Kinetics of interleukin-1 and tumor necrosis factor secretion by rabbit macrophages recovered from the peritoneal cavity after surgery

Macrophages play a crucial role in wound healing after surgical injury, both as scavenger cells responsible for wound debridement and as cells that secrete soluble factors such as interleukin-1 (IL-1) and tumor necrosis factor (TNF). IL-1 and TNF alter many of the biological activities of cells that appear in postsurgical wounds. In this study, we determined the kinetics of IL-1 and TNF production by rabbit macrophages harvested from postsurgical peritoneal exudate (postsurgical macrophages) at several time points after peritoneal surgery. To further characterize the level of functional activities of postsurgical macrophages, the IL-1 and TNF levels were determined with or without stimulating the cells with lipopolysaccharide (LPS) and phorbol myristate acetate (PMA). After surgery, the number of macrophages harvested by peritoneal lavage increased, reached peak levels on postsurgical day 3, and then decreased. IL-1 levels secreted by macrophages cultured without stimuli were elevated on postsurgical day 14 compared to the values on day 3 and 7. TNF concentrations peaked on days 1 and 14. In the conditioned culture media from LPS-PMA-stimulated macrophages, the levels of both IL-1 and TNF peaked on postsurgical days 3 and 14. These data suggest that the susceptibility of postsurgical macrophages to stimuli changes during the wound healing process with maximum sensitivity to the stimuli present during the early phase of peritoneal repair (day 3).     

Production of protease inhibitors by postsurgical macrophages

The deposition and lysis of fibrin are important processes in normal peritoneal healing. Since macrophages secrete a neutral plasminogen activator, we studied the production of plasminogen-dependent, fibrinolytic activity by postsurgical macrophages. Peritoneal exudate macrophages were collected from rabbits after resection and reanastomosis of their ileum. Intracellular plasminogen activator (PA) activity of resident (nonsurgical) macrophages was 8.4 +/- 1.8 milli-Plough units (mPU)/10(6) cells. Postsurgical Day 1 macrophages had significantly less activity (0.33 +/- 0.056 mPU/10(6) cells) compared to resident cells. Thereafter, the PA activity gradually increased and reached control levels by Postsurgical Day 7. The PA activity secreted by postsurgical macrophages into serum-free medium after 48 hr of culture was also determined. Conditioned medium from macrophages collected on Postoperative Days 1-5 exhibited less PA activity than buffer controls. PA activity was detected after acid treatment of the conditioned medium to remove acid-labile inhibitors. The activities of PA in acid-treated conditioned medium increased gradually and reached nonsurgical levels by Postsurgical Day 7. In spent medium from macrophages collected on Postsurgical Days 1-3, high levels of urokinase inhibitory activities were secreted; production gradually decreased during the later postoperative period. This inhibitory activity of macrophage-conditioned medium on urokinase-like PA activity was partially diminished by acidification of the media. These results support the hypothesis that macrophages in the postsurgical exudate may play an important role in the fibrinolytic process during peritoneal wound healing, perhaps through production and secretion of plasminogen activator as well as acid-labile and resistant protease inhibitors.     

Modulation of postsurgical macrophage function by postsurgical polymorphonuclear leukocytes]

Rapid and transient influx of polymorphonuclear leukocytes (PMN) is observed prior to accumulation of macrophages after surgical trauma. Rabbits underwent intestinal reanastomosis and at various times peritoneal exudate cells were collected and separated using a Percoll gradient. Postsurgical macrophages were incubated with PMN spent media obtained from various postsurgical periods. Macrophage release of O2- had already increased at 2 hr after surgery, reached peak levels at 6 hr and decreased by 24 hr. PMN spent media from 6 hr postsurgical cells functioned as a suppressor, whereas 12 or 24 hr PMN spent media increased the O2- release from the macrophages harvested at 6 and 12 hr after surgery. Plasminogen activator (PA) activity in the macrophage spent medium was elevated at 24 hr after surgery by exposure to PMN spent media, however no effects were observed on macrophages harvested within 12 hr after surgery. PA inhibitory activity was reduced at 2 hr after surgery, and gradually increased, but no effects of PMN spent media on the PA inhibitory activity was observed. Thus, soluble factors secreted into the medium by PMN may modulate macrophage metabolism in stages as the macrophages differentiate and promote wound repair.     

