene was used as an internal standard.
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Figure 1. Temporally distinct expression of KC and MIP-2 at sites of surgical injury. C57Bl/6 mice were subjected to surgical incision, and 6 or 16 h later, the mice were killed, and the tissue from the injury site was harvested and used to prepare protein extracts. KC and MIP-2 levels were analyzed by ELISA and are presented as the means of results from four mice ± SEM. Similar results were obtained in three separate experiments.
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Figure 2. Analysis of KC expression at sites of surgical injury by in situ hybridization. C57Bl/6 mice were subjected to surgical incision as described in Materials and Methods. After 6 h, animals were killed, and full-thickness sections of the abdominal skin and muscle were obtained and fixed in buffered formalin. Sections were cut and processed as described. (A) An H&E stain illustrating the four tissue layers of the abdominal wall: epidermis (E), dermis (D), subcutaneous adipose (SA), abdominal muscle (AM). S, Suture within the wound. Original magnification, 50x. (B) Low-power (50x, original magnification) view of a section hybridized with KC antisense cRNA and counterstained with hematoxylin. The wound margins transect the section from top to bottom. The boxed areas indicate the location of high-power views shown in C and D. (C) High-power view (400x, original magnification) from the section shown in (B). Arrows identify cells positive for KC mRNA based on overlying grain density. (D) High-power view (400x, original magnification) of the section shown in (B) focusing on an area containing epidermis (e) and dermis (d). Arrows identify positive cells. (E) Low-power (50x, original magnification) view of a section hybridized with KC antisense cRNA, counterstained with hematoxylin. The boxed area identifies the region examined in (F). (F) High-power (400x, original magnification) view of the section shown in (E). Abdominal muscle is illustrated with KC expression evident in endothelial cells lining the microvasculature. Arrows identify positive cells. Similar results were obtained in two separate hybridization experiments.
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Figure 3. MIP-2 expression at sites of surgical injury. C57Bl6 mice were operated as described in the legend to Figure 2
and killed 16 h later. Sections of the injury site were excised and processed for detection of MIP-2 mRNA by in situ hybridization. (A) Low-power view (50x, original magnification) of a section of wound hybridized with MIP-2 cRNA and counterstained with hematoxylin. The boxed area identifies the region of the section examined in (B). The wound margins can be seen in the lower left quadrant of the section. There are collections of inflammatory neutrophils and monocytes within and around the boxed area. (B) High-power view (400x, original magnification) showing the boxed region outlined in (A). Arrows identify positive cells. Cell morphology within the section is consistent with polymorphonuclear leukocytes. (C) High-power view (400x, original magnification) of a section hybridized with antisense cRNA for MIP-2. The section contains a cluster of inflammatory leukocytes in the region separating dermis (D) and abdominal muscle layers (M). Arrows identify positive cells. (D) High-power view (400x, original magnification) of a section hybridized with MIP-2 sense-strand cRNA showing lack of signal over inflammatory cell infiltrate. Similar results were obtained in two separate hybridization experiments.
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(10 ng/ml) or LPS (10 ng/ml) for 4 h. The use of LPS or IL-1 as stimuli was based on prior experience with expression of KC and MIP-2 in these cell types. Total RNA was prepared and used to assess MIP-2 and KC mRNA expression by Northern hybridization. In dermal fibroblasts, KC mRNA expression was markedly induced by IL-1 treatment, but MIP-2 expression could only be detected following long exposure times (Fig. 4A
). When RNA from H5V endothelial cells, neutrophils, and macrophages was compared for KC expression, MIP-2 mRNA levels were significantly higher in the myeloid cell populations as compared with H5V endothelial cells, and KC levels in endothelial cells exceeded or equaled those measured in myeloid cells (Fig. 4B)
.
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Figure 4. KC and MIP-2 mRNA expression in cultured cell populations. Confluent 100 mm Petri dishes of primary dermal fibroblasts and H5V mouse endothelial cells were prepared as described in Materials and Methods. Peritoneal exudate neutrophils were harvested 6 h following i.p. injection of TG broth, and macrophages were obtained after 72 h. Cells (1x107) were plated in 100 mm Petri dishes and were not treated (NT) or stimulated with recombinant IL-1 (100 ng/ml) or LPS (10 ng/ml) for 6 h (dermal fibroblasts) or as indicated (H5V endothelial cells, neutrophils, macrophages) before preparation of total RNA and analysis of specific mRNA levels by Northern hybridization with radiolabled cDNAs encoding KC, MIP-2, or glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as indicated. Similar results were obtained in three separate experiments.
