- Email: [email protected]
Macrophage Colony-Stimulating Factor Accelerates Wound Healing and Upregulates TGF-β1 mRNA Levels through Tissue Macrophages
JOURNAL OF SURGICAL RESEARCH ARTICLE NO.
72, 162–169 (1997)
JR975178
Macrophage Colony-Stimulating Factor Accelerates Wound Healing and Upregulates TGF-b1 mRNA Levels through Tissue Macrophages1 Liancun Wu, M.D.,*,2 Yung L. Yu, M.D.,‡ Robert D. Galiano, M.D.,* Sanford I. Roth, M.D.,† and Thomas A. Mustoe, M.D., FACS*,3 *Division of Plastic Surgery and †Department of Pathology, Northwestern University Medical School, Chicago, Illinois 60611; and ‡Department of Surgery, The Chicago Medical School, Chicago, Illinois 60608 Submitted for publication February 27, 1997
Macrophage colony-stimulating factor (M-CSF) is produced by many cell types involved in wound repair, yet it acts specifically on monocytes and macrophages. The monocyte-derived cell is thought to be important in wound healing, but the importance of the role of tissue macrophages in wound healing has not been well defined. Dermal ulcers were created in normal and ischemic ears of young rabbits. Either rhM-CSF (17 m g/wound) or buffer was applied to each wound. Wounds were bisected and analyzed histologically at Days 7 and 10 postwounding. The amounts of epithelial growth and granulation tissue deposition were measured in all wounds. The level of increase of TGF- b1 mRNA level in M-CSFtreated wounds was examined using competitive RT-PCR. M-CSF increased new granulation tissue formation by 37% (N Å 21, P õ 0.01) and 50% (P õ 0.01) after single and multiple treatments, respectively, in nonischemic wounds. TGF- b1 mRNA levels in rhM-CSF-treated wounds increased 5.01-fold (N Å 8) over vehicle-treated wounds under nonischemic conditions. In contrast, no effect could be detected in ischemic wounds treated with rhM-CSF, and these wounds only showed a 1.66-fold increase in TGF- b 1 mRNA levels when compared to ischemic wounds treated with vehicle alone. GAPDH, a housekeeping gene, showed no change. As mesenchymal cells lack receptors for M-CSF, the improved healing of wounds treated with topical rhM-CSF must reflect a generalized enhancement of activation and function of tissue macrophages, as demonstrated by upregulation of TGF- b . The lack of effect under ischemic conditions suggests that either macrophage activity and/or response to M-CSF is ad1 This work was supported in part by NIH Grant GM 41303 and was presented at the Fifth Wound Healing Society Meeting at Minneapolis, MN, April 1995. Recombinant human M-CSF was supplied by Chiron Corporation, Emeryville, CA. 2 Current address: Department of Anesthesiology, Medical College of Virginia Hospital, Richmond, VA 23220. 3 To whom correspondence and reprint requests should be addressed at Division of Plastic Surgery, Northwestern University Medical School, 707 N. Fairbanks Court, Suite 811, Chicago, IL 60611-3008.
0022-4804/97 $25.00 Copyright q 1997 by Academic Press All rights of reproduction in any form reserved.
AID
JSR 5178
/
6n26$$$241
versely affected under those conditions; this may suggest the pathogenesis of impaired wound healing at the cellular level. q 1997 Academic Press
INTRODUCTION
Many peptide growth factors have been shown to increase wound healing under normal and impaired conditions [1], but none of these stimulators has been accepted as a pharmacological adjunct for the treatment of acute and chronic wounds. One of the reasons may be that wound healing is an extremely complex process involving the interaction of numerous cell types, multiple growth factors, and the extracellular matrix present within the wound at the cellular and molecular levels. Although the precise molecular and cellular events in wound healing are under active investigation [2], it is generally recognized that the wound macrophage may be a major player in wound repair, with several studies suggesting a critical role for macrophages throughout wound healing [3–5]. None of these studies, however, has elucidated the precise role of local tissue macrophages in wound healing versus bloodderived monocytes. Activated macrophages produce over 100 different growth factors and interleukins [6] and more than 20 of these have been shown to be involved in wound healing [1]. Therefore, one strategy to pharmacologically enhance repair is by attracting and/or activating tissue macrophages to subsequently release a physiological combination of growth factors. Possible macrophage stimulators include charged beads [7–8], macrophageactivating substance [9], and growth factors such as platelet-derived growth factor type BB (PDGF-BB) and granulocyte-macrophage colony-stimulating factor (GM-CSF) [10, 11]. Macrophage colony-stimulating factor (M-CSF), also known as CSF-1, is a glycoprotein [12, 13] which selectively stimulates the survival, proliferation, and activation of macrophages [14, 15]. A biologically active M-CSF can be detected in the circulation [16] and intravenous administration of recombinant human M-CSF (rhM-CSF) in mice elevates the
162
10-08-97 10:33:42
srga
163
WU ET AL.: M-CSF AND WOUND HEALING
concentration of tissue macrophages [17] and stimulates the recovery of radiation-impaired and autologous bone marrow transplant recipients [18, 19]. The osteopetrotic mutant mouse, a spontaneously occurring M-CSF gene knockout [20], is deficient in circulating monocytes and tissue macrophages [21]. In this mutant murine model, tissue macrophages are absent and dermal development is impaired. This finding suggests that the tissue macrophage subpopulation requires MCSF for development. While the role of M-CSF in wound repair has not been well studied, several observations suggest that it may be an important growth factor to study. M-CSF is produced by many cell types involved in wound repair including fibroblasts, myoblasts, endothelial cells, keratinocytes, and vascular smooth muscle cells [22–24]. M-CSF has been detected in the wound fluid during the early phase of wound healing at significantly higher levels compared to the basal serum level in unwounded animals [25]. Furthermore, rhM-CSF has been shown to indirectly stimulate angiogenesis [26]. In addition, it has been documented both in vivo and in vitro that hypoxia induces angiogenic factors and increases neovascularization [27], but the response of macrophages to growth factors in ischemic wound healing has not been fully investigated. It is crucial to evaluate the behavior of a potential vulnerary agent such as M-CSF in clinically pertinent models of impaired wound healing [28]. Previous studies from our laboratory have revealed a difference in the behavior of growth factors under ischemic conditions [29]. The behavior of other growth factors such as M-CSF in the same model merits further investigation. MATERIALS AND METHODS Recombinant human macrophage colony-stimulating factor, supplied by Chiron Corporation (Emeryville, CA), was expressed in E. coli and purified as previously described [30]. The rhM-CSF was then suspended in phosphate-buffered saline solution (PBS) at a concentration of 1.7 mg/ml and was assayed for endotoxin and biological activity prior to use. Eighteen young adult female, New Zealand white rabbits (New Franken Inc., New Franken, WI), weighing 2.7–3.4 kg, were acclimated and housed individually under standard conditions. After they were anesthetized with ketamine (60 mg/kg) and xylazine (5 mg/kg), the surgical procedures were performed under sterile conditions as previously described [29, 31]. This procedure was approved by the Animal Care Committee of Northwestern University (Chicago, IL). Briefly, four 6 mm diameter full-thickness circular wounds were made down to bare cartilage in nonischemic wounds. For the ischemic model, three wounds were made on each ear after two of the
three arteries that supplied the rabbit ear were divided. This ischemic procedures decrease the ear tissue oxygen tension by 50% (55 { 3 mm Hg for normal tissue, 25 { 3 mm Hg for ischemic ear) and impairs the new granulation tissue deposition more than 80% at Day 7 postwounding [29, 31, 32, and unpublished data]. The wounds on one ear were treated at the time of wounding with 17 mg/wound of rhM-CSF; in one set of rabbits the same dose of rhM-CSF was applied on Days 0, 1, and 2 postwounding. The contralateral ear served as a paired control and was treated with the same amount of vehicle alone (PBS). All the wounds were covered with an occlusive polyurethane dressing (Tegaderm, 3M, Minneapolis, MN). The wounds were bisected and analyzed histologically at 7 days postwounding for the nonischemic groups and at 10 days postwounding for the ischemic groups as previously described [29, 33]. Two independent histological analyses were performed on each sample with the observers blinded to treatment. Reepithelialization and new granulation tissue formation were measured based on minimal contraction of these ulcers [33]. The wounds were harvested in a paired fashion, and the paired Student’s t-test (two-tailed) was used to compare the results (Epistat Service, Richardson, TX). Cellular infiltration into the wound consisting mainly of macrophages and proliferative fibroblasts [34, 35] in the