Zymosan-induced luminol- response of circulating and · 2019. 8. 1. · 3. Baskurt OK, Temiz A,...

This study examines a concurrent profiling ofcirculating and extravasated polymorphonuclear leu-kocytes (PMNs) in a rat model of experimental sepsis.Fecal peritonitis was induced in Wistar male rats byintraperitoneal instillation of a fecal suspension insaline (1:1 w/v). Blood and peritoneal fluid werecollected 8 h following fecal inoculation for theevaluation of inflammatory response of PMNs usingzymosan-induced luminol-dependent chemilumines-cence. Fifty microliters of pre-diluted blood or peri-toneal fluid samples were mixed with 150 ml ofreaction mixture (4�/10�4 M luminol�/50 mg opso-nized zymosan�/0.1% gelatin in Hank’s balanced saltsolution) and the chemiluminescence signal wasmeasured in a luminometer at 378C. Fecal peritonitiscaused a significant leukocytopenia (35409/297mm�3 versus control value of 75259/711 mm�3,pB/ 0.001) accompanied by massive infiltration ofPMNs in the peritoneal cavity (347009/4006 versus73259/425 mm�3, pB/ 0.001). The phagocytic activityof circulating blood PMNs was down-regulatedwhereas a significant up-regulation was observed inthe activity of PMNs from peritoneal fluid. In conclu-sion, this study clearly demonstrates sepsis-inducedalterations in both blood and peritoneal fluid PMNsand their quantitative assessment may be helpful indisease evaluation and designing effective therapies.

Key words: Septicemia, Peritonitis, Leukocytes, Phagocy-tosis, Chemiluminescence, Rat

Mediators of Inflammation, 13(2), 123�/125 (Aptil 2004)

Zymosan-induced luminol-dependent chemiluminescenceresponse of circulating andextravasated leukocytes inexperimental sepsis

Haseeb Ahmad KhanCA

Research Center, Riyadh Al Kharj Hospital Program,P.O. Box 7897 (T-835), Riyadh 11159, Saudi Arabia

CACorresponding authorTel: �/966 1 4777714, ext. 3809E-mail: [email protected]

Introduction

Peritonitis is a serious infection that often leads tomultiple organ failure, septicemia and mortality. Thecascade of events initiating from infection to septicshock and organ failure is poorly understood.Phagocytosis is the first line of host defense againstforeign antigens including bacteria. Polymorphonuc-lear leukocytes (PMNs) such as neutrophils andmacrophages are responsible for inactivating and

clearing bacteria and microbial-related noxious pro-ducts using the process of phagocytosis. The recruit-ment and activation of PMNs at the site of infectionare essential for effective host defense.1 However,prolonged activation of PMNs results in the excessivegeneration of pro-inflammatory cytokines and reac-tive oxygen species (ROS) that can lead to progres-sive damage of host tissue.2 Activated PMNs have alsobeen implicated in oxidant stress and altered bio-chemistry of red blood cells in sepsis.3

Recently, Holzer et al .4 suggested that immuno-modulation to down-regulate circulating PMNs and toup-regulate emigrated PMNs might offer a valuableprotection against complicated peritonitis. Thus, aconcurrent evaluation of the phagocytic response ofPMNs in blood and peritoneal fluid could help in

grading septicemia and planning more effectivepharmacotherapies. In this investigation, an attempthas been made to evaluate phagocytic response ofPMNs in the blood and peritoneal fluid of ratssubjected to fecal peritonitis.

Materials and methods

Animals

Wistar male rats (2309/10 g) were divided into twogroups of five animals each. The rats were housed ina temperature-controlled room maintained in 12 hlight/dark cycles. The standard laboratory food andwater were freely available ad libitum except for anovernight fasting before inducing experimental sepsisin rats. The study protocol was supported by theResearch and Ethics Committee of Riyadh Al-KharjHospital Program, Saudi Arabia.

Experimental sepsis model

A small incision was made in the abdomen of ether-anesthetized rats for intraperitoneal instillation offecal suspension at a dosage of 1 ml/kg body weightof animals. The fecal suspension was prepared by

Short Communication

ISSN 0962-9351 print/ISSN 1466-1861 online/04/20123-03 – 2004 Taylor & Francis LtdDOI: 10.1080/09629350410001688558

123

dissolving fresh feces (1:1 w/v in normal saline)obtained surgically from the caecum of non-fastedhealthy rats, and was used within 2 h. Controlanimals received pre-autoclaved (1358C for 1 h) fecalsuspension. The wound was closed and the animalsreturned to their home cages. The surgery wasperformed aseptically. This procedure results in afecal peritonitis septicemia.5

Blood and peritoneal fluid samples

The specimens of blood and peritoneal fluid werecollected at 8 h following fecal inoculation. Thesamples were diluted either 500-fold (blood) or100-fold (peritoneal fluid) in Hank’s balanced saltsolution (HBSS) containing 0.1% gelatin, and werekept on ice until promptly analyzed.

