Meconium stimulates a pro-inflammatory response in peritoneal macrophages: Implications for meconium peritonitis

Meconium Stimulates a Pro-Inflammatory Response in Peritoneal Macrophages: Implications for Meconium Peritonitis By Kevin

P. Lally,

John

R. Mehall,

Houston,

Background/Purpose: Although meconium peritonitis is a rare condition, the mortality rate can be as high as 40%. Meconium peritonitis is a result of intestinal perforation in utero, which leads to dense inflammation in the peritoneal cavity. The fetus has relatively immature peritoneal defense mechanisms, so the cause of this dense inflammation is unclear. The peritoneal macrophage is a key cell in the peritoneal inflammatory response in adults. The purpose of this investigation was to determine if sterile meconium had a direct stimulatory effect on the peritoneal macrophage. Methods:Peritoneal macrophages were harvested from adult CBH/HEN mice. The cells were placed in microtiter wells at IO5 cells per well. Sterile human meconium was diluted in media and placed in the wells at varying concentrations for 8 hours. Lipopolysaccharide (LPS) (IO pg/mL) served as a positive control. Supernatants were harvested and assayed for tumor necrosis factor alpha (TNF-(Y) using a commercial ELISA kit. Separate cells were assayed for TNF-01 message using polymerase chain reaction (PCR). In another series of experiments, procoagulant activity (PCA) was determined on freeze-thawed cells using a two-stage amidolytic assay. To test for the role of protein kinase C (PKC) in the PCA response H7, a PKC inhibitor, was used as well.

I

NTESTINAL PERFORATION in utero with subsequent spillage of meconium into the peritoneal cavity occurs approximately once in every 35,000 live births and results in a severe, sterile peritonitis.’ This peritonitis is characterized by a marked inflammatory response with significant dense adhesions and a mortality rate as high as 40%.’ Bendel and Michel? first defined meconium peritonitis in 1953 as a nonbacterial, chemical, and foreign body peritonitis occurring during intrauterine or early neonatal life as a result of an abnormal communication between the bowel lumen and the peritoneal cavity.’ Martin3 and others”7 have classified meconium peritonitis into adhesive, infected, giant cystic meconium peritonitis, and meconium ascites, based on clinical and surgical findings. These subtypes preferentially develop From the Department of Surgery The Universi@ of Texas-Houston and Hermann Children k Hospital, Houston, 7X. Presented at the 29th Annual Meeting of the American Pediatric Surgical Association, Hilton Head, South Carolina, May 10-13, 1998. Address reprint requests to Kevin F! Lall~~ MD, Division of Pediatric Surgev, 6431 Farmin. 5.258, Houston. 7X 77030. Copyright o 1999 by N!B. Saunders Company 0022.3168/99/3101-00-13$03.00/O

214

Hasen

Xue,

and

Julie

Thompson

Texas

Results: Meconium stimulation resulted in a significant increase in TNF-(U compared with negative controls with a peak at 0.1% meconium (121 pg/mL v 11 pg/mL, P< .05). There was a significant increase in PCA, with a IO-fold increase with 1% meconium compared with controls (P < .05). This response was limited to less than 5% by PKC inhibition. Conc/usions:Sterile meconium results in a marked proinflammatory response in the peritoneal macrophage with elevations of both PCA and TNF-a. The TNF response is likely mediated at a pretranscriptional level because there is a marked increase in TNF mRNA. These data suggest that the PCA response is regulated by a PKC mechanism similar to LPS. Stimulation of the peritoneal macrophage by meconium is a possible cause of the marked inflammation seen in meconium peritonitis. J fediatr Surg 34:274-277. Copyright 1~ 7999 by W.B. Saunders Company.

INDEX WORDS: tis, macrophage, factor.

