Modulation of peritoneal re-epithelialization by postsurgical macrophages

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Modulation of Peritoneal Re-epithelialization by Postsurgical Macrophages KATHLEEN Livingston

Reproductive

Biology Laboratory, School of Medicine,

E. RODGERS AND GERE S. DIZEREGA’ Department of Obstetrics and Gynecology, University of Southern 1321 North Mission Road, Los Angeles, California 90033 Submitted

for publication

Re-epithelialization of peritoneal defects after surgery occurs over the entire surface of the injured site simultaneously [ 11.Accordingly, the surface of a relatively large injury will re-epithelize as rapidly as a smaller wound. In this review, functions of the epithelial cells found at the site of peritoneal trauma and the modulation of these functions by macrophages and macrophage secretory products are discussed. Throughout this review, mesothelial and other epithelial cells collected from the site of peritoneal injury at various times after trauma are referred to as tissue repair cells (TRC). When fibroblasts are referred to in this review, it will be to present information from the literature for comparison. In order to study the re-epithelialization of peritoneal injury and the changing character of these cells in our laboratory, a standardized excision of rabbit parietal peritoneum was used to initiate peritoneal repair. The cells on the surface of the parietal defect were harvested and grown for 4-8 days in culture to allow for the generation of a confluent layer of adherent cells with few leukocytes. These TRC are the basis for a series of studies performed to characterize the function and responsivity of these epithelial-repair cells to macrophages, macrophage secretory products, and individual growth factors throughout the postoperative interval. Results of these studies are reviewed here and summarized with respect to general concepts of postsurgical peritoneal repair. At 4 days after peritoneal injury, the surface of a peritoneal injury contains TRCs proliferating throughout the wound base which actively secrete connective tissue matrix [l-3]. The activity of TRCs, as measured by protein and collagen synthesis, also increases after surgery reaching peak levels on Postsurgical Days 5-7 [3]. When cultured in vitro these cells proliferate and form a syncytium. TRCs are not “fibroblasts” 1 To whom reprint requests productive Biology Laboratory, geles, CA 90033. oozz-4804/92 Copyright All rights

should be addressed at Livingston Re1321 North Mission Road, Los An-

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$4.00 0 1992 by Academic Press, of reproduction in any form

Inc.

reserved.

July

California

8, 1991

as they respond to a variety of stimuli, including monokines, in a manner distinctly different from that of established fibroblast cell lines (discussed below). ROLE

OF MACROPHAGES

IN TRC

PROLIFERATION

To study the role of macrophages in the repair of injured peritoneum, we evaluated the influence of peritoneal macrophages on mesothelial re-epithelialization at an injured site using a postsurgical rabbit model. The number of macrophages present on the surface of the healing peritoneum after surgical injury is insufficient to characterize the metabolic events which occur during repair. Therefore, we utilized the cellular elements within the exudative fluid that accumulate in the peritoneal cavity after surgical injury. Since cellular elements in the peritoneal exudate are in close proximity to the site of re-epithelialization and enter the peritoneal cavity with kinetics similar to that of tissue repair leukocytes, we hypothesized that this cellular constituency closely reflects the postsurgical peritoneal surface. Studies were initially conducted to examine the effects of postsurgical macrophages on morphology and function of TRC. TRC are morphologically transformed and activated to proliferate when cocultured with postsurgical macrophages [4]. The mitogenic activity of TRC cocultured with postsurgical macrophages is greater than that measured when they are cultured with nonsurgical macrophages. Similarly, proliferation of TRC exposed to medium from cultures of macrophages collected on Postsurgical Days 4 and 7 was elevated compared to TRC maintained in spent medium from nonsurgical macrophage cultures (Fig. 1) [4]. These findings suggest that a macrophage-derived mitogenic factor(s) is secreted following postsurgical activation. Indeed, fibroblast proliferation can be stimulated by a variety of monokines following macrophage activation in vitro [5-131. How do postsurgical macrophages modulate the prolif-