hematoxylin and eosin stained slides was rated on a scale of 0–4 (0 for lowest cellularity, 4 for highest cellularity) in a blinded fashion using a Nikon microscope as previously described [36]. Differences in cellularity were compared using the Wilcoxson rank test (Epistat Service). The wound granulation tissue from each rabbit was harvested with a 7-mm biopsy punch under the operating microscope after each ulcer was bisected for histological analysis. The harvested tissue was immediately placed into liquid nitrogen. The total cellular RNA from each ear’s wounds (which consisted of three pooled wounds for the ischemic model and four pooled wounds for the nonischemic model) was extracted with a guanidine thiocyanate/phenol-based reagent (TRI Reagents, Cincinnati, OH) as previously described [37]. All reverse transcription (RT) reactions were performed simultaneously using a master mix to eliminate variability of reverse transcriptase efficiency between different reaction tubes. Acceptable purity was determined spectrophotometrically (Pharmacia LKB, Cambridge, England), and only RNA samples having an A260/A280 absorbence ratio greater than 1.8 were utilized. Five micrograms of total RNA was then converted to cDNA by using Moloney murine leukemia virus reverse transcriptase (M-MLV RT) and random primers (Gibco BRL, Grand Island, NY). Specific PCR primers for rabbit glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and TGF-b1 were designed based on the conserved complete and partial cDNA sequences of various species published in GenBank (Table 1). The PCR products were confirmed as rabbit TGF-b1 and GAPDH by inserting the PCR products into a TA cloning vector (Invitrogen, San Diego, CA) and sequencing this insert [38, 39]. Competitive RT-polymerase chain reaction (PCR) fragments (mimics) were created by attaching the primer sequences of rabbit TGFb1 or GAPDH to the ends of a nonhomologous DNA fragment of a different size using a commercial kit (Clontech Inc., Palo Alto, CA) [40]. Serial dilution of a known quantity of the nonhomologous mimic fragments was coamplified with a constant amount of the cDNA sample [41]. This results in differential band intensities depending on which template is in excess (the target gene product cDNA or the nonhomologous fragments) in the reaction tube. The reaction products were run out on a 2% agarose gel (FMC BioProducts, Rock-
TABLE 1 Primer Sequences and PCR Cycle Information Primers
GAPDH
TGF-b1
Upper primer Lower primer Cycling
5-CCA TGT TCG TCA TGG GTG TGA ACC A-3 5-CAT GAG TCC TTC CAC GAT ACC AAA G-3 947C, 45 sec; 557C, 45 sec; 727C, 75 sec 25 cycles
5*-CTT CAG CTC CAC AGA GAA GAA CTG C-3 5*-CAC GAT CAT GTT GCA CAC TGC TCC-3* 947C, 45 sec; 557C, 45 sec; 727C, 90 sec 25 cycles
AID
JSR 5178
/
6n26$$$241
10-08-97 10:33:42
srga
164
JOURNAL OF SURGICAL RESEARCH: VOL. 72, NO. 2, OCTOBER 1997
FIG. 1. New granulation tissue formation. Four 6-mm-diameter ulcer wounds were made on each rabbit ear. The wounds on one ear were treated at the time of wounding with 17 mg/wound of rhM-CSF (single treatment); in one set of rabbits the same dose of rhM-CSF was applied on Days 0, 1, and 2 postwounding (multiple treatment). The contralateral ear treated was with vehicle and served as matched controls. The wounds were bisected at Day 7 postwounding and analyzed histologically. New granulation tissue formations were measured in all wounds.
FIG. 2. Reepithelialization and new granulation tissue formation under ischemic conditions. The rabbit ear was made ischemic and then three 6-mm ulcer wounds were created on each ear. One ear was treated with rhM-CSF and contralateral ear treated with vehicle. The wounds were harvested at Day 10 postwounding and new epithelium (NE) growth and granulation tissue growth (NGTV) were determined histologically. There is no difference between rhM-CSFtreated wounds and their matched controls.
land, ME). The gel was stained with ethidium bromide (Sigma, St. Louis, MO) and photographed under ultraviolet light. The photographs were then scanned and the band intensities were quantified using densitometry imaging (Imaging Densitometer GS-670, BioRad, Richmond, CA). The ratio of gene products to mimic was plotted against the known quantity of the mimic concentration. At a ratio of one, each curve gives the corresponding concentration of gene product of each cDNA. Results were confirmed by repeating experiments within the same cDNA, by another run of the same RNA extraction, and by using multiple rabbits. The level of the housekeeping gene GAPDH in each cDNA sample was determined using the same method. Two-tailed paired and unpaired Student’s t tests were used to compare TGF-b1 and GAPDH levels between control and rhM-CSF-treated wounds (Epistat Service). Quantitative PCR techniques have been criticized in the past due to the high degree of variability in the amplification process. The use of a competitive internal standard with homologous priming sequences, however, greatly reduces the intrinsic variation seen in other techniques [40, 41]. Competitive RT-PCR has proven to be both reproducible and sensitive in estimating mRNA expression and has been used to detect wound mRNA change for over three years [42, 43]. In order to limit the variability of RT-PCR, we use multiple rabbits and run two sets of reaction per sample, the relative ratio of the change seen in mRNA level was then averaged. Although competitive RT-PCR has limitations in the ability to quantify mRNA numbers, the ratios expressed here between treated and controls have been highly reproducible. In particular, GAPDH levels are consistent with 20% of a ratio of one, and TGF-b1 mRNA levels have been extremely reproducible and consistent with the in vitro study utilizing PCR and Northern blot [44, 45]. The small amounts of RNA available from the wounds have precluded methods other than RT-PCR.
thelialization from their paired controls (data not shown). However, new granulation tissue formation was significantly increased in wounds treated with rhM-CSF compared with matched controls on the opposite ear under the same conditions (Fig. 1). This is the first growth factor we have examined that has a stimulatory effect on granulation tissue only and not on epithelial regrowth. Under ischemic conditions, in which the tissue oxygen tension decreases from the nonischemic level of 55 to 25 mm Hg [32, Wu and Mustoe, unpublished data], rhM-CSF failed to promote both reepithelialization and new granulation tissue formation (Fig. 2). Histologic analysis of the wounds on Day 7 postwounding showed an increase of new granulation tissue formation in rhM-CSF-treated wounds compared with matched controls. More mononuclear cells, mixed with macrophages and fibroblasts as demonstrated by previous studies [34, 35], could be seen at the wound margins and in the new granulation tissue when compared with controls (Fig. 3). The wounds in rhM-CSFtreated animals showed an increase in cellularity compared with wounds treated with vehicle alone (P õ 0.01). No differences in wound cellularity were found under ischemic conditions between rhM-CSF treated wounds and their controls (Fig. 4). GAPDH levels in all samples were nearly equal between controls and rhM-CSF-treated wounds (Fig. 5a). To assure that GAPDH did not show significant variability among different wound samples, 22 cDNA sam-
RESULTS
Under nonischemic conditions, rhM-CSF-treated wounds did not show a significant difference in reepi-
FIG. 3. H&E stained slides (401). There is an increase in new granulation tissue in rhM-CSF-treated wounds (b) compared to their controls (a) and there are more mononuclear cells in rhM-CSF-treated wounds compared to vehicle-treated wounds.
AID
JSR 5178
/
6n26$$$241
10-08-97 10:33:42
srga
10-08-97 10:33:42
srga
166
JOURNAL OF SURGICAL RESEARCH: VOL. 72, NO. 2, OCTOBER 1997
GAPDH level between different wounds when the same technique was used [42, 43]. For the measurement of TGF-b1 levels, wounds from the eight rabbits, which included four nonischemic and four ischemic wounds, were harvested for total cellular RNA extraction. Two cDNA samples were reverse-transcribed from each RNA sample and TGF-b1 mRNA levels were measured by competitive RT-PCR (Fig. 6a). There was a mean 5.01-fold increase over controls in ulcers treated with a single dose of rhM-CSF, with a variable ranging from 3.1-fold to a 9.0-fold increase (5.01 { 0.865, P õ 0.00001, N Å 8). For the ischemic group, the 1.66-fold increase over control wounds found in rhM-CSF-treated wounds (1.662 { 0.215) was not statistically significant (P Å 0.12, N Å 8) (Fig. 6b). There was, however, a significant difference between the nonischemic and ischemic groups in upregulation of TGF-b1 mRNA levels (P Å 0.01). FIG. 4. Cellularity. There is an increase in cellularity in rhMCSF-treated wounds compared to controls with nonischemic wounds; no difference can be detected under ischemic conditions between rhM-CSF-treated wounds and controls. Single refers to single treatment with rhM-CSF at the time of wounding, and multiple refers to multiple treatment with rhM-CSF at the time of wounding, postwounding Day 1 and Day 2. Ischemic refers to ischemic ear wounds.