Regulation of proliferation of peritoneal tissue repair cells by peritoneal macrophages

Macrophages produce soluble mediators which modulate fibroblast growth during tissue repair. Interaction between tissue repair fibroblasts (TRC) and regulatory proteins from surgically elicited macrophages is important for peritoneal reepithelialization. In this study, we compared the effects of an extract from postsurgical macrophage spent medium with those of known growth factors on TRC collected from injured peritoneum to evaluate certain characteristics of macrophage secretory products on peritoneal healing. Rabbits underwent a midline laparotomy followed by resection and reanastomosis of the ileum or abrasion of the abdominal wall. TRC were then collected at various times after surgery. Peritoneal macrophages recovered from nonsurgical or postsurgical rabbits were cultured for 2 days in vitro. The peak of thymidine incorporation by TRC occurred on day 5 after surgery; this gradually decreased with extended postsurgical times. Fibroblast growth factor and epidermal growth factor stimulated, whereas TGF-beta inhibited, [3H]thymidine incorporation into TRC. Maximal thymidine incorporation occurred when TRC from Postsurgical Day 5 were cultured with an extract from postsurgical macrophage spent medium. However, when TRC recovered from Postsurgical Days 2 and 10 were cultured with an extract of postsurgical macrophage spent medium, they showed greater stimulation than Day 5 TRC. These data suggest that postsurgical macrophages may produce an array of factors that stimulate fibroblast growth and differentiation and may in turn affect tissue repair throughout the wound healing process.     

Effects of interleukin-1 (IL-1) on postsurgical macrophage secretion of protease and protease inhibitor activities.

Although peritoneal macrophages secrete a variety of inflammatory mediators and proteases during postsurgical repair of the peritoneum, regulation of this secretion is poorly understood. Here, the responsivity of peritoneal macrophages to interleukin-1 (IL-1) stimulation in vitro, measured by the secretion of protease and protease inhibitor activities, was evaluated as a function of postsurgical time. Macrophages were harvested at various times after peritoneal sidewall abrasion, isolated by discontinuous density centrifugation and cultured with varying concentrations of IL-1. IL-1 increased the secretion of plasminogen activator (PA) activity by peritoneal macrophages in a concentration-dependent manner on postsurgical Days 0, 3, 10, and 14. Macrophages harvested on postsurgical Day 1 after surgery responded only to high concentration of IL-1, while on Days 5 and 7 all doses of IL-1 stimulate PA. On Days 7, 10, and 14 after surgery, the secretion of PA activity (after acid treatment) by postsurgical macrophages was generally high and increased with IL-1 treatment. The level of PA activity after inactivation of acid labile inhibitors (PAI) also increased in a dose-dependent manner on Days 0, 3, and 5. Although Day 1 macrophages expressed the highest PAI activity of all groups, they had relatively low responsivity to IL-1 with regards to PAI secretion. The level of elastase activity by postsurgical macrophages was lowest on Day 1, highest on Day 7, and decreased thereafter. All concentrations of IL-1 inhibited elastase activity of macrophages on Day 7.(ABSTRACT TRUNCATED AT 250 WORDS)     

A role of postsurgical macrophage activation by peritoneal injury]

At a site of peritoneal injury after abdominal surgery, macrophages are thought to be a principle type of inflammatory cells. Therefore, we determined the metabolic activities of postsurgical peritoneal exudate macrophages using standardized rabbit model. Rabbits underwent midline laparotomy followed by resection and reanastomosis of the ileum. At various days after surgery, peritoneal exudate macrophages were recovered from lavage fluid. Postsurgical Day-5 macrophages expressed significantly high potential to produce superoxide anion even without PMA stimulation compared to non-surgical control macrophages, although an activity of Day-10 macrophages was similar to control. The conditioned media from postsurgical Day-1 macrophage culture strongly inhibited urokinase type plasminogen activator (PA) and this plasminogen activator inhibitor (PAI) activities decreased following the extension of postsurgical time. Conversely, PA activities of macrophage-conditioned media decreased by day 1 and then gradually increased reaching control levels by day 10. Elastase activities of macrophage-conditioned media gradually decreased until postsurgical day 10. These data suggest that surgical injury activates postsurgical exudate macrophages. However, a time course of metabolic activities of these cells was dependent upon each secretory products. This differential secretion might express the stage of activation and differentiation of postsurgical macrophages. Moreover, postsurgical activated macrophages may control the tissue repair through a digestion of injured matrix and fibrinolytic process.     