RODGERS THYMIDINE

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FIG. 1. Effect of different pulse times on the incorporation of [3H]thymidine into TRCs. TRCs were preincubated with resident (+), Postsurgical Day 4 (w), and Postsurgical Day 7 macrophage-spent media (+), and fresh medium as control for 48 hr. TRCs were then pulsed with [3H]thymidine and fresh medium for 3-24 hr. Each data point expresses percentage of control incorporation. Each point represents the mean of three experiments (4).

eration of TRC during peritoneal healing? Following stimulation in viva or in vitro, macrophages produce “factors” which stimulate the proliferation and expression of differentiated functions of a variety of cell types [14-201. Interleukin-1 (IL-l), which is produced by stimulated macrophages, enhances the proliferation and differentiation of fibroblasts [15, 21-231. Jimenez de Asua et al. reported that prostaglandin F,, also stimulates the proliferation of fibroblasts [24, 251. Inhibition of fibroblast mitogenic activity by prostaglandin E, (PGE,) was reported by other investigators [26-291. Thus, macrophage secretory products have the potential to modulate the proliferation of fibroblasts by both stimulation [macrophage derived growth factor, (MDGF), IL-l, PGF,,, transforming growth factor-p (TGF-P)] and inhibition [PGE, , fibroblast growth inhibitor, TGF& interferon-a (IFN-a)]). However, which product(s) secreted by postsurgical macrophages is the dominant regulator(s) of TRC proliferation during the tissue repair process is dependent upon the amount of factor produced as well as the responsivity of TRC to these factors. The secretory products of macrophages recovered from peritoneal exudate at various times after surgery initially suppress (during the initial 48 hr of culture) and later enhance (following 48-54 hr of incubation) the incorporation of radiolabeled thymidine into TRC (Fig. 2) [30]. This suppression by postsurgical macrophages is significantly less than that observed with resident (nonsurgical) macrophages [30, 311. Therefore, modulation of TRC proliferation by macrophages appears to be a complex process involving elements of both suppression and stimulation. These data suggest a lag time during which TRC are either refractory to proliferative signals from postsurgical macrophages or controlled by inhibitors of proliferation. Alternatively, TRC may initially not be responsive to macrophage-derived growth fac-

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tar(s), but the inhibitory signal(s), which is presumably also macrophage-derived, initially predominates. Since the inhibition of TRC proliferation is reduced at later times in culture, the inhibitory signals may (a) be shortlived (as prostaglandins are thought to be), (b) themselves be overcome (or inactivated) by a proliferative factor(s), or (c) not affect TRC during culture acquired alterations in TRC response. It is well known that macrophages are critical in the final resolution of tissue debris and completion of healing, a process that ends in the formation of the connective tissue matrix and mesothelial syncytium [32, 331. Factors secreted by activated macrophages, such as MDGF, IL-l, and tumor necrosis factor (TNF), in nonsurgical systems can stimulate fibroblast proliferation as well as the secretion of connective tissue proteins, such as fibronectin, proteoglycans, collagen, and proteases, such as collagenase and elastase. Proper coordination of TRC proliferation, secretion of extracellular matrix, and tissue remodelling is necessary for repair. Accordingly, the rate and extent that these factors regulate wound healing may be amenable to modulation by extrinsic intervention. Postsurgical macrophages appear to revert to macrophages that are functionally similar to resident macrophages after the cessation of peritoneal repair. This is shown by the observation that proliferative activity is increased on Postsurgical Days 4 and 7 (Fig. 1) [4] and decreases to resident levels on Day 28 [30]. However, there is also an inhibition of TRC proliferation when compared to TRC cultured in medium supplemented with only serum [30, 311. Candidates for suppressors of proliferation include prostaglandins [341, interferons [35], arginase [36], complement cleavage products [37], and TGF-/3. Calderon et al. found that macrophages produce a dialyzable substance that inhibits the prolifera-

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FIG. 2. Incorporation of [3H]thymidine by TRC (fresh medium pulse). TRC were preincubated with resident (O), Postsurgical Day 4 (I) and Postsurgical Day 7 macrophage-spent media (A), and fresh medium as control. Each data point represents the percentage of control incorporation. *P x 0.05, **P < 0.01, ***P < 0.001 compared to control. “P < 0.001, bP < 0.05 compared to group treated with spent media from resident macrophages (30).