ples, five rabbits for nonischemic wounds and six rabbits for ischemic wounds, were scanned and analyzed; the range of variability was only 76 to 107% of the control GAPDH mRNA level (N Å 11). No statistical differences in their mRNA levels were found between rhM-CSF-treated wounds and their controls under both ischemic and nonischemic conditions (Fig. 5b). A 7.2% difference was shown when data were pooled and compared to each other (1.00 versus 0.928). This result was consistent with other studies showing a constant
DISCUSSION
Most tissue macrophages (80–90%) are derived from circulating monocytes, and it is accepted that these play an important role in wound healing [3–5, 11, 46]. Previous studies using GM-CSF have indicated that stimulation of macrophages even in nonimpaired wounds could augment healing substantially [11]. However, GM-CSF also affects neutrophils and mesenchymal cells [27, 47, 48], making it difficult to isolate the effects resulting solely from macrophage activation. In this study, M-CSF, which is known to act specifically on macrophages, caused a significant increase (37– 50%) in granulation tissue formation. The wound macrophages in this study were most likely maximally stimulated by the M-CSF based on in vitro dose response studies, and findings in a mouse study that showed similar effects using a broad range of doses
FIG. 5. Gel photo of GAPDH competitive RT-PCR and GAPDH mRNA level results. (a) GAPDH gel photo. The left side is a control wound and the right side is an rhM-CSF-treated wound. We used 100 bp DNA ladders. The mimic size should be 271 bp, and GAPDH product should be 149 bp. (b) GAPDH mRNA level results. Total of 11 paired randomly selected cDNA samples were examined. There was no difference in GAPDH mRNA levels between rhM-CSF-treated wounds and their matched controls.
AID
JSR 5178
/
6n26$$$241
10-08-97 10:33:42
srga
167
WU ET AL.: M-CSF AND WOUND HEALING
FIG. 6. Gel photo of TGF-b1 competitive RT-PCR and TGF-b1 mRNA level results. (a) The left side is control and the right side is rhM-CSF-treated wounds. 100 bp DNA ladders were used in this study and 6-fold mimic was skipped to get the best performance in competitive RT-PCR. The mimic length should be 426 bp and TGF-b1 should be 300 bp. (b) Control wounds are regarded as baseline and rhM-CSF significantly upregulates TGF-b1 mRNA levels under nonischemic conditions (5.01-fold). In contrast, it is only upregulated 1.66fold in TGF-b1 mRNA levels under ischemic conditions.
[49]. However, members from the major growth factor families (PDGF, TGF, and FGF) showed a substantially greater increase in wound healing in this model [29, 33, 42, 50, 51]. Therefore macrophage stimulation and activation, although important in wound healing, are not rate limiting, and further stimulation of mesenchymal tissue is possible. Chen et al. [52] found that M-CSF receptors mediate at least some of their intracellular effects through a phospholipase c pathway. It has also been documented that hypoxia significantly decreases phospholipase c activity on cell membranes [53]. Therefore, it is possible that the M-CSF receptor activity on macrophages may be decreased under ischemic conditions, which would explain why rhM-CSF failed to upregulate wound TGFb1 mRNA levels and failed to induce new granulation tissue formation in the ischemic wounds. Other possible explanations include an alternation in the M-CSF signal transduction pathway at another step, or more intriguing, a general depression of macrophage activation or activities under ischemic conditions. There is now substantial evidence that hypoxia is a significant event for cells rather than merely impacting oxidative metabolism [54]. In wound healing the reasons for impaired healing under ischemic conditions are undoubtedly complex, but this work suggests that on the cellular level, macrophage function plays a role in the pathogenesis of impaired wound healing. In conclusion, we have demonstrated that topical rhM-CSF treatment increases dermal ulcer wound healing in rabbits by activating and stimulating the wound macrophages. Low tissue oxygen tension diminishes the effect of rhM-CSF. This study indicates at cellular levels that tissue macrophages play an important role in wound healing and points out the possible pathogenesis of impaired wounds.
AID
JSR 5178
/
6n26$$$241
10-08-97 10:33:42
ACKNOWLEDGMENTS We thank Sharon Lang for her assistance in histology and Pat Lee for manuscript editing. We also thank Chiron Corp. for the supply of recombinant human M-CSF.
REFERENCES 1. Pierce, G. F., and Mustoe, T. A. Pharmacologic enhancement of wound healing. Annu. Rev. Med. 46: 467, 1995. 2. Clark, R. A. F. Wound Repair: Overview and general considerations. In R. A. F. Clark (Eds.), The Molecular and Cellular Biology of Wound Repair. New York, Plenum, 1995, Pp. 3–55. 3. Leibovich, S. J., and Ross, R. The role of the macrophage in wound repair. A study with hydrocortisone and antimacrophage serum. Am. J. Pathol. 78: 71, 1975. 4. Cohen, B. J., Danon, D., and Roth, G. S. Wound repair in mice as influenced by age and antimacrophage serum. J. Gerontol. 42: 295, 1987. 5. Danon, D., Kowatch, M. A., and Roth, G. S. Promotion of wound repair in old mice by local injection of macrophages. Proc. Natl. Acad. Sci. USA 86: 2018, 1989. 6. Rappolee, D. A., and Werb, Z. Macrophage-derived growth factors. Curr. Top. Microbiol. Immunol. 181: 87, 1992. 7. Mustoe, T. A., Weber, D. A., and Krukowski, M. Enhanced healing of cutaneous wounds in rats using beads with positively charged surfaces. Plast. Reconst. Surg. 89: 891, 1992. 8. Galiano, R. D., Jyung, R. W., Krukowski, M., and Mustoe, T. A. Efficacy of positively charged ion exchange beads in radiationimpaired models of wound healing. Ann. Plast. Surg. 36: 608, 1996. 9. Hatz, R. A., Kelley, S. F., and Ehrlich, H. P. The tetrachlorodecaoxygen complex reverses the effect of cortisone on wound healing. Plast. Reconst. Surg. 84: 953, 1989. 10. Pierce, G. F., Mustoe, T. A., Lingelbech, J., Masakowski, V. R., Gramates, P., and Deuel, T. F. Transforming growth factor beta reverses the glucorticoid-induced wound healing deficit in rats: Possible regulation in macrophages by platelet-derived growth factor. Proc. Natl. Acad. Sci. USA 86: 2229, 1989. 11. Jyung, R. W., Wu, L., Pierce, G. F., and Mustoe, T. A. Granulo-
srga
168
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
26.
27.
28.