Modulation of postsurgical macrophage function by early postsurgical polymorphonuclear leukocytes.

Surgical trauma to the peritoneum, in the absence of infection, elicits a rapid and transient influx of polymorphonuclear leukocytes (PMNs) into the peritoneal cavity prior to the accumulation of macrophages. The aim of this study was to characterize the effects of these PMNs on macrophage function in the early postsurgical period. Rabbits underwent intestinal reanastomosis and peritoneal exudate cells were collected at various times after surgery. Macrophage-enriched preparations were incubated with spent media from cultures of PMNs obtained at the corresponding times after surgery. Superoxide anion (O2-) release by macrophages in response to phorbol myristate acetate was determined by cytochrome c reduction. Fibrinolytic and protease inhibitory activities in macrophage-spent media were also evaluated. The release of O2- had already increased at 2 hr, reached peak levels at 6 hr, and decreased by 24 hr after surgery. Spent media from PMNs harvested 6 hr after surgery suppressed, whereas spent media from postsurgical 12- or 24-hr PMNs increased O2- release from macrophages harvested at 6 and 12 hr after surgery. PMN-spent media had no effect on the secretion of plasminogen activator (PA) from macrophages harvested within 12 hr after surgery. In contrast, PA activity in the spent media from macrophages harvested 24 hr after surgery was elevated after exposure to PMN-spent media. PA inhibitory activity was reduced in macrophage-spent media at 2 hr after surgery and increased by 24 hr, while PMN-spent media had no effect on the level of PA inhibitory activity. Thus, soluble factors secreted into the culture medium by PMNs modulate macrophage function as soon as 6-12 hr after surgery.     

Superoxide anion production by postsurgical macrophages

In order to characterize further the metabolic activity of postsurgical macrophages, we examined their potential to produce superoxide anion (O2-) in response to phorbol myristate acetate. Rabbits underwent resection and reanastomosis of their ileum after which peritoneal exudative cells (PEC) were collected at various times after surgery. Macrophages were isolated from the PEC with a discontinuous Percoll gradient. Thereafter, O2- release by these cells in response to phorbol myristate acetate (PMA) was determined by cytochrome c reduction. Macrophages recovered on postsurgical Days 3-7 expressed three- to fourfold greater O2- release than resident (nonsurgical) macrophages (P less than 0.001). In a time-course study of O2- release, maximum release occurred at 6 hr postsurgery, dropped to half of peak levels by Day 1, increased to peak levels again on Day 4, and remained increased until Day 7. The ability of macrophages to release O2- than gradually decreased, reaching control levels by Day 13. These data suggest that resident peritoneal macrophages are rapidly primed to produce enhanced amounts of superoxide anion in response to appropriate stimuli after surgery, and that macrophages subsequently recruited into the peritoneal cavity as a result of surgical trauma may also be primed for enhanced O2- release as they differentiate into modulator macrophages for tissue repair.     

Modulation of peritoneal re-epithelialization by postsurgical macrophages.

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.     

In vivo administration of tolmetin in hyaluronic acid modulates protease levels in postsurgical macrophage-conditioned media.

Tolmetin sodium in a hyaluronic acid carrier (tolmetin-HA) was previously shown to reduce adhesion formation and alter the kinetics and levels of cellular influx into the peritoneal cavity after surgery. In this study, the effect of tolmetin-HA on the level of protease activity in macrophage-conditioned media was determined. The level of collagenase activity in macrophage-conditioned media was suppressed at 12 and 24 h after administration of tolmetin-HA. Alternatively, the peak level of elastase activity measured in macrophage-conditioned media was unchanged after tolmetin-HA treatment, but the kinetics of expression of maximal protease activity was delayed from 12 h in the control surgical rabbits to 24 h in tolmetin-HA-treated rabbits. Elevated plasminogen activator activity was detected in acid-treated conditioned media from the tolmetin-HA-treated rabbits when compared to control levels. However, no alteration in the level of plasminogen activator inhibitor activity was present in conditioned media of macrophages harvested from tolmetin-HA-treated rabbits compared to controls. These data suggest that tolmetin-HA treatment altered the levels of neutral protease activity secreted by postsurgical macrophages and may therefore elevate the fibrinolytic potential of the peritoneal cavity after surgery.     