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could be removed upon dialysis. In fact, following dialysis, medium from postsurgical macrophages were quite efficacious at enhancing TRC proliferation.

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FIG. 3. Effect of growth factors and an extract of macrophagespent medium on 13H]thymidine incorporation into TRC. Incorporation of tritiated thymidine was determined. Postsurgical Day 10 TRC previously cultured for 8 days were treated with various concentrations of the extract of macrophage spent medium (A), FGF (B), EGF (C), and PDGF (D). Each data point represents the mean k SE (cpm/ well). C, media control (39).

tion of cells in vitro [38]. The manifestation of these complex signals appears to be a function of (a) the period of time after surgery; and (b) the length of TRC preincubation with macrophage-spent media prior to study (Fig.

Gospodarowicz [40] demonstrated that fibroblasts were transformed from a resting state (by contact inhibition) into a “mobile” state. Microscopic examination of TRC cultured with spent macrophage media indicates that an alteration in the morphology of TRC from a flatoval shape to a more spindly appearance occurs together with the macrophage-induced inhibition of TRC proliferation [4]. Interestingly, this morphological change seems to be associated with the inhibition of proliferation; that is, the time required for a reduction in thymidine incorporation was the same as that required for morphological changes. Alterations in the morphology of TRC cultured with macrophage-spent media indicate a complex change in cell metabolism to maximize the potential for migration. In this regard, Postsurgical Day 4 TRC are more responsive to spent media from postsurgical macrophages than Day 8 TRC, although the mitogenie activity of Day 4 TRC cultured with postsurgical macrophage spent media is less than that of Postsurgical Day 8 TRC. This dissociation between basal proliferative activity and responsiveness to stimuli (“transformation”) might be related to a time constraint for the cell to return to the original nonmigratory state prior to division. This time constraint may be related to the TRC’s ability to proliferate or not proliferate at the site of injury [41]. EFFECT

OF CULTURE

TIME

ON TRC

FUNCTION

2) [301.

Recent studies by Fukasawa et al. showed that the suppressive activity in postsurgical macrophage-spent media is dialyzable (Fig. 3) [39]. In this study, spent medium was harvested from serum-free cultures of macrophages from Days 5 to 7 after surgery, the media were dialyzed and lyophilized, and the effect on proliferation of TRC was tested. The proliferation of the TRC harvested at Days 2,5,7, and 10 after surgery, in response to this extract of macrophage-conditioned medium, was greater than that in medium alone. Since previous studies showed that macrophage-conditioned medium suppressed TRC proliferation compared to medium alone, this study suggeststhat the suppressive factor was dialyzable. In addition, when this extract of postsurgical macrophage-conditioned medium was compared with purified growth factors on a weight basis, this extract was more potent at stimulating TRC proliferation (Fig. 3). In summary, secretory products of postsurgical macrophages were able to enhance or suppress TRC proliferation when compared to the spent medium from resident macrophages or fresh medium, respectively. The suppressive factor in postsurgical macrophage-spent media

The time course of proliferative and functional activities of TRC was determined in culture to elucidate how in vitro metabolic activity of TRC directly collected from injured peritoneum reflects activation and differentiation of TRC in uiuo. Although initially TRC rapidly proliferate after recovery from injured peritoneum, this activity gradually decreases during culture. In addition, after Day 5, the TRC harvested from the initial site have a lower rate of basal proliferation at early time points of culture. Collagen production (as measured by [3H]proline incorporation) by TRC also decreases during culture [42]. Since sulfate is found mainly in glycosaminoglycans, [35S]sulfate was used to monitor the production of glycosaminoglycans by TRC. Interestingly, [35S]sulfate incorporation into TRC gradually increases during culture [42]. Studies by other investigators report that TGF-P stimulates production of extracellular matrix by fibroblasts but does not stimulate the proliferation of fibroblasts [43]. Thus, mitogenic and secretory activities of TRC are not always concurrent events. From the foregoing observation, the following hypothesis was developed: the mobilization and prolifera-

RODGERS

AND

DIZEREGA:

PERITONEAL

loo100 10' 102 103 EGF CONCENTRATION (pg/ml)

FIG. 4. Dose-response of postsurgical TRC to EGF. [3H]Thymidine incorporation was determined. Data represent percentage of media control culture (means k SEM). (0) Postsurgical Day 2 TRC; (@) Postsurgical Day 5 tissue repair cells (TRC); (A) Postsurgical Day 7 TRC; (a) Postsurgical Day 10 TRC (54).

tion of TRC are responsive to factors secreted by postsurgical macrophages. Although resident macrophages may function as negative modulators of TRC proliferation, the postsurgical macrophage secretes substances which induce migration and proliferation of TRC. The effect of postsurgical macrophages on TRC function may thus be dependent upon the (a) responsivity of TRC to these substances and (b) populations of macrophages (resident or suppressor vs activated or helper macrophages) present. This concept is supported by the observation that after surgical trauma in vivo, migration and proliferation of TRC are accelerated and then stop once tissue repair is complete. In addition, postsurgical macrophages can modulate the proliferation, morphology, and secretory products of postsurgical TRC in vitro. Thus, the macrophage can initiate and regulate the migration and proliferation of TRC. Accordingly, peritoneal wound healing may be controlled by the regulation of either macrophage migration and/or TRC proliferation.

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compartments in which tissue repair occurs, i.e., serum, wound fluid, platelets, macrophages, and TRC. FGF, which is produced by macrophages [46], stimulates the proliferation of fibroblasts and endothelial cells [48]. Epidermal growth factor (EGF), initially isolated from the submaxillary gland, is present in serum [49]. Insulin-like growth factor-I (IGF-I)/Somatomedin C was isolated from fibroblasts [50]. TGF-/3 is found in platelets, wound fluid, and macrophages [51-531. Since these factors may directly modulate the growth of TRC during postsurgical healing, the proliferative activities of TRC collected from injured peritoneum in response to growth factors at various times after surgery was determined. EGF and FGF stimulate the incorporation of thymidine into TRC [54]. Interestingly, the responsivity of TRC to EGF increases during the postsurgical period with Postsurgical Day 10 TRC demonstrating the greatest response to this factor (Fig. 4) [54]. PDGF also stimulates the incorporation of thymidine into TRC, but the stimulation is only 30% of control values for Postsurgical Day 10 TRC [54] (Fig. 5). PDGF is known to stimulate the proliferation of fibroblasts, especially under conditions of confluent culture. The effect of PDGF on proliferation is thought to induce the entry of G,arrested cells into the proliferative phase of the cell cycle [55]. TRC may be undergoing a mitotic cycle and, therefore, do not manifest as great a response to the addition of PDGF. On the other hand, EGF and FGF, which affected the proliferation of TRC to a greater extent, may function as competence factors involved in the transition of cells from G, to S phase [55]. IL-2 stimulates the healing of skin wounds [56]; however, IL-2 does not affect proliferation of fibroblasts. IL2 did not stimulate the incorporation of thymidine into TRC. IL-2 may indirectly affect the growth of TRC in vivo through stimulation of other cellular elements. For example, macrophages have cell surface receptors for IL-2 and IL-2 can stimulate the respiratory burst of macrophage in the absence of other stimuli [57]. IL-l also

FACTORS

A large number of factors have been shown to modulate the growth of fibroblasts. However, since TRCs are not fibroblasts, the effects of some of these factors on the proliferation of TRC at harvested various postoperative times were examined. At the site of peritoneal injury, TRC are in contact with other types of cells (i.e., lymphocytes, PMN, platelets, endothelial cells) which may modulate the in situ proliferative and functional activities of TRC through cell-cell interaction or the secretion of cytokines [44, 451. For example, platelet derived growth factor (PDGF), isolated from platelets, stimulates the proliferation of normal skin fibroblasts and established fibroblast cell lines. PDGF also functions as a chemoattractant for fibroblasts [46]. Ross and his colleagues reported the production of a PDGF-like factor by macrophages [47]. Many factors can be found in the

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FIG. 5. Response of postsurgical TRC to growth factors measured by [3H]thymidine incorporation. TRC were cultured for 8 days prior to the assay. Data represent percentage of control culture media (means -t SEM). (0) PDGF; (0) 1 rig/ml EGF, (a) 100 ng FGF; (0) 0.1 ng TGFp (54).