Measurement of phagocytosis

A sensitive procedure based on zymosan-inducedluminol-enhanced chemiluminescence (CL) was usedto measure the phagocytic response of leukocytes.6

Fifty microliters of pre-diluted blood or peritonealfluid samples were mixed with 150 ml of reactionmixture (4�/10�4 M luminol�/50 mg opsonizedzymosan�/0.1% gelatin in HBSS) in the well of anopaque cliniplate (Labsystems, Helsinki, Finland).The CL signal produced by phagocytosing leukocyteswas measured in a luminometer (Model Luminoscan-RT; Labsystems) at 378C. Thirty cycles of measure-ments using a 5 sec counting time and a 70 secinterval time were performed for each sample.Leukocytes were counted in all the samples using ahemocytometer.

Statistics

The statistical significance between the control andsepsis groups was analyzed by independent samplet -test. p B/0.05 was considered statistically significant.

Results

The leukocyte count was significantly decreased inthe blood and increased in the peritoneal fluid ofanimals that underwent peritoneal fecal instillation

(Table 1). The peak CL response during the entirecounting time was not significantly different betweenthe control and peritonitis groups (Table 2), whereasa significant decrement in the integrated CL signalswas observed in the peritonitis group as comparedwith the CL response from the same volume (100 nl)of control blood. The normalization of CL to 1000leukocytes also showed reduced phagocytic re-sponse in the peritonitis group but without reachinga significance level (Table 2). In contrast to bloodanalysis, all the CL measurements (peak value,integral value, normalized value) were significantlyhigher in the peritoneal fluid of rats with septicemiaas compared with respective control values (Table 2).

Discussion

A significant reduction in blood leukocyte countfollowing experimental sepsis is in agreement withearlier studies.3,7 Transient leukocytopenia has alsobeen reported in patients with sepsis who developedrespiratory failure, suggesting that the disappearanceof PMNs from the blood stream and entrapment ofcells in lung capillaries is an early event of acuterespiratory distress syndrome.8 On the other hand,fecal peritonitis caused a significant increase in theleukocyte count within the peritoneal fluid, indicat-ing massive infiltration of PMNs towards the bacterialload. PMN migration into the peritoneal cavity inresponse to fecal peritonitis is an important mechan-ism of host defense against bacterial invasion as theexudative PMNs localize and contain infection byphagotizing and killing bacteria.9

The PMN migration to the peritoneal cavityshowed a highly significant up-regulation of theirphagocytic activity as opposed to a concurrent down-regulation of phagocytic activity of blood PMNs(Table 2). These findings are supported by an earlierstudy showing the ability of ascitic PMNs to producesignificantly high levels of ROS, including super-oxide, hydrogen peroxide and myeloperoxidase, ascompared with blood PMNs following inducedperitonitis in rats.10 As the ROS have a tendency ofproducing CL in the presence of luminol, a high CLsignal from migrated PMNs during sepsis indicatesexcessive generation of ROS that might trigger path-ways for host damage.2,10

Activated PMNs are considered the primary med-iators of remote organ injury in peritonitis.9,11 Thesevere systemic inflammation caused by sepsis mayalso lead to multiple organ failure, a condition that isoften fatal to the host.12 The natural resolution ofacute inflammation involves bulk clearance of extra-vasated inflammatory cells in an ordered manner.However, an imbalance between the clearance andinfiltration of PMNs and/or disruption of equilibrium

Table 1. Effect of experimental peritonitis on leukocytescount in blood and peritoneal fluid

Experimental group(n�/5)

Leukocyte count (mm�3)

Blood Peritoneal fluid

Control 75259/711 73259/425Peritonitis 35409/297* 34,7009/4006*

* pB/0.001 versus the respective control groups (t-test).

H. A. Khan

124 Mediators of Inflammation � Vol 13 � 2004

between bacterial load and extent of phagocytosingPMNs could be deleterious to host cells. Thereforethe role of PMNs should be evaluated in the light ofboth host defense as well as host injury, whileapproaches to control PMN infiltration to the infectedsite and regulation of their activity have improved theanimals’ survival and protected them against remoteorgan injury following experimental peritonitis.13�15

In conclusion, this study clearly showed differen-tial changes in the blood and ascitic PMNs followingfecal peritonitis. However, the real clinical applica-tion of these parameters in defining septicemia is yetto be established.