AID
JOURNAL OF SURGICAL RESEARCH: VOL. 72, NO. 2, OCTOBER 1997 cyte-macrophage colony-stimulating factor and granulocyte colony-stimulating factor: Differential action on incisional wound healing. Surgery 115: 325, 1994. Stanley, E. R., and Heard, P. M. Factors regulating macrophage production and growth. Purification and some properties of the colony stimulating factor from medium conditioned by mouse L cells. J. Biol. Chem. 252: 4305, 1977. Das, S. K., and Stanley, E. R. Structure-function studies of a colony stimulating factor (CSF-1). J. Biol. Chem. 257: 13679, 1982. Stanley, E. R., Guilbert, L. J., Tushinski, R. J., and Bartelmez, S. H. CSF-1 a mononuclear phagocyte lineage-specific hemopoietic growth factor. J. Cell. Biochem. 21: 151, 1983. Tushinski, R. J., Oliver, I. T., Guilbert, L. J., Tynan, P. W., Warne, J. R., and Stanley, E. R. Survival of mononuclear phagocytes depends on a lineage-specific growth factor that the differentiated cells selectively destroy. Cell 28: 71, 1982. Bartocci, A., Mastrogiannis, D. S., Migliorati, G., Stockert, R. J., Wolkoff, A. W., and Stanley, E. R. Macrophages specifically regulate the concentration of their own growth factor in the circulation. Proc. Natl. Acad. Sci. USA 84: 6179, 1987. Hume, D. A., Pavli, P., Donahue, R. E., and Fidler, I. J. The effect of human recombinant macrophage colony-stimulating factor (CSF-1) on the murine mononuclear phagocyte system in vivo. J. Immunol. 141: 3405, 1988. Blazar, B. R., Aukerman, S. L., and Vallera, D. A. Effect of recombinant human macrophage colony-stimulating factor in irradiated murine recipients of T-cell-depleted allogeneic or nondepleted syngeneic bone marrow transplants. Blood 79: 1636, 1982. Khwaja, A., Yong, K., Jones, H. M., Chopra, R., McMillan, A. K., Goldstone, A. H., Patterson, K. G., Matheson, C., Ruthven, K., Abramson, S. B., et al. The effect of macrophage colony-stimulating factor on haemopoietic recovery after autologous bone marrow transplantation. Br. J. Haematol. 81: 288, 1992. Marks, Jr., S. C., and Lane, P. W. Osteopetrosis: A new recessive sketetal mutation on chromosome 12 of the mouse. J. Hered. 67: 11, 1976. Cecchini, M. G., Dominguez, M. G., Mocci, S., Wetterwald, A., Felix, R., Fleisch, H., Chisholm, O., Hofstetter, W., Pollard, J. W., and Stanley, E. R. Role of colony stimulating factor-1 in the establishment and regulation of tissue macrophages during postnatal development of the mouse. Development 120: 1357, 1994. Ralph, P., and Sampson-Johannes, A. Macrophage growth and stimulating factor, M-CSF. In P. Ralph and A. Sampson-Johannes (Eds.), Hematopoietic Growth Factor in Transfusion Medicine. New York: A. R. Liss, 1990. Pp. 43–63. Aukerman, S. L., Middleton, S., Sampson-Johannes, A., and Ralph, P. Biological and preclinical activity of macrophage colony-stimulating factor, M-CSF. In M. Symann, P. Qwesen, and G. Morstyn. (Eds.), Growth Factors and Interleukins: Biology and Clinical Applications. Macclesfield, England: Caldwell Communications, 1992. Pp. 79–93. Rosenfeld, M. E., Yla-Herttuala, S., Lipton, B. A., Ord, V. A., Witztum, J. L., and Steinberg, D. Macrophage colony-stimulating factor mRNA and protein in atherosclerotic kesuibs of rabbits and humans. Am. J. Pathol. 140: 291, 1992. Ford, H. R., Hoffman, R. A., Wing, E. J., Magee, D. M., McIntyre, L., and Simmons, R. L. Characterization of wound cytokines in the sponge matrix model. Arch. Surg. 124: 1422, 1989. Phillips, G. D., Aukerman, A. L., Whitehead, R. A., and Knighton, D. R. Macrophage colony-stimulating factor induces indirect angiogenesis in vivo. Wound Rep. Reg. 1: 3, 1993. Knighton, D. R., Hunt, T. K., Scheuenstuhl, H., Halliday, B. J., Werb, Z., and Banda, M. J. Oxygen tension regulates the expression of angiogenesis factor by macrophages. Science 221: 1283, 1983. Stromberg, K., Chapekar, M. S., Goldman, B. A., Chambers,
JSR 5178
/
6n26$$$241
10-08-97 10:33:42
29.
30.
31.
32.
33.
34.
35.
36.
37.
38.
39.
40.
41. 42.
43.
44.
45.
46.
47.
W. A., and Cavagnaro, J. A. Government affairs article: Regulatory concerns in the development of topical recombinant ophthalmic and cutaneous wound healing biologies. Wound Rep. Reg. 2: 155, 1994. Wu, L., Pierce, G. F., Ladin, D. A., Zhao, L. L., Rogers, D., and Mustoe, T. A. Effects of oxygen on wound response to growth factors: Kaposi’s FGF, but not basic FGF stimulates repair in ischemic wounds. Growth Factors 12: 29, 1995. Halenbeck, R., Kawasaki, E., Wrin, J., and Koths, K. Renaturation and purification of biologically active recombinant human macrophage colony-stimulating factor expression in E. coli. BioTechnology 7: 710, 1989. Ahn, S. T., and Mustoe, T. A. Effects of ischemia on ulcer wound healing: A new model in the rabbit ear. Ann. Plast. Surg. 24: 17, 1995. Siddiqui, A., Davidson, J. D., and Mustoe, T. A. Ischemic tissue oxygen capacitance after hyperbaric oxygen therapy: A new physiologic concept. Plast. Reconst. Surg. 99: 148, 1997. Mustoe, T. A., Pierce, G. F., Morishima, C., and Deuel, T. F. Growth factor-induced acceleration of tissue repair through direct and inductive activities in a rabbit dermal ulcer model. J. Clin. Invest. 87: 694, 1991. Pierce, G. F., Brown, G., and Mustoe, T. A. Quantitative analysis of inflammatory cell influx, procollagen type I synthesis and collagen cross-linking in incisional wounds: Influence of growth factor therapy. J. Lab. Clin. Med. 117: 373, 1991. Porras-Reyes, B. H., Blair, H. C., Jeffrey, J. J., and Mustoe, T. A. Collagenase production at the border of granulation issue in a healing wound: Macrophage and interstitial collagenase production in vivo. Connect. Tissue Res. 27: 63, 1991. Wu, L., Kumar, S. B., Porras-Reyes, B. H., Gramates, P. H., and Mustoe, T. A. Platele-derived growth factor-BB accelerates wound closure in a new mesentery culture model without macrophages. Ann. Plast. Surg. 33: 155, 1994. Chomczynski, P. A reagent for the single-step simultaneous isolation of RNA, DNA and proteins from cell and tissue samples. BioTechnology 15: 532, 1993. Byravan, S., Foster, L. M., Phan, T., Verity, A. N., and Campagnoni, A. T. Murine oligodendroglial cells express nerve growth factor. Proc. Natl. Acad. Sci. USA 91: 8812, 1994. Taglialatela, M., Wible, B. A., Caporaso, R., and Brown, A. M. Specification of pore properties by the carboxyl terminus of inwardly rectifying K/ channels. Science 264: 844, 1994. Siebert, P. D., and Larrick, J. W. PCR MIMICS: Competitive DNA fragments for use as internal standards in quantitative PCR. BioTechnology 14: 244, 1993. Siebert, P. D., and Larrick, J. W. Competitive PCR. Nature 359: 557, 1992. Wu, L., Siddiqui, A., Morris, D. E., Cox, D. A., Roth, S. I., and Mustoe, T. A. Transforming growth factor b3 (TGFb3) accelerates wound healing without alteration of scar prominence: Histologic and competitive RT-PCR studies. Arch. Surg. 132, 1997. Brucker, M. J., Gruskin, E., Farrell, C. L., Siddiqui, A., and Mustoe, T. A. Differential expression of PDGF receptor in an aging model of wound repair. Wound Rep. Reg. 4: 219, 1996. Siddiqui, A., Galiano, R. D., Connors, D., Gruskin, E., Wu, L., and Mustoe, T. A. Differential effects of oxygen on human dermal fibroblasts: Acute versus chronic hypoxia. Wound Rep. Reg. 4: 211, 1996. Falanga, V., Qian, S. W., Danielpour, D., Katz, M. H., Roberts, A. B., and Sporn, M. B. Hypoxia upregulates the synthesis of TGF-beta 1 by human dermal fibroblasts. J. Invest. Dermatol. 97: 634, 1991. Brower, W., Williams, D., Lucore, P., Pretus, H., Jones, E., and McNamee, R. Effect of enhanced macrophage function on early wound healing. Surgery 104: 224, 1988. Vaillant, P., Muller, V., Martinet, Y., and Martinet, N. Human granulocyte- and granulocyte macrophage-colony stimulating
srga
WU ET AL.: M-CSF AND WOUND HEALING factors are chemotactic and ‘‘competence’’ growth factors for human mesenchymal cells. Biochem. Biophy. Res. Commun. 192: 879, 1993. 48. Bober, L. A., Grace, M. J., Pugliese-Sivo, C., Rojas-Triana, A., Waters, T., Sullivan, L. M., and Narula, S. K. The effect of GMCSF and G-CSF on human neutrophil function. Immunopharmacology 29: 111, 1995. 49. Middleton, S. C., and Aukerman, S. L. The effects of macrophage colony stimulating factor (M-CSF) on wound healing in normal and genetically diabetic (db/db) mice (abstract). J. Cell. Biochem. 15F: 197, 1991. 50. Zhao, L. L., Davidson, J. D., Wee, S. C., Roth, S. I., and Mustoe, T. A. Effect of hyperbaric oxygen and growth factors on rabbit ear ischemic ulcers. Arch. Surg. 29: 1043, 1994.