In Vivo Administration of Tolmetin in Hyaluronic Acid Modulates Protease Levels in Postsurgical Macrophage-Conditioned Media

Tolmetin sodium in a hyaluronic acid carrier (tolmetin-HA) was previously shown to reduce adhesion formation and alter the kinetics and levels of cellular influx into the peritoneal cavity after surgery. In this study, the effect of tolmetin-HA on the level of protease activity in macrophage-conditioned media was determined. The level of collagenase activity in macrophage-conditioned media was supressed at 12 and 24 h after administration of tolmetin-HA. Alternatively, the peak level of elastase activity measured in macrophage-conditioned media was unchanged after tolmetin-HA treatment, but the kinetics of expression of maximal protease activity was delayed from 12 h in the control surgical rabbits to 24 h in tolmetin-HA-treated rabbits. Elevated plasminogen activator activity was detected in acid-treated conditioned media from the tolmetin-HA-treated rabbits when compared to control levels. However, no alteration in the level of plasminogen activator inhibitor activity was present in conditioned media of macrophages harvested from tolmetin-HA-treated rabbits compared to controls. These data suggest that tolmetin-HA treatment altered the levels of neutral protease activity secreted by postsurgical macrophages and may therefore elevate the fibrinolytic potential of the peritoneal cavity after surgery.     

Modulation of proline and glucosamine incorporation into tissue repair cells by peritoneal macrophages

The purpose of this study was to determine the patterns of [14C]proline and [14C]glucosamine incorporation by tissue repair cells (TRC) as modulated by postsurgical macrophages. Rabbits underwent a midline laparotomy followed by resection (2.0 cm) and reanastomosis of their ileum. Another group of rabbits underwent peritoneal wall abrasion with sterile gauze until punctate bleeding developed. Postoperative (1-28 days) exudate cells (PEC) were recovered from the peritoneal cavity after reanastomosis, and (TRC) were obtained directly from the injured peritoneal surface after abrasion. Since the postsurgical exudate was composed mainly of macrophages, we examined the effect of postsurgical macrophage-spent media on the incorporation of [14C]proline, [14C]glucosamine, and [3H]thymidine by TRC. After 7 days of culture, Postsurgical Day 7 TRC were incubated with spent media from postsurgical PEC (greater than 90% macrophages). When TRC were cultured with macrophage-spent media, the number of TRC increased significantly compared to that of fresh medium-treated controls. The incorporation of [3H]thymidine by TRC was also enhanced by macrophage-spent media. The incorporation of [14C]proline and [14C]glucosamine by TRC was also enhanced when incubated with macrophage-spent medium. However, when data were expressed on a per cell basis, incorporation of [14C]proline and [14C]glucosamine by TRC cultured with macrophage-spent media was the same or less than that by cells incubated with fresh medium. These data suggest that the increase in incorporation of glucosamine and proline into connective tissue protein by postsurgical repair cells may be directly modulated by macrophages recruited in response to surgical injury and that this increase is due to the fibroproliferative effect of postsurgical macrophages.     