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produced comparable levels of KC protein as determined by ELISA. LPS was an effective but substantially less potent inducer of KC in both cell types. Only modest levels of MIP-2 were detected in supernatants of either cell type regardless of the stimulus used. In LPS-stimulated neutrophils and macrophages, production of MIP-2 protein exceeded that of KC protein in the myeloid cells by approximately twofold. |
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Table 1. In Vitro Production of KC and MIP-2
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and TNF- have also been shown to be potent stimuli of chemokine expression in endothelial cells; in both cases, KC levels markedly exceeded MIP-2 levels. Mouse macrophages and neutrophils exhibit very limited sensitivity for production of these chemokines in response to IL-1 or TNF-. It is interesting that several laboratories have reported that fibrinogen is a good inducer of chemokine expression in inflammatory leukocytes [31
, 32
]. Although H5V endothelial cells showed only modest response to fibrinogen, neutrophils showed a strong response, confirming prior reports. The expression patterns were, however, always consistent with the cell type bias for individual chemokine expression as seen previously. A variety of growth factors including PDGF, EGF, FGF, VEGF, and transforming growth factor-ß was also tested but showed very low or no chemokine production in either cell type. These results suggest that the pattern of chemokine expression observed in vivo following injury reflects the differential capacity of individual cell types to produce specific chemokines rather than a selective response to different stimuli. |
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Table 2. Different Stimuli Promote Comparable Cell Type-Specific Expression of KC and MIP-2
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Figure 5. Cell type-specific expression of KC and MIP-2 mRNA is mediated by differential transcription and not mRNA decay. (A) H5V endothelial cells were stimulated with LPS (10 ng/ml) for 3 h before the addition of ActD (5 µg/ml). Individual cultures were harvested at the indicated times for determination of KC and MIP-2 mRNA levels by Northern hybridization and autoradiography. The autoradiographs were quantified using NIH Image software, and values for KC and MIP-2 normalized to GAPDH values. Data are presented as percent remaining mRNA at each time point. (B) Cultures of H5V endothelial cells or TG-elicited peritoneal macrophages were stimulated with LPS (10 ng/ml) for the indicated times. Nuclei were isolated, and primary transcripts initiated in vivo were allowed to complete in the presence of radiolabeled UTP before hybridization with slot-blotted cDNAs encoding KC, MIP-2, and tubulin (Tub). (C) Cultures of H5V endothelial cells were stimulated with LPS (10 ng/ml) or IL-1 (10 ng/ml) for the indicated times before assessment of KC, MIP-2, and tubulin transcripts as described above. Similar results in each case were obtained in two separate experiments.
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were compared (Fig. 5C) . Although IL-1 or LPS enhanced MIP-2 transcription, KC gene transcription exceeded that from the MIP-2 gene by more than fivefold in response to both stimuli. These results demonstrate that differential transcriptional induction of the two genes is mechanistically responsible for the difference in mRNA levels. |
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As many cell types have the capacity to produce KC or MIP-2 in vitro in response to a diverse collection of stimuli, we reasoned that the distinct temporal profiles of KC and MIP-2 expression could result from differential participation of distinct cells types and/or stimuli at different times following injury. The results of the present study support the hypothesis that this pattern of KC and MIP-2 expression is a consequence of cell type-specific bias for individual chemokine gene expression. This conclusion is supported by the following observations. In situ hybridization analysis demonstrates that KC and MIP-2 are expressed in nonoverlapping cell populations at sites of surgical injury. Early KC production is restricted to cells in the dermal and subdermal layers of the skin that include fibroblasts and endothelial cells. In contrast, MIP-2 expression was largely restricted to infiltrating inflammatory neutrophils and monocytes that appear in highest numbers between 16 and 24 h after injury. Following stimulation in vitro, primary dermal fibroblasts and H5V endothelial cells produce five to 20 times more KC than MIP-2, and peritoneal exudate neutrophils and macrophages are biased toward production of MIP-2 over KC. This cell type-specific pattern of chemokine expression is independent of the stimulus used.
Many cell types including keratinocytes, fibroblasts, endothelial cells, epithelial cells, monocytes/macrophages, and neutrophils have been shown to express one or both of these chemokines in vitro [17 , 20 , 21 , 33 , 41 ]. Few studies have, however, compared the quantity of the individual chemokines produced at the same time in different cell populations. Both genes are expressed detectably in most cell types, but the same stimulation produces markedly more (five- to 20-fold) KC than MIP-2 in the nonmyeloid cell populations, and MIP-2 mRNA and protein exceed KC mRNA and protein levels by two- to threefold in primary peritoneal exudate neutrophils or macrophages. This bias for expression of specific chemokine genes appears to be an inherent characteristic of the different cell types (i.e., stromal, endothelial, and epithelial cells favor KC, and myeloid leukocytes favor MIP-2). Although multiple stimuli were able to induce chemokine gene expression in the different cell populations examined, the pattern of response did not vary with the stimulus but rather reflected the cell type bias uniformly. The cell populations used in vitro may not, of course, accurately represent the character of the cell types that appear to be responsible for differential chemokine expression at the site of surgical injury. Although H5V endothelial cells retain many characteristics of primary endothelial cells, they are immortalized and have been maintained in culture for multiple generations. Peritoneal exudate macrophages and neutrophils are primary, infiltrating, inflammatory leukocytes but have entered a distinct tissue site in response to a different set of stimuli and hence may exhibit significant differences from cells that may enter the skin following surgical injury. Nevertheless, these cell populations represent a reasonable approximation of the cells involved in the injury model, and their responses provide support for the hypothesis that cell type-specific properties determine the magnitude of specific chemokine expression.