AID
JSR 5178
/
6n26$$$241
10-08-97 10:33:42
169
51. Wu, L., Pierce, G. F., Galiano, R. D., and Mustoe, T. A. KGF induces granulation tissue in ischemic dermal wounds: The importance of epithelial-mesenchymal cell interaction. Arch. Surg. 131: 660, 1996. 52. Chen, B. D-M., Chou, T. H., and Sensenbrenner, L. Downregulation of M-CSF receptors by lipopolysaccharide in murine peritoneal exudate macrophages is mediated through a phospholipase C dependent pathway. Exp. Hematol. 21: 623, 1993. 53. Schwertz, D. W., and Halverson, J. Changes in phosphoinositide-specific phospholipase C and phospholipase A2 activity in ischemic and reperfused rat heart. Basic Res. Cardiol. 87: 113, 1992. 54. Bunn, H. F., and Poyton, R. O. Oxygen sensing and molecular adaptation to hypoxia [Review]. Physiol. Rev. 76: 839, 1996.
srga
72, 162–169 (1997)
JR975178
Macrophage Colony-Stimulating Factor Accelerates Wound Healing and Upregulates TGF-b1 mRNA Levels through Tissue Macrophages1 Liancun Wu, M.D.,*,2 Yung L. Yu, M.D.,‡ Robert D. Galiano, M.D.,* Sanford I. Roth, M.D.,† and Thomas A. Mustoe, M.D., FACS*,3 *Division of Plastic Surgery and †Department of Pathology, Northwestern University Medical School, Chicago, Illinois 60611; and ‡Department of Surgery, The Chicago Medical School, Chicago, Illinois 60608 Submitted for publication February 27, 1997
Macrophage colony-stimulating factor (M-CSF) is produced by many cell types involved in wound repair, yet it acts specifically on monocytes and macrophages. The monocyte-derived cell is thought to be important in wound healing, but the importance of the role of tissue macrophages in wound healing has not been well defined. Dermal ulcers were created in normal and ischemic ears of young rabbits. Either rhM-CSF (17 m g/wound) or buffer was applied to each wound. Wounds were bisected and analyzed histologically at Days 7 and 10 postwounding. The amounts of epithelial growth and granulation tissue deposition were measured in all wounds. The level of increase of TGF- b1 mRNA level in M-CSFtreated wounds was examined using competitive RT-PCR. M-CSF increased new granulation tissue formation by 37% (N Å 21, P õ 0.01) and 50% (P õ 0.01) after single and multiple treatments, respectively, in nonischemic wounds. TGF- b1 mRNA levels in rhM-CSF-treated wounds increased 5.01-fold (N Å 8) over vehicle-treated wounds under nonischemic conditions. In contrast, no effect could be detected in ischemic wounds treated with rhM-CSF, and these wounds only showed a 1.66-fold increase in TGF- b 1 mRNA levels when compared to ischemic wounds treated with vehicle alone. GAPDH, a housekeeping gene, showed no change. As mesenchymal cells lack receptors for M-CSF, the improved healing of wounds treated with topical rhM-CSF must reflect a generalized enhancement of activation and function of tissue macrophages, as demonstrated by upregulation of TGF- b . The lack of effect under ischemic conditions suggests that either macrophage activity and/or response to M-CSF is ad1 This work was supported in part by NIH Grant GM 41303 and was presented at the Fifth Wound Healing Society Meeting at Minneapolis, MN, April 1995. Recombinant human M-CSF was supplied by Chiron Corporation, Emeryville, CA. 2 Current address: Department of Anesthesiology, Medical College of Virginia Hospital, Richmond, VA 23220. 3 To whom correspondence and reprint requests should be addressed at Division of Plastic Surgery, Northwestern University Medical School, 707 N. Fairbanks Court, Suite 811, Chicago, IL 60611-3008.
0022-4804/97 $25.00 Copyright q 1997 by Academic Press All rights of reproduction in any form reserved.
AID
JSR 5178
/
6n26$$$241
versely affected under those conditions; this may suggest the pathogenesis of impaired wound healing at the cellular level. q 1997 Academic Press
INTRODUCTION
Many peptide growth factors have been shown to increase wound healing under normal and impaired conditions [1], but none of these stimulators has been accepted as a pharmacological adjunct for the treatment of acute and chronic wounds. One of the reasons may be that wound healing is an extremely complex process involving the interaction of numerous cell types, multiple growth factors, and the extracellular matrix present within the wound at the cellular and molecular levels. Although the precise molecular and cellular events in wound healing are under active investigation [2], it is generally recognized that the wound macrophage may be a major player in wound repair, with several studies suggesting a critical role for macrophages throughout wound healing [3–5]. None of these studies, however, has elucidated the precise role of local tissue macrophages in wound healing versus bloodderived monocytes. Activated macrophages produce over 100 different growth factors and interleukins [6] and more than 20 of these have been shown to be involved in wound healing [1]. Therefore, one strategy to pharmacologically enhance repair is by attracting and/or activating tissue macrophages to subsequently release a physiological combination of growth factors. Possible macrophage stimulators include charged beads [7–8], macrophageactivating substance [9], and growth factors such as platelet-derived growth factor type BB (PDGF-BB) and granulocyte-macrophage colony-stimulating factor (GM-CSF) [10, 11]. Macrophage colony-stimulating factor (M-CSF), also known as CSF-1, is a glycoprotein [12, 13] which selectively stimulates the survival, proliferation, and activation of macrophages [14, 15]. A biologically active M-CSF can be detected in the circulation [16] and intravenous administration of recombinant human M-CSF (rhM-CSF) in mice elevates the
162
10-08-97 10:33:42
srga
163
WU ET AL.: M-CSF AND WOUND HEALING
concentration of tissue macrophages [17] and stimulates the recovery of radiation-impaired and autologous bone marrow transplant recipients [18, 19]. The osteopetrotic mutant mouse, a spontaneously occurring M-CSF gene knockout [20], is deficient in circulating monocytes and tissue macrophages [21]. In this mutant murine model, tissue macrophages are absent and dermal development is impaired. This finding suggests that the tissue macrophage subpopulation requires MCSF for development. While the role of M-CSF in wound repair has not been well studied, several observations suggest that it may be an important growth factor to study. M-CSF is produced by many cell types involved in wound repair including fibroblasts, myoblasts, endothelial cells, keratinocytes, and vascular smooth muscle cells [22–24]. M-CSF has been detected in the wound fluid during the early phase of wound healing at significantly higher levels compared to the basal serum level in unwounded animals [25]. Furthermore, rhM-CSF has been shown to indirectly stimulate angiogenesis [26]. In addition, it has been documented both in vivo and in vitro that hypoxia induces angiogenic factors and increases neovascularization [27], but the response of macrophages to growth factors in ischemic wound healing has not been fully investigated. It is crucial to evaluate the behavior of a potential vulnerary agent such as M-CSF in clinically pertinent models of impaired wound healing [28]. Previous studies from our laboratory have revealed a difference in the behavior of growth factors under ischemic conditions [29]. The behavior of other growth factors such as M-CSF in the same model merits further investigation. MATERIALS AND METHODS Recombinant human macrophage colony-stimulating factor, supplied by Chiron Corporation (Emeryville, CA), was expressed in E. coli and purified as previously described [30]. The rhM-CSF was then suspended in phosphate-buffered saline solution (PBS) at a concentration of 1.7 mg/ml and was assayed for endotoxin and biological activity prior to use. Eighteen young adult female, New Zealand white rabbits (New Franken Inc., New Franken, WI), weighing 2.7–3.4 kg, were acclimated and housed individually under standard conditions. After they were anesthetized with ketamine (60 mg/kg) and xylazine (5 mg/kg), the surgical procedures were performed under sterile conditions as previously described [29, 31]. This procedure was approved by the Animal Care Committee of Northwestern University (Chicago, IL). Briefly, four 6 mm diameter full-thickness circular wounds were made down to bare cartilage in nonischemic wounds. For the ischemic model, three wounds were made on each ear after two of the