References

1. Mercer-Jones MA, Heinzelmann M, Peyton JC, Wickel D, Cook M,Cheadle WG. Inhibition of neutrophil migration at the site of infectionincreases remote organ neutrophil equestration and injury. Shock 1997;8: 193�/199.

2. Goya T, Morisaki T, Torisu M. Immunologic assessment of host defenseimpairment in patients with septic multiple organ failure: relationshipbetween compliment activation and changes in neutrophil function.Surgery 1994; 115: 145�/155.

3. Baskurt OK, Temiz A, Meiselman HJ. Red blood cell aggregation inexperimental sepsis. J Lab Clin Med 1997; 130: 183 �/190.

4. Holzer K, Richter A, Konietzny P, Schubel F, Wilhelm K, Henrich D.Functions of circulating and intra-abdominal polymorphonuclearleukocytes during human secondary peritonitis. Zentralbl Chir 2003;128: 291 �/297.

5. Noronha-Blob N, Lowe VC, Otterbein L, Steranka L, Burch RM. NPC15669 reduces mortality associated with sepsis in rats. J Pharmacol ExpTher 1993; 267: 664�/669.

6. Lilius E, Waris M. A very sensitive and rapid chemiluminescence methodfor the measurement of phagocytosis. Analytical Applications ofBioluminescence and Chemiluminescence . London: Academic Press,1984: 401�/404.

7. Jacobi CA, Ordemann J, Halle E, Volk HD, Muller JM. Impact oflaparoscopy with carbon dioxide versus helium on local and systemicinflammation in an animal model of peritonitis. J Laparoendosc SurgTech A 1999; 9: 305�/312.

8. Thommasen HV, Russel JA, Boyko WJ, Hogg JC. Transient leukopeniaassociated with adult respiratory distress syndrome. Lancet 1984; 2:809�/812.

9. Frode TS, Ferreira SI, Medeiros YS. Analysis of local and systemicinflammatory responses induced by polymicrobial peritonitis in mice.Mediat Inflamm 2001; 10: 237 �/243.

10. Shen SC, Nakao A, Kishimoto W, Harada A, Nonami T, Nakano M,Takagi H. The ability of polymorphonuclear leukocytes to produceactive oxygen in a model of peritonitis in rats. Surg Today 1993; 23:603�/608.

11. Wickel D, Cheadle W, Mercer Jones M, Garrison R. Poor outcome fromperitonitis is caused by disease acuity and organ failure, not recurrentperitoneal infection. Ann Surg 1997; 225: 744 �/753.

12. Bone RC. Immunologic dissonance: a continuing evolution in ourunderstanding of the systemic inflammatory response syndrome (SIRS)and the multiple organ dysfunction syndrome. Ann Intern Med 1996;125: 680�/687.

13. Gardinali M, Borrelli E, Chiara O, et al . Inhibition of CD11-CD18complex prevents acute lung injury and reduces mortality afterperitonitis in rabbits. Am J Respir Crit Care Med 2000; 161: 1022�/1029.

14. Tariq M, Al Moutaery A, Arshaduddin M, Khan HA, Price Evans D, JacobsS. Fluconazole attenuates lung injury and mortality in rat peritonitismodel. Intensive Care Med 2003; 29: 2043�/2049.

15. Perdue PW, Kazarian KK, Nevola J, Law WR, Williams T. The use of localand systemic antibiotics in rat fecal peritonitis. J Surg Res 1994; 57: 360�/365.

Received 2 December 2003Accepted 12 January 2004

Table 2. Phagocytic activity of leukocytes in blood and peritoneal fluid, measured by zymosan-induced luminol-dependentchemiluminescence response

Experimental group(n�/5)

Peak RLU(�/10�3)

Integral RLU(30 min)

RLU/1000leukocytes

BloodControl 4.009/0.91 4.5769/1.61 6.1609/2.26Peritonitis 3.009/0.54 0.8959/0.42* 2.9589/1.52

Peritoneal fluidControl 12.509/4.80 18.259/7.60 5.219/2.16Peritonitis 269.759/74.57** 397.059/112.37** 22.569/5.93*

RLU, relative light unit, an arbitrary unit of chemiluminescence measurement.* pB/0.05 and ** pB/0.01 versus the respective control group (t-test).

Phagocytosis in experimental sepsis

Mediators of Inflammation � Vol 13 � 2004 125

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