The predominance of KC production compared with MIP-2 production by endothelial cells was reflected in the accumulation of specific mRNAs. This could result from differences in the transcription or decay of the message. Nuclear run-on analysis of gene transcription definitively demonstrated that KC is transcribed with greater frequency than MIP-2 in H5V endothelial cells stimulated with IL-1 or LPS. In macrophages, the transcriptional initiation frequency for the two genes favors MIP-2 over KC. The promoters of KC and MIP-2 have been examined [42
, 43
] and share a strong dependence on a nuclear factor-
B site within the first 100 nucleotides of the upstream region. This promoter structure would not predict the highly differential, transcriptional response seen in nonmyeloid cells. This suggests that sites that confer cell-type (myeloid) specificity, which are not found in the KC gene, regulate the MIP-2 gene. In addition, KC and MIP-2 mRNAs possess adenosine and uridine-rich regions in their 3'-UTR [15
, 18
]. Although these sequences can promote rapid mRNA decay and confer sensitivity to stabilization in response to IL-1 [33
, 44
], this does not appear to contribute to the differential expression pattern.
A similar (although not identical), temporal pattern of KC and MIP-2 expression has been demonstrated recently in several models [22 , 45 , 46 ]. For example, in a model of ocular onchocerciasis (river blindness) developing in response to parasite antigen injection into murine corneal stroma, KC expression precedes MIP-2 [45 ]. Tateda et al. [46 ] also demonstrated a temporal difference in KC versus MIP-2 expression in a murine model of pneumonia at 24 versus 48 h following Legionella pneumophila inoculation [46 ]. Other models of acute inflammatory response have reported overlapping expression patterns for KC versus MIP-2 [11 , 37 , 47 , 48 ]. These include intratracheal instillation of LPS, i.p. injection of TG, and ischemia/reperfusion injury in liver and kidney. These differences suggest that cell-type participation and/or the nature of injury or stimulus may result in different patterns.
KC and MIP-2, like many chemokines, exhibit overlapping, functional activities. The existence of multiple, functionally similar genes within the genome provides the opportunity to differentially control expression of the same function in different physiologic settings. The demonstration of temporally distinct patterns of expression and cell type-specific control of expression supports this hypothesis and provides the means to generate comparable activities in different anatomic locations and/or different time frames. Individual chemokines might, however, be responsible for different functions when expressed at different times during inflammatory stimulation [49 50 51 ]. Although a major role for ELR + CXC chemokines may be to recruit inflammatory neutrophils, they also appear to function in regulating the movement and/or activity of other cell types during wound-healing and development. Increased understanding of the nonchemotaxis-related functions of these chemokines will be central to fully understanding their contribution to the inflammatory process.
Received August 6, 2003; revised November 11, 2003; accepted December 3, 2003.
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granule proteins J. Biol. Chem. 264,4133-4137
selectively inhibits lipopolysaccharide-inducible JE/monocyte chemoattractant protein-1 and KC/gro/melanoma growth-stimulating activity gene expression in mouse peritoneal macrophages J. Immunol. 153,2204-2212[Abstract]-mediated recruitment of neutrophils is required for elicitation of contact hypersensitivity Eur. J. Immunol. 29,3485-3495[CrossRef][Medline] and macrophage inflammatory protein-2 attenuates renal ischemia/reperfusion injury Am..J..Pathol. 159,2137-2145 and IFN-ß independently stimulate the expression of lipopolysaccharide-inducible genes in murine peritoneal macrophages J. Immunol. 142,2325-2331[Abstract]B transcription factors in mononuclear phagocytes J. Immunol. 161,1462-1470, MCP-1, IP-10, and Mig are sequentially and differentially expressed during phase-specific infiltration of leukocyte subsets in human wound healing Am. J. Pathol. 153,1849-1860 induces cell type and tissue-specific expression of chemoattractant cytokines in vivo Am. J. Pathol. 142,861-870[Abstract]B sequence motifs cooperatively control LPS-induced KC gene transcription in mouse macrophages J. Immunol. 155,3593-3600[Abstract], and MIP-1ß, members of the chemokine family of pro-inflammatory cytokines J. Immunol. 150,4996-5003[Abstract] promotes spinal cord oligodendrocyte precursor proliferation J. Neurosci. 18,10457-10463This article has been cited by other articles:
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J. L. Lomas-Neira, C.-S. Chung, P. S. Grutkoski, E. J. Miller, and A. Ayala CXCR2 inhibition suppresses hemorrhage-induced priming for acute lung injury in mice J. Leukoc. Biol., July 1, 2004; 76(1): 58 - 64. [Abstract] [Full Text] [PDF]
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