three arteries that supplied the rabbit ear were divided. This ischemic procedures decrease the ear tissue oxygen tension by 50% (55 { 3 mm Hg for normal tissue, 25 { 3 mm Hg for ischemic ear) and impairs the new granulation tissue deposition more than 80% at Day 7 postwounding [29, 31, 32, and unpublished data]. The wounds on one ear were treated at the time of wounding with 17 mg/wound of rhM-CSF; in one set of rabbits the same dose of rhM-CSF was applied on Days 0, 1, and 2 postwounding. The contralateral ear served as a paired control and was treated with the same amount of vehicle alone (PBS). All the wounds were covered with an occlusive polyurethane dressing (Tegaderm, 3M, Minneapolis, MN). The wounds were bisected and analyzed histologically at 7 days postwounding for the nonischemic groups and at 10 days postwounding for the ischemic groups as previously described [29, 33]. Two independent histological analyses were performed on each sample with the observers blinded to treatment. Reepithelialization and new granulation tissue formation were measured based on minimal contraction of these ulcers [33]. The wounds were harvested in a paired fashion, and the paired Student’s t-test (two-tailed) was used to compare the results (Epistat Service, Richardson, TX). Cellular infiltration into the wound consisting mainly of macrophages and proliferative fibroblasts [34, 35] in the hematoxylin and eosin stained slides was rated on a scale of 0–4 (0 for lowest cellularity, 4 for highest cellularity) in a blinded fashion using a Nikon microscope as previously described [36]. Differences in cellularity were compared using the Wilcoxson rank test (Epistat Service). The wound granulation tissue from each rabbit was harvested with a 7-mm biopsy punch under the operating microscope after each ulcer was bisected for histological analysis. The harvested tissue was immediately placed into liquid nitrogen. The total cellular RNA from each ear’s wounds (which consisted of three pooled wounds for the ischemic model and four pooled wounds for the nonischemic model) was extracted with a guanidine thiocyanate/phenol-based reagent (TRI Reagents, Cincinnati, OH) as previously described [37]. All reverse transcription (RT) reactions were performed simultaneously using a master mix to eliminate variability of reverse transcriptase efficiency between different reaction tubes. Acceptable purity was determined spectrophotometrically (Pharmacia LKB, Cambridge, England), and only RNA samples having an A260/A280 absorbence ratio greater than 1.8 were utilized. Five micrograms of total RNA was then converted to cDNA by using Moloney murine leukemia virus reverse transcriptase (M-MLV RT) and random primers (Gibco BRL, Grand Island, NY). Specific PCR primers for rabbit glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and TGF-b1 were designed based on the conserved complete and partial cDNA sequences of various species published in GenBank (Table 1). The PCR products were confirmed as rabbit TGF-b1 and GAPDH by inserting the PCR products into a TA cloning vector (Invitrogen, San Diego, CA) and sequencing this insert [38, 39]. Competitive RT-polymerase chain reaction (PCR) fragments (mimics) were created by attaching the primer sequences of rabbit TGFb1 or GAPDH to the ends of a nonhomologous DNA fragment of a different size using a commercial kit (Clontech Inc., Palo Alto, CA) [40]. Serial dilution of a known quantity of the nonhomologous mimic fragments was coamplified with a constant amount of the cDNA sample [41]. This results in differential band intensities depending on which template is in excess (the target gene product cDNA or the nonhomologous fragments) in the reaction tube. The reaction products were run out on a 2% agarose gel (FMC BioProducts, Rock-
TABLE 1 Primer Sequences and PCR Cycle Information Primers
GAPDH
TGF-b1
Upper primer Lower primer Cycling
5-CCA TGT TCG TCA TGG GTG TGA ACC A-3 5-CAT GAG TCC TTC CAC GAT ACC AAA G-3 947C, 45 sec; 557C, 45 sec; 727C, 75 sec 25 cycles
5*-CTT CAG CTC CAC AGA GAA GAA CTG C-3 5*-CAC GAT CAT GTT GCA CAC TGC TCC-3* 947C, 45 sec; 557C, 45 sec; 727C, 90 sec 25 cycles
AID
JSR 5178
/
6n26$$$241
10-08-97 10:33:42
srga
164
JOURNAL OF SURGICAL RESEARCH: VOL. 72, NO. 2, OCTOBER 1997
FIG. 1. New granulation tissue formation. Four 6-mm-diameter ulcer wounds were made on each rabbit ear. The wounds on one ear were treated at the time of wounding with 17 mg/wound of rhM-CSF (single treatment); in one set of rabbits the same dose of rhM-CSF was applied on Days 0, 1, and 2 postwounding (multiple treatment). The contralateral ear treated was with vehicle and served as matched controls. The wounds were bisected at Day 7 postwounding and analyzed histologically. New granulation tissue formations were measured in all wounds.
FIG. 2. Reepithelialization and new granulation tissue formation under ischemic conditions. The rabbit ear was made ischemic and then three 6-mm ulcer wounds were created on each ear. One ear was treated with rhM-CSF and contralateral ear treated with vehicle. The wounds were harvested at Day 10 postwounding and new epithelium (NE) growth and granulation tissue growth (NGTV) were determined histologically. There is no difference between rhM-CSFtreated wounds and their matched controls.
land, ME). The gel was stained with ethidium bromide (Sigma, St. Louis, MO) and photographed under ultraviolet light. The photographs were then scanned and the band intensities were quantified using densitometry imaging (Imaging Densitometer GS-670, BioRad, Richmond, CA). The ratio of gene products to mimic was plotted against the known quantity of the mimic concentration. At a ratio of one, each curve gives the corresponding concentration of gene product of each cDNA. Results were confirmed by repeating experiments within the same cDNA, by another run of the same RNA extraction, and by using multiple rabbits. The level of the housekeeping gene GAPDH in each cDNA sample was determined using the same method. Two-tailed paired and unpaired Student’s t tests were used to compare TGF-b1 and GAPDH levels between control and rhM-CSF-treated wounds (Epistat Service). Quantitative PCR techniques have been criticized in the past due to the high degree of variability in the amplification process. The use of a competitive internal standard with homologous priming sequences, however, greatly reduces the intrinsic variation seen in other techniques [40, 41]. Competitive RT-PCR has proven to be both reproducible and sensitive in estimating mRNA expression and has been used to detect wound mRNA change for over three years [42, 43]. In order to limit the variability of RT-PCR, we use multiple rabbits and run two sets of reaction per sample, the relative ratio of the change seen in mRNA level was then averaged. Although competitive RT-PCR has limitations in the ability to quantify mRNA numbers, the ratios expressed here between treated and controls have been highly reproducible. In particular, GAPDH levels are consistent with 20% of a ratio of one, and TGF-b1 mRNA levels have been extremely reproducible and consistent with the in vitro study utilizing PCR and Northern blot [44, 45]. The small amounts of RNA available from the wounds have precluded methods other than RT-PCR.
thelialization from their paired controls (data not shown). However, new granulation tissue formation was significantly increased in wounds treated with rhM-CSF compared with matched controls on the opposite ear under the same conditions (Fig. 1). This is the first growth factor we have examined that has a stimulatory effect on granulation tissue only and not on epithelial regrowth. Under ischemic conditions, in which the tissue oxygen tension decreases from the nonischemic level of 55 to 25 mm Hg [32, Wu and Mustoe, unpublished data], rhM-CSF failed to promote both reepithelialization and new granulation tissue formation (Fig. 2). Histologic analysis of the wounds on Day 7 postwounding showed an increase of new granulation tissue formation in rhM-CSF-treated wounds compared with matched controls. More mononuclear cells, mixed with macrophages and fibroblasts as demonstrated by previous studies [34, 35], could be seen at the wound margins and in the new granulation tissue when compared with controls (Fig. 3). The wounds in rhM-CSFtreated animals showed an increase in cellularity compared with wounds treated with vehicle alone (P õ 0.01). No differences in wound cellularity were found under ischemic conditions between rhM-CSF treated wounds and their controls (Fig. 4). GAPDH levels in all samples were nearly equal between controls and rhM-CSF-treated wounds (Fig. 5a). To assure that GAPDH did not show significant variability among different wound samples, 22 cDNA sam-
RESULTS
Under nonischemic conditions, rhM-CSF-treated wounds did not show a significant difference in reepi-
FIG. 3. H&E stained slides (401). There is an increase in new granulation tissue in rhM-CSF-treated wounds (b) compared to their controls (a) and there are more mononuclear cells in rhM-CSF-treated wounds compared to vehicle-treated wounds.
AID
JSR 5178
/
6n26$$$241
10-08-97 10:33:42
srga
10-08-97 10:33:42
srga
166
JOURNAL OF SURGICAL RESEARCH: VOL. 72, NO. 2, OCTOBER 1997
GAPDH level between different wounds when the same technique was used [42, 43]. For the measurement of TGF-b1 levels, wounds from the eight rabbits, which included four nonischemic and four ischemic wounds, were harvested for total cellular RNA extraction. Two cDNA samples were reverse-transcribed from each RNA sample and TGF-b1 mRNA levels were measured by competitive RT-PCR (Fig. 6a). There was a mean 5.01-fold increase over controls in ulcers treated with a single dose of rhM-CSF, with a variable ranging from 3.1-fold to a 9.0-fold increase (5.01 { 0.865, P õ 0.00001, N Å 8). For the ischemic group, the 1.66-fold increase over control wounds found in rhM-CSF-treated wounds (1.662 { 0.215) was not statistically significant (P Å 0.12, N Å 8) (Fig. 6b). There was, however, a significant difference between the nonischemic and ischemic groups in upregulation of TGF-b1 mRNA levels (P Å 0.01). FIG. 4. Cellularity. There is an increase in cellularity in rhMCSF-treated wounds compared to controls with nonischemic wounds; no difference can be detected under ischemic conditions between rhM-CSF-treated wounds and controls. Single refers to single treatment with rhM-CSF at the time of wounding, and multiple refers to multiple treatment with rhM-CSF at the time of wounding, postwounding Day 1 and Day 2. Ischemic refers to ischemic ear wounds.