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did not affect the proliferation of TRC although the proliferation of rat NRK cells was enhanced by in vitro exposure to IL-l [54]. Although IL-l does not stimulate proliferation of TRC, IL-l stimulates protein synthesis by TRC. IL-l may function as an initiation factor similar to the role played by PDGF or may be more important for the production of extracellular matrix. IGF-I did not affect the incorporation of tritiated thymidine into TRC [54]. IGF-I/somatomedin C is known to stimulate the proliferation of fibroblasts, by effecting the transition from the S-G, phase to the M phase [55]. Since the effect of IGF-I is thought to be important on the M phase, and since this factor may be produced by fibroblasts, the growth factors involved in G,-S phase of the cell cycle may be more important for the enhancement of TRC proliferation [58]. TGF-/3 stimulates the anchorage-independent proliferation of fibroblasts but not anchorage-dependent growth [43, 511. In our studies, TGF-P was found to inhibit the incorporation of thymidine into TRC [54] (Fig. 6). TGF-P stimulates the production of extracellular matrix (collagen and fibronectin) by fibroblasts [59]. Incubation of TRC with TGF-fi enhances the incorporation of radiolabeled proline (to measure protein synthesis); in contrast TGF-P inhibits the proliferation of TRC (Fig. 7) [54]. In this context, TGF-/3 may function as a modulator of TRC differentiation in that it may induce these mesothelial cells to enter a functional (secretory) stage rather than a proliferative stage. Cromack reported that increase in TGF-P levels in wound fluid occur late after surgery, not during the early phase of tissue repair [53]. Fukasawa et al. [39] showed that after dialysis and lyophilization macrophage-spent media is more potent at stimulating the proliferation of TRC collected 10 days after surgery (Fig. 3) [39]. These results suggest postsurgical macrophages are capable of secreting a combination of stimulatory and inhibitory factors which may act in an additive or synergistic fashion in appropriate pro-

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TGF, CONCENTRATION

hg/ml)

FIG. 7. Effect of TGF/3 on protein synthesis by Postsurgical Day 10 TRC cultured for 8 days in u&o. Protein synthetic activity was determined using incorporation of acid-precipitable tritiated proline. Data represent means t SEM (cpm/well). (0) [3H]Thymidine incorporation into TRC; (m) [sH]proline incorporation into TRC; (0) [3H]proline incorporation into TCA-precipitable supernatant; (A) [sH]proline incorporation into trypsin-labile protein from cultured cells (54).

portions to maximize the proliferation of TRC. These data should be kept in mind when individual growth factors are examined for their ability to stimulate tissue repair [601. SUMMARY

The studies reviewed here show that postsurgical macrophages are capable of modulating the proliferation of TRC. That is, macrophages either suppress or enhance the proliferation of TRC depending on the culture time and the medium used as a comparison, i.e., culture medium with only serum or spent medium from cultures of resident peritoneal macrophages. Postsurgical macrophages also modulate the morphology of (spindly or rounded appearance) and the secretion of extracellular matrices by TRC. The responsivity of TRC to control by postsurgical macrophage-spent media or growth factors changes as a function of postsurgical and/or culture time. In addition, cells harvested from the site of peritoneal trauma (TRC) did not respond to growth factors in a fashion entirely the same as fibroblasts. This indicates that cells harvested from the site of peritoneal injury are unique. Lastly, after removal of a suppressive factor from postsurgical macrophage-spent media by dialysis, the factors secreted by postsurgical macrophages are more potent in enhancing TRC proliferation than growth factors individually. REFERENCES 1.

FIG. 6. Dose-response of TGFfl on thymidine incorporation by postsurgical TRC. Mitogenic activity was determined. Data represent percentages of control culture media (0) Day 2 TRC; (6) Day 5 TRC; (A) Day 7 TRC; (A) Day 10 TRC (54).

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