Peritonitis, meconium, meconium procoagulant, cytokine, tumor

peritoninecrosis

based on the timing of the perforation relative to birth, and whether the perforation seals spontaneously. Despite the various clinical manifestations, all cases of meconium peritonitis share a common etiology: the stimulation of inflammation by the presence of meconium in the peritoneal cavity. The peritoneal inflammatory response in the infant is poorly described. In adults, the peritoneal macrophage plays a central role in mediating the initial peritoneal inflammatory response.* The inflammatory response to meconium centers around chemical irritation and removal of foreign material. Macrophages likely play a role in clearing meconium from the peritoneal cavity. In addition, a predominance of mononuclear cells has been noted in the peritoneal white cell counts of survivors.’ The macrophage responds in a number of ways to an inflammatory stimulus. Secretion of tumor necrosis factor alpha (TNF-or) by the peritoneal macrophage is a known response. TNF-a promotes thrombosis by causing endothelial cells to lose their usual anticoagulant properties and enhances fibrin deposition. Moreover, TNF promotes adherence of polymorphonuclear cells to the endothelium and causes them to degranulate and form JournalofPediatricSurgery,

Vol 34, No 1 (January),

1999: pp 214-217

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reactive oxygen intermediates.’ Another aspect of the macrophage inflammatory response is the expression of tissue factor or procoagulant activity (PCA). Tissue factor expression has been linked to disseminated intravascular coagulation, transplant rejection, and death from sepsis.“’ There is a paucity of published manuscripts evaluating the mechanism of meconium-induced peritonitis. The purpose of this study was to evaluate the effects of meconium on the peritoneal macrophage. We hypothesized that meconium would induce a pro-inflammatory response in the peritoneal macrophage. MATERIALS

8000

1

7000 1

2000

I”W

0 LPS C.)

Msonrum concenmtion (S,

LPS (+I

1.0

AND METHODS

Human mrconium was harvested from neonates using sterile technique. cultured to confirm sterility. and frozen at -20°C until used. Sample\ were confirmed to be endotoxin free using a limulus amebocyte asay. Resident peritoneal macrophages were harvested from 7- to X-week-old C3H/HEN mice by cold lavage with RPM1 1630 (GIBCO. Inc). The lavage fluid was retrieved. centrifuged, and the pellet resuspended in Dulbecco’< Modified Eagle Medium (DMEM) with 10% fetal calf serum. Cell viability was confirmed using trypan blue exclusion. Materials were confirmed to be endotoxin free using a limulus amebocyte assay. The cells were then placed in plastic wells at IO5 cell< per well and allowed to adhere for 1 hour. Nonadherent cell\ were washed off. Cells were confirmed to be macrophages using nonspecific erterase \raining. Macrophages were then stimulated with meconium at concentrations of 1.0. 0.1, 0.01. 0.001. and 0.0001% in media for 8 hours. Stimulation with IO pg/mL of lipopolysaccharidr f LPS. ~.s~~hrri~~hicr chili 055:BS) served as a positive control and media alone ah a negative control for all experiments. After stimulation. the supematants were removed, and the cells were frozen at -70°C in media. ProLen. thawed cells were then sonicated and assayed for procoagulant activity using a two-stage amidolytic assay.” Eight milligrams of rabbit brain thromboplaatin (Sigma, St Loui\. MO) was assigned 3 standard of 100.000 mU. then serially diluted to ehtabliah a standard curve. A toral of six sets of macrophages were evaluated. To detcrmmc if PCA stimulation was mediated via protein kinase C (PKC). four sets of macrophage\ were pretreated for 15 minutes with the PKC inhibitor H-7 at 50 mmol/L before meconium stimulation. After pretreatment. macrophages were stimulated with I % meconium for 8 hours. Lipopolysaccharide again served as a positive control and media ;L\ J negative control. Frozen thawed cells were sonicated and assayed for PCA ah described. Macrophage TNF-cu wa\ assessed by plating six sets of macrophage\ and stimulating with sterile meconium in the above concentration\, After 2 hours of stimulation, mRNA was harvested and transcripted to murine. cDNA. TNF-cu DNA was then amplified by polymerase chain reaction (PCR) using murine TNF-cu PCR primer (RT-PCR kit: Clontech Laborntories, Palo Alto. CA ). In a separate serie\ of experiments. sets of macrophages at a concentration of IO5 were stimulated for 8 hour? with varying concentrationh of meconium. Po?tstirnulation supernatants were assayed for TNF-cc 3ctiVity using a commercial ELISA kit (Genzyme. Cambridge. MA,. Data were analyzed using an analysis of variance (ANOVA) and Studenl’\ t test to compare data between groups with P less than .O5 considered signiiicant.