ples, five rabbits for nonischemic wounds and six rabbits for ischemic wounds, were scanned and analyzed; the range of variability was only 76 to 107% of the control GAPDH mRNA level (N Å 11). No statistical differences in their mRNA levels were found between rhM-CSF-treated wounds and their controls under both ischemic and nonischemic conditions (Fig. 5b). A 7.2% difference was shown when data were pooled and compared to each other (1.00 versus 0.928). This result was consistent with other studies showing a constant
DISCUSSION
Most tissue macrophages (80–90%) are derived from circulating monocytes, and it is accepted that these play an important role in wound healing [3–5, 11, 46]. Previous studies using GM-CSF have indicated that stimulation of macrophages even in nonimpaired wounds could augment healing substantially [11]. However, GM-CSF also affects neutrophils and mesenchymal cells [27, 47, 48], making it difficult to isolate the effects resulting solely from macrophage activation. In this study, M-CSF, which is known to act specifically on macrophages, caused a significant increase (37– 50%) in granulation tissue formation. The wound macrophages in this study were most likely maximally stimulated by the M-CSF based on in vitro dose response studies, and findings in a mouse study that showed similar effects using a broad range of doses
FIG. 5. Gel photo of GAPDH competitive RT-PCR and GAPDH mRNA level results. (a) GAPDH gel photo. The left side is a control wound and the right side is an rhM-CSF-treated wound. We used 100 bp DNA ladders. The mimic size should be 271 bp, and GAPDH product should be 149 bp. (b) GAPDH mRNA level results. Total of 11 paired randomly selected cDNA samples were examined. There was no difference in GAPDH mRNA levels between rhM-CSF-treated wounds and their matched controls.
AID
JSR 5178
/
6n26$$$241
10-08-97 10:33:42
srga
167
WU ET AL.: M-CSF AND WOUND HEALING
FIG. 6. Gel photo of TGF-b1 competitive RT-PCR and TGF-b1 mRNA level results. (a) The left side is control and the right side is rhM-CSF-treated wounds. 100 bp DNA ladders were used in this study and 6-fold mimic was skipped to get the best performance in competitive RT-PCR. The mimic length should be 426 bp and TGF-b1 should be 300 bp. (b) Control wounds are regarded as baseline and rhM-CSF significantly upregulates TGF-b1 mRNA levels under nonischemic conditions (5.01-fold). In contrast, it is only upregulated 1.66fold in TGF-b1 mRNA levels under ischemic conditions.
[49]. However, members from the major growth factor families (PDGF, TGF, and FGF) showed a substantially greater increase in wound healing in this model [29, 33, 42, 50, 51]. Therefore macrophage stimulation and activation, although important in wound healing, are not rate limiting, and further stimulation of mesenchymal tissue is possible. Chen et al. [52] found that M-CSF receptors mediate at least some of their intracellular effects through a phospholipase c pathway. It has also been documented that hypoxia significantly decreases phospholipase c activity on cell membranes [53]. Therefore, it is possible that the M-CSF receptor activity on macrophages may be decreased under ischemic conditions, which would explain why rhM-CSF failed to upregulate wound TGFb1 mRNA levels and failed to induce new granulation tissue formation in the ischemic wounds. Other possible explanations include an alternation in the M-CSF signal transduction pathway at another step, or more intriguing, a general depression of macrophage activation or activities under ischemic conditions. There is now substantial evidence that hypoxia is a significant event for cells rather than merely impacting oxidative metabolism [54]. In wound healing the reasons for impaired healing under ischemic conditions are undoubtedly complex, but this work suggests that on the cellular level, macrophage function plays a role in the pathogenesis of impaired wound healing. In conclusion, we have demonstrated that topical rhM-CSF treatment increases dermal ulcer wound healing in rabbits by activating and stimulating the wound macrophages. Low tissue oxygen tension diminishes the effect of rhM-CSF. This study indicates at cellular levels that tissue macrophages play an important role in wound healing and points out the possible pathogenesis of impaired wounds.
AID
JSR 5178
/
6n26$$$241
10-08-97 10:33:42
ACKNOWLEDGMENTS We thank Sharon Lang for her assistance in histology and Pat Lee for manuscript editing. We also thank Chiron Corp. for the supply of recombinant human M-CSF.
REFERENCES 1. Pierce, G. F., and Mustoe, T. A. Pharmacologic enhancement of wound healing. Annu. Rev. Med. 46: 467, 1995. 2. Clark, R. A. F. Wound Repair: Overview and general considerations. In R. A. F. Clark (Eds.), The Molecular and Cellular Biology of Wound Repair. New York, Plenum, 1995, Pp. 3–55. 3. Leibovich, S. J., and Ross, R. The role of the macrophage in wound repair. A study with hydrocortisone and antimacrophage serum. Am. J. Pathol. 78: 71, 1975. 4. Cohen, B. J., Danon, D., and Roth, G. S. Wound repair in mice as influenced by age and antimacrophage serum. J. Gerontol. 42: 295, 1987. 5. Danon, D., Kowatch, M. A., and Roth, G. S. Promotion of wound repair in old mice by local injection of macrophages. Proc. Natl. Acad. Sci. USA 86: 2018, 1989. 6. Rappolee, D. A., and Werb, Z. Macrophage-derived growth factors. Curr. Top. Microbiol. Immunol. 181: 87, 1992. 7. Mustoe, T. A., Weber, D. A., and Krukowski, M. Enhanced healing of cutaneous wounds in rats using beads with positively charged surfaces. Plast. Reconst. Surg. 89: 891, 1992. 8. Galiano, R. D., Jyung, R. W., Krukowski, M., and Mustoe, T. A. Efficacy of positively charged ion exchange beads in radiationimpaired models of wound healing. Ann. Plast. Surg. 36: 608, 1996. 9. Hatz, R. A., Kelley, S. F., and Ehrlich, H. P. The tetrachlorodecaoxygen complex reverses the effect of cortisone on wound healing. Plast. Reconst. Surg. 84: 953, 1989. 10. Pierce, G. F., Mustoe, T. A., Lingelbech, J., Masakowski, V. R., Gramates, P., and Deuel, T. F. Transforming growth factor beta reverses the glucorticoid-induced wound healing deficit in rats: Possible regulation in macrophages by platelet-derived growth factor. Proc. Natl. Acad. Sci. USA 86: 2229, 1989. 11. Jyung, R. W., Wu, L., Pierce, G. F., and Mustoe, T. A. Granulo-
srga
168
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
26.
27.
28.
AID
JOURNAL OF SURGICAL RESEARCH: VOL. 72, NO. 2, OCTOBER 1997 cyte-macrophage colony-stimulating factor and granulocyte colony-stimulating factor: Differential action on incisional wound healing. Surgery 115: 325, 1994. Stanley, E. R., and Heard, P. M. Factors regulating macrophage production and growth. Purification and some properties of the colony stimulating factor from medium conditioned by mouse L cells. J. Biol. Chem. 252: 4305, 1977. Das, S. K., and Stanley, E. R. Structure-function studies of a colony stimulating factor (CSF-1). J. Biol. Chem. 257: 13679, 1982. Stanley, E. R., Guilbert, L. J., Tushinski, R. J., and Bartelmez, S. H. CSF-1 a mononuclear phagocyte lineage-specific hemopoietic growth factor. J. Cell. Biochem. 21: 151, 1983. Tushinski, R. J., Oliver, I. T., Guilbert, L. J., Tynan, P. W., Warne, J. R., and Stanley, E. R. Survival of mononuclear phagocytes depends on a lineage-specific growth factor that the differentiated cells selectively destroy. Cell 28: 71, 1982. Bartocci, A., Mastrogiannis, D. S., Migliorati, G., Stockert, R. J., Wolkoff, A. W., and Stanley, E. R. Macrophages specifically regulate the concentration of their own growth factor in the circulation. Proc. Natl. Acad. Sci. USA 84: 6179, 1987. Hume, D. A., Pavli, P., Donahue, R. E., and Fidler, I. J. The effect of human recombinant macrophage colony-stimulating factor (CSF-1) on the murine mononuclear phagocyte system in vivo. J. Immunol. 141: 3405, 1988. Blazar, B. R., Aukerman, S. L., and Vallera, D. A. Effect of recombinant human macrophage colony-stimulating factor in irradiated murine recipients of T-cell-depleted allogeneic or nondepleted syngeneic bone marrow transplants. Blood 79: 1636, 1982. Khwaja, A., Yong, K., Jones, H. M., Chopra, R., McMillan, A. K., Goldstone, A. H., Patterson, K. G., Matheson, C., Ruthven, K., Abramson, S. B., et al. The effect of macrophage colony-stimulating factor on haemopoietic recovery after autologous bone marrow transplantation. Br. J. Haematol. 81: 288, 1992. Marks, Jr., S. C., and Lane, P. W. Osteopetrosis: A new recessive sketetal mutation on chromosome 12 of the mouse. J. Hered. 67: 11, 1976. Cecchini, M. G., Dominguez, M. G., Mocci, S., Wetterwald, A., Felix, R., Fleisch, H., Chisholm, O., Hofstetter, W., Pollard, J. W., and Stanley, E. R. Role of colony stimulating factor-1 in the establishment and regulation of tissue macrophages during postnatal development of the mouse. Development 120: 1357, 1994. Ralph, P., and Sampson-Johannes, A. Macrophage growth and stimulating factor, M-CSF. In P. Ralph and A. Sampson-Johannes (Eds.), Hematopoietic Growth Factor in Transfusion Medicine. New York: A. R. Liss, 1990. Pp. 43–63. Aukerman, S. L., Middleton, S., Sampson-Johannes, A., and Ralph, P. Biological and preclinical activity of macrophage colony-stimulating factor, M-CSF. In M. Symann, P. Qwesen, and G. Morstyn. (Eds.), Growth Factors and Interleukins: Biology and Clinical Applications. Macclesfield, England: Caldwell Communications, 1992. Pp. 79–93. Rosenfeld, M. E., Yla-Herttuala, S., Lipton, B. A., Ord, V. A., Witztum, J. L., and Steinberg, D. Macrophage colony-stimulating factor mRNA and protein in atherosclerotic kesuibs of rabbits and humans. Am. J. Pathol. 140: 291, 1992. Ford, H. R., Hoffman, R. A., Wing, E. J., Magee, D. M., McIntyre, L., and Simmons, R. L. Characterization of wound cytokines in the sponge matrix model. Arch. Surg. 124: 1422, 1989. Phillips, G. D., Aukerman, A. L., Whitehead, R. A., and Knighton, D. R. Macrophage colony-stimulating factor induces indirect angiogenesis in vivo. Wound Rep. Reg. 1: 3, 1993. Knighton, D. R., Hunt, T. K., Scheuenstuhl, H., Halliday, B. J., Werb, Z., and Banda, M. J. Oxygen tension regulates the expression of angiogenesis factor by macrophages. Science 221: 1283, 1983. Stromberg, K., Chapekar, M. S., Goldman, B. A., Chambers,
JSR 5178
/
6n26$$$241
10-08-97 10:33:42
29.