Modulation of fibroblast proliferation by postsurgical macrophages

Macrophages and fibroblasts are major components of postsurgical peritoneal repair. In order to understand the interaction between these two cell types, we studied the effects of spent macrophage culture media on fibroblast proliferation. Rabbits underwent resection and reanastomosis of their small intestine. Peritoneal exudative cells (PEC) were then collected from these animals on postoperative Days 4, 7, and 28 and from nonsurgical controls. PEC (5 X 10(5) cells/ml) were cultured in M-199 with 3% fetal calf serum. After 48 hr the spent media from the cultured PEC were harvested, centrifuged (200g for 10 min), and stored (medium: M-D0, D4, D7, D28). A second group of rabbits underwent peritoneal wall abrasion followed by collection of fibroblasts directly from the site of injury on postoperative Days 1, 4, and 7. After Day 7 of culture, fibroblasts were resuspended and seeded into dishes (1 X 10(5) cells in 1 ml medium), to which was added 1 ml of spent PEC culture medium. After 24 hr of incubation, 1 muCi [3H]thymidine was added for an additional 18 hr. Fibroblasts were then collected and the amount of [3H]thymidine incorporated into trichloroacetic acid-precipitable material was quantitated. In one protocol, fresh M-199 with 3% fetal calf serum was used, and in another protocol, "U-medium" (which was previously incubated for 48 hr with fibroblasts) was used. The cytology of the PEC was determined by Wright's staining, nonspecific esterase activity, and phagocytosis. At least 80% of the peritoneal exudative cells were identified as macrophages. Postsurgical Day 7 fibroblasts demonstrated greater [3H]thymidine incorporation compared to fibroblasts from postoperative Days 1 and 4.(ABSTRACT TRUNCATED AT 250 WORDS)     

The mitogenic activity of peritoneal tissue repair cells: control by growth factors

The purpose of this study was to determine the proliferative activity of tissue repair fibroblasts recovered directly from injured peritoneum at various times after surgery and to test the mitogenic response of tissue repair cells (TRC) to growth factors. Rabbits underwent bilateral peritoneal abrasion (5 X 5 cm) with sterile gauze until punctate bleeding developed. Postsurgical (Days 2, 5, 7, and 10) tissue repair cells were recovered from the injured peritoneum by scraping with a scalpel blade. Although tissue repair cells consisted of a mixed cell type after 4 days in culture, recovered cells were essentially fibroblasts. These TRC were then pulsed with [3H]thymidine after 4 days in culture. The incorporation of thymidine into Postsurgical Day 5 TRC increased significantly compared to that of Day 2 TRC (P less than 0.05). Incorporation then decreased with time following surgery. Fibroblast growth factor (FGF) and epidermal growth factor (EGF) stimulated the incorporation of thymidine into TRC. However, the response of Postsurgical Day 7 and 10 TRCs to 1 microgram/ml EGF was significantly greater than those of Postsurgical Day 2 and 5 TRCs (Day 2 TRC, 166 +/- 7.4; Day 10 TRC, 420 +/- 96% of control cells without EGF, P less than 0.05). Platelet-derived growth factor (PDGF, 10 ng/ml) also stimulated the incorporation of thymidine into Day 10 TRCs, but this stimulatory activity (129.9 +/- 8.5% of control) was less than EGF or FGF. IL-1 alpha and IL-2 did not stimulate the incorporation of thymidine into TRC at a concentration of 100 pg/ml, but these cytokines did stimulate protein synthesis by TRC.(ABSTRACT TRUNCATED AT 250 WORDS)     

Temporal response of leukocyte accumulation in the thoracic cavity after two types of surgical injury

To characterize pleural healing, we quantitated leukocyte accumulation in the pleural cavity and histological changes after two types of thoracic surgery. Rabbits underwent intercostal thoracotomy followed by abrasion of the parietal pleura and ligation and resection of the right middle lobe of the lung (group A), or only abrasion of the parietal pleura (group B). After surgery, the influx of leukocytes into the pleural exudate was characterized by an increase in the number of polymorphonuclear neutrophils (PMNs) followed by monocytes/macrophages. In group A, the total number of leukocytes reached maximum levels on days 5-7 after surgery, 80% of which were monocytes/macrophages. In group B, the total number of leukocytes reached peak levels on postsurgical day 3, 85% of which were monocytes/macrophages. Histologically, we observed a relative delay in pleural healing in group A compared with group B. An inflammatory response including appearance of fibrinous exudates and infiltration of acute inflammatory cells occurred in group A on days 1-3 after surgery. On days 5-7, an increase in submesothelial connective tissue was seen. An increase in cellularity was observed in this layer (fibroplasia) and the wound surface was covered by macrophagelike cells. In group B, disappearance of fibrinous exudates and fibroplasia occurred by day 3. In both groups, these histological changes from inflammatory phase to proliferative phase occurred on the day when the number of monocytes/macrophages in the pleural cavity reached peak levels. These data demonstrate that different types of thoracic injury alter the kinetics of leukocyte accumulation in the pleural cavity and the healing process of parietal pleura, suggesting that macrophages that accumulate in the pleural cavity may be implicated in postsurgical repair.     