Fig 1. stimulation. .05 versus

0.01

O.WI

Peritoneal macrophage PCA in response to meconium Meconium concentrations are noted on the X axis. *P < negative controls.

peak response at 1% meconium (P < .05, Fig 1). Pretreatment with the PKC inhibitor H-7 limited procoagulant activity to less than 5% of controls in response to meconium stimulation (Fig 2). H-7 inhibition was statistically significant for control stimulation with LPS; however, meconium stimulation only reached marginal significance (P = .06). Meconium stimulation resulted in marked increase in TNF-ol mRNA as well as supernatant levels of TNF-CY (P < .05). with a maximal response at 0.1% meconium (Figs 3 and 4). DISCUSSION

Meconium peritonitis is a result of an in utero intestinal perforation. The subsequent inflammatory response leads to significant adhesions throughout the peritoneal cavity. While meconium is sterile, it is clearly capable of eliciting a dose-dependent pro-inflammatory response, as evidenced by increased macrophage TNF-a production and procoagulant activity. The meconium-stimulated production of these pro-inflammatory mediators suggests that they play an important role in the development of the

RESULTS

Procoagulant activity was increased significantly by meconium stimulation in a dose-dependent fashion with a

0.1

LPS (-,

Fig 2. H7. *PC

Peritoneal .05 versus

US(+)

H-7+LPS

1%’ Mcc.

macrophage PCA activity with stimulated positive controls.

H-7+1%

Met.

the PKC inhibitor

216

LALLY

ET AL

700 650 6OO 550 500 450 2 2il la

Jo0 350 300 250 200 150 loo 50 0

LPS l-1

LPS (+I

1.0 0.1 0.01 Mecomum Concentrat1onl70)

Fig 3. Peritoneal macrophageTNF tion, supernatant levels in pg/mL.

response

to maconium negative

*P < .05 versus

0.001

stimulacontrols.

inflammation and adhesions seen at laparotomy. Procoagulant activity on the surface of the macrophage has been shown to be an important event in the inflammatory cascade with expression of tissue factor correlating with transplant rejection and other sequelae of acute inflammation.‘0.12,‘3 Inhibition or blockade of tissue factor causes a significant improvement in survival rate in animal models.14 We have shown that the macrophage responds to meconium stimulation; however, the mechanism of the meconium-induced response is unclear. Other investigators have demonstrated that induction of macrophage procoagulant activity involves protein kinase C.15-17Our data show that meconium-stimulated PCA activity was limited to less than 5% with inhibition of PKC, confirming the likely role of PKC in regulating meconiuminduced PCA activity. Other pro-inflammatory responses of the macrophage include the rapid production of tumor necrosis factor. Meconium clearly stimulates the peritoneal macrophage to produce TNF-a. Endotoxin-induced TNF-a production is in part mediated through the receptor CD14 with expression of TNF-a mRNA regulated by nuclear factor

LPS (-) LPS (+)

0.1

0.01

0.001

Meconium Concentration (%) Fig 4. Peritoneal macrophaga TNF mRNA in response to meconium stimulation. The concentration of maconium is noted at the bottom.

Kappa B.18 There is no known meconium receptor, and meconium may be activating the macrophage through several different mechanisms. Meconium is a complex mixture of bile salts, cell debris, and proteins, and several of these constituents have been shown to activate immune cells.19~‘U Meconium is clearly a strong pro-inflammatory mediator as evidenced by in vitro stimulation and clinical disease.2i-23We demonstrated procoagulant activity and TNF-(x production by macrophages to be increased significantly in response to meconium stimulation. PCA (in response to meconium and other stimulants) appears to involve a protein kinase C mechanism. Because macrophages mediate peritoneal inflammation, this response to meconium is a likely cause of the dense inflammation seen clinically in meconium peritonitis.

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