30.
31.
32.
33.
34.
35.
36.
37.
38.
39.
40.
41. 42.
43.
44.
45.
46.
47.
W. A., and Cavagnaro, J. A. Government affairs article: Regulatory concerns in the development of topical recombinant ophthalmic and cutaneous wound healing biologies. Wound Rep. Reg. 2: 155, 1994. Wu, L., Pierce, G. F., Ladin, D. A., Zhao, L. L., Rogers, D., and Mustoe, T. A. Effects of oxygen on wound response to growth factors: Kaposi’s FGF, but not basic FGF stimulates repair in ischemic wounds. Growth Factors 12: 29, 1995. Halenbeck, R., Kawasaki, E., Wrin, J., and Koths, K. Renaturation and purification of biologically active recombinant human macrophage colony-stimulating factor expression in E. coli. BioTechnology 7: 710, 1989. Ahn, S. T., and Mustoe, T. A. Effects of ischemia on ulcer wound healing: A new model in the rabbit ear. Ann. Plast. Surg. 24: 17, 1995. Siddiqui, A., Davidson, J. D., and Mustoe, T. A. Ischemic tissue oxygen capacitance after hyperbaric oxygen therapy: A new physiologic concept. Plast. Reconst. Surg. 99: 148, 1997. Mustoe, T. A., Pierce, G. F., Morishima, C., and Deuel, T. F. Growth factor-induced acceleration of tissue repair through direct and inductive activities in a rabbit dermal ulcer model. J. Clin. Invest. 87: 694, 1991. Pierce, G. F., Brown, G., and Mustoe, T. A. Quantitative analysis of inflammatory cell influx, procollagen type I synthesis and collagen cross-linking in incisional wounds: Influence of growth factor therapy. J. Lab. Clin. Med. 117: 373, 1991. Porras-Reyes, B. H., Blair, H. C., Jeffrey, J. J., and Mustoe, T. A. Collagenase production at the border of granulation issue in a healing wound: Macrophage and interstitial collagenase production in vivo. Connect. Tissue Res. 27: 63, 1991. Wu, L., Kumar, S. B., Porras-Reyes, B. H., Gramates, P. H., and Mustoe, T. A. Platele-derived growth factor-BB accelerates wound closure in a new mesentery culture model without macrophages. Ann. Plast. Surg. 33: 155, 1994. Chomczynski, P. A reagent for the single-step simultaneous isolation of RNA, DNA and proteins from cell and tissue samples. BioTechnology 15: 532, 1993. Byravan, S., Foster, L. M., Phan, T., Verity, A. N., and Campagnoni, A. T. Murine oligodendroglial cells express nerve growth factor. Proc. Natl. Acad. Sci. USA 91: 8812, 1994. Taglialatela, M., Wible, B. A., Caporaso, R., and Brown, A. M. Specification of pore properties by the carboxyl terminus of inwardly rectifying K/ channels. Science 264: 844, 1994. Siebert, P. D., and Larrick, J. W. PCR MIMICS: Competitive DNA fragments for use as internal standards in quantitative PCR. BioTechnology 14: 244, 1993. Siebert, P. D., and Larrick, J. W. Competitive PCR. Nature 359: 557, 1992. Wu, L., Siddiqui, A., Morris, D. E., Cox, D. A., Roth, S. I., and Mustoe, T. A. Transforming growth factor b3 (TGFb3) accelerates wound healing without alteration of scar prominence: Histologic and competitive RT-PCR studies. Arch. Surg. 132, 1997. Brucker, M. J., Gruskin, E., Farrell, C. L., Siddiqui, A., and Mustoe, T. A. Differential expression of PDGF receptor in an aging model of wound repair. Wound Rep. Reg. 4: 219, 1996. Siddiqui, A., Galiano, R. D., Connors, D., Gruskin, E., Wu, L., and Mustoe, T. A. Differential effects of oxygen on human dermal fibroblasts: Acute versus chronic hypoxia. Wound Rep. Reg. 4: 211, 1996. Falanga, V., Qian, S. W., Danielpour, D., Katz, M. H., Roberts, A. B., and Sporn, M. B. Hypoxia upregulates the synthesis of TGF-beta 1 by human dermal fibroblasts. J. Invest. Dermatol. 97: 634, 1991. Brower, W., Williams, D., Lucore, P., Pretus, H., Jones, E., and McNamee, R. Effect of enhanced macrophage function on early wound healing. Surgery 104: 224, 1988. Vaillant, P., Muller, V., Martinet, Y., and Martinet, N. Human granulocyte- and granulocyte macrophage-colony stimulating
srga
WU ET AL.: M-CSF AND WOUND HEALING factors are chemotactic and ‘‘competence’’ growth factors for human mesenchymal cells. Biochem. Biophy. Res. Commun. 192: 879, 1993. 48. Bober, L. A., Grace, M. J., Pugliese-Sivo, C., Rojas-Triana, A., Waters, T., Sullivan, L. M., and Narula, S. K. The effect of GMCSF and G-CSF on human neutrophil function. Immunopharmacology 29: 111, 1995. 49. Middleton, S. C., and Aukerman, S. L. The effects of macrophage colony stimulating factor (M-CSF) on wound healing in normal and genetically diabetic (db/db) mice (abstract). J. Cell. Biochem. 15F: 197, 1991. 50. Zhao, L. L., Davidson, J. D., Wee, S. C., Roth, S. I., and Mustoe, T. A. Effect of hyperbaric oxygen and growth factors on rabbit ear ischemic ulcers. Arch. Surg. 29: 1043, 1994.
AID
JSR 5178
/
6n26$$$241
10-08-97 10:33:42
169
51. Wu, L., Pierce, G. F., Galiano, R. D., and Mustoe, T. A. KGF induces granulation tissue in ischemic dermal wounds: The importance of epithelial-mesenchymal cell interaction. Arch. Surg. 131: 660, 1996. 52. Chen, B. D-M., Chou, T. H., and Sensenbrenner, L. Downregulation of M-CSF receptors by lipopolysaccharide in murine peritoneal exudate macrophages is mediated through a phospholipase C dependent pathway. Exp. Hematol. 21: 623, 1993. 53. Schwertz, D. W., and Halverson, J. Changes in phosphoinositide-specific phospholipase C and phospholipase A2 activity in ischemic and reperfused rat heart. Basic Res. Cardiol. 87: 113, 1992. 54. Bunn, H. F., and Poyton, R. O. Oxygen sensing and molecular adaptation to hypoxia [Review]. Physiol. Rev. 76: 839, 1996.
srga