Role of macrophage polarisation in skin wound healing

Skin wound healing is a complex pathophysiological change that is driven by macrophages and their secreted related factors. Depending on the stimuli, macrophages can be polarised into two subtypes of macrophages with completely different phenotypes and functions, namely M1 and M2. The aim of this study was to explore the role of M1 and M2 macrophages in skin healing in order to develop new drugs for the treatment of refractory wounds. Primary bone marrow-derived macrophages (BMDMs) were isolated from rats and expanded in vitro using macrophage colony stimulating factor. In addition, the BMDMs were polarised into the M1 and M2 subtypes using lipopolysaccharides (LPS) and interleukin-4 (IL-4), respectively. Cytokine levels in the culture supernatants were measured by an enzyme linked immunosorbent assay. Epidermal wounds were made on the dorsal surface of rats, and treated with M1 or M2 cell suspensions or phosphate buffered saline. Wound healing was recorded on days 1, 3, 7, 10, and 14 after stamping, and the wound healing rate was measured by haematoxylin-eosin and Masson staining. A total of 3 to 4 × 10⁷ bone marrow cells were extracted from each rat femur. The BMDM culture had 87.1% CD45⁺ cells, 89.2% CD68⁺ cells, and 86.5% CD45⁺CD68⁺ cells. Furthermore, IL-12 (P < .05) and IL-10 (P ≥ .05) levels, respectively, increased and decreased in the culture supernatants of the M1 cells after LPS stimulation compared with those in the M0 (unstimulated) group. Likewise, IL-4 stimulation led to a significant increase in IL-10 levels (P < .01) in the conditioned media of M2 cells, while that of IL-12 decreased slightly (P ≥ .05). In the rat model, the infusion of M2 cells accelerated wound healing and tissue regeneration, whereas the M1 cells delayed the recruitment of inflammatory cells, granulation growth, and collagen deposition, which impaired wound healing. Macrophage polarisation and activation are critical for skin wound healing. While exogenous M1 cell infusion delayed wound healing, the M2 cells promoted wound healing in a rat model.     

Mitogenic and protein synthetic activity of tissue repair cells: control by the postsurgical macrophage

It is well known that fibroblasts are a main source of extracellular matrix synthesis necessary for tissue repair. In addition, macrophages secrete products that are known to modulate synthesis of extracellular matrix. Accordingly, we studied the incorporation of [3H]thymidine, [3H]proline, and [35S]sulfate into macromolecules produced by fibroblasts recovered from the site of peritoneal tissue repair cultured with and without spent media from postsurgical peritoneal macrophages. Rabbits underwent resection and reanastomosis of their small intestines. Peritoneal exudative cells (PEC) were then collected on postsurgical day 5 and day 10 as well as from nonsurgical controls, separated by discontinuous Percoll gradient centrifugation, and cultured for 48 h. A second group of rabbits underwent peritoneal wall abrasion from which fibroblast tissue repair cells (TRC) were collected from the site of injury at postsurgical day 7 and maintained in culture for varying times. Incorporation of radiolabeled precursors into DNA, collagen, and sulfated proteoglycans was determined. Incorporation of [3H]thymidine and [3H]proline into untreated TRC gradually decreased with culture duration. Conversely, [35S]sulfate incorporation gradually increased during prolonged culture. Macrophage spent media increased the levels of [3H]thymidine incorporation by the TRC. [3H]Proline and [35S]sulfate incorporation into TRC were also stimulated by macrophage spent media. However, this stimulation may be due to the enhanced proliferation of TRC by macrophage spent media. In conclusion, tissue repair fibroblasts are activated for postsurgical repair at the site of injury by many factors including secretory products from postsurgical macrophages.     

The hemostatic effect of deacetylated chitin membrane on peritoneal injury in rabbit model

In this study, we determined the effect of 80% deacetylated chitin (DAC-80) membrane on postsurgical bleeding after visceral and parietal peritoneal abrasion. Japanese white rabbits underwent a midline laparotomy followed either by a bilateral peritoneal sidewall abrasion (4×4 cm) or an abrasion of liver surface (3×2 cm). The injured surface was then covered with a 0.2 mm thick DAC-80 membrane. On postsurgical day 2, the rabbits were sacrificed and the amounts of postsurgical bleeding was determined by quantitating the number of red blood cells recovered in 50 ml peritoneal lavage fluid. The DAC-80 membrane was found to reduce postsurgical bleeding after the abrasion of liver surface (treated with DAC-80 membrane: 2.9±0.8; control: 24.6±5.9×10⁸ cells/peritoneal cavity, P<0.005). This same hemostatic activity was not observed after application in the peritoneal sidewall abrasion model. We also measured plasminogen activator activity (PA) and urokinase inhibitory (PAI) activity in the spent culture media of macrophages recovered from the postsurgical peritoneal exudate. The DAC-80 membrane reduced the PA secretion from postsurgical macrophages after liver surface abrasion (treated with DAC-80: 2.8±0.7; control: 3.9±0.9 mPU/ml). The DAC-80 membrane also showed similar effects on PA secretion after peritoneal sidewall abrasion. No significant effects were found in the secretion of PAI by postsurgical macrophages in both surgical models.These findings suggest that the DAC-80 membrane may have hemostatic activity through the modulation of fibrinolytic activity of peritoneal exudative macrophages.     

A simplified method for monitoring cytokines in wound fluid

Cytokines in wound fluid are used as surrogates for wound healing in clinical research. The current methods used to collect and process wound fluid are noninvasive but not optimal. The aim of this prospective study was to evaluate a method (NovaSwab) by which wound fluid is collected by a surface swab and eluted in a physiological buffer for subsequent cytokine analysis. Wound fluid from 12 patients with leg ulcers was assessed by NovaSwab at the start (Day 0) and at the end of a 23-h collection period of wound fluid retained by foam oblates beneath an occlusive film dressing (Day 1). GM-CSF, IL-1α, IL-1β, IL-6, IL-8, PDGF-AA, TNF-α and VEGF levels were measured by multiplex and electrochemiluminescence assays. IL-1α (2.4×), IL-1β (2.0×) and IL-8 (1.8×) levels increased from Day 0 to Day 1 as detected by NovaSwab, indicating local production of these polypeptides in the wounds. On Day 1, the NovaSwab method yielded higher levels of IL-1α (4.0×), IL-1β (2.7×) and IL-6 (2.7×), and 35% lower levels of VEGF than those in wound fluid accumulated for 23 h in foam oblates (on average, 5 ml of wound fluid). In vitro experiments showed that the investigated cytokines in cell-free wound fluid were recovered in a quantitative manner by the NovaSwab method. We conclude that the method presented here is a promising research tool to study the kinetics of soluble cytokines over the course of wound healing. More studies are needed to determine the interobserver variation and reproducibility of the NovaSwab method.     

Trauma-hemorrhage delays wound healing potentially by increasing pro-inflammatory cytokines at the wound site.

BACKGROUND: Studies indicate impaired wound healing after trauma. The underlying mechanism remains unknown. METHODS: Mice were subjected to midline laparotomy, and polyvinyl alcohol sponges were implanted subcutaneously before hemorrhage (35 +/- 5 mmHg for 90 minutes, resuscitated) or sham operation. Wound exudate cells from the sponges were harvested on the first, third, and fifth postoperative day and cultured for 24 hours. Interleukin (IL)-1 beta, IL-6, monocyte chemotactic protein-1 (MCP-1), macrophage inflammatory protein-2 (MIP-2), and transforming growth factor (TGF)-beta were determined in the supernatants. IL-1 beta and IL-6 were measured in the wound fluid. RESULTS: Hemorrhage decreased collagen deposition in the wound. TGF-beta release was significantly decreased on the first and third postoperative days after hemorrhage, whereas IL-1 beta and IL-6 release was increased at 3 and 5 days after hemorrhage. Similarly, IL-1 beta and IL-6 in the wound fluid were significantly increased at 3 days after hemorrhage. CONCLUSIONS: Because increased levels of pro-inflammatory cytokines and decreased amounts of TGF-beta have been reported to impair the process of wound healing, the increased release of IL-1 beta and IL-6 and the decreased release of TGF-beta after hemorrhage might contribute to the decreased collagen production in those animals. Thus, attempts to locally change the ratio of those cytokines in trauma victims might be useful for improving wound healing in those patients.