Alterations in rat peripheral blood neutrophil function as a consequence of colitis

Digestive Diseases and Sciences. Vol. 40, No. 1 (January 19951, pp. 192-197

Alterations in Rat Peripheral Blood Neutrophil Function as a

Consequence of Colitis LISA CARTER, PhD and JOHN L. WALLACE, PhD

Altered peripheral neutrophil function is a feature of IBD that may contribute to the chronicity and extragastrointestinal manifestations of this disease, but clinical evidence for such alterations is confounded by variations in patient characteristics, disease onset, and use of therapeutics that can influence neutrophil function. The use of a rat model of colitis has permitted us to characterize, in a controlled manner, the causal relationship between colitis and altered peripheral neutrophil function. At various times after induction of colitis with trinitrobenzene sulfonic acid (TNBS), peripheral neutrophils were isolated and assays of phagocytosis, chemotaxis, leukotriene B 4 (LTB4) synthesis, and superoxide production were performed using a variety of stimuli. Circulating neutrophil numbers increased about fourfold within 12 hr of TNBS administration and returned to normal levels over the following two weeks. LTB4 synthesis in response to calcium ionophore decreased at 12 hr after induction of colitis, then returned to control levels. The chemotactic responses of peripheral neutrophils to LTB 4 and FMLP in vitro and to LTB 4 and IL-8 in vivo were profoundly suppressed through the two-week study period. Phagocy- tosis of nitroblue tetrazolium was significantly enhanced (ca. threefold) at 12 hr after induction of colitis and remained elevated throughout the study period. Superoxide production was also significantly elevated in the early phase of colitis (by ca. fourfold), but was not different from control levels at seven and 14 days. These results demonstrate that colonic inflammation profoundly influences peripheral blood neutrophil function, although the direction and magnitude of the alteration varied among the various functions assessed. The prolonged depression of chemotactic activity may represent a physiological reaction to limit the inflammatory response.

KEY WORDS: neutrophils; inflammatory bowel disease; chemotaxis; leukotriene B4; superoxide; phagocytosis.

Neutrophil infiltration into the lamina propria is a common feature of both Crohn's disease and ulcerative colitis, and probably accounts for significant nonspecific injury in these diseases (1-3). Current

Manuscript received October 15, 1993; revised manuscript received June 7, 1994; accepted July 28, 1994.

From the Intestinal Disease Research Unit, Faculty of Medi- cine, University of Calgary, Calgary, Alberta, T2N 4N1, Can- ada.

Address for reprint requests: Dr. J. L. Wallace, Department of Pharmacology & Therapeutics, University of Calgary, 3330 Hos- pital Drive NW, Calgary, Alberta, T2N 4N1, Canada.

therapies for IBD, including corticosteroids and aminosalicylates, may produce their beneficial effects, in part through inhibitory actions on neutrophil function. A plethora of clinical studies over the past three decades has provided compelling evidence of peripheral neutrophil dysfunction in IBD (4-8), and it has been argued that such dysfunction may contribute to the chronicity of these diseases and to the generation of extragastrointestinal manifestations. Neutrophil dysfunction may also be a contributing factor in the development of IBD in

192 Digestive Diseases and Sciences, VoL 40, No. 1 (January 1995) 0163-2116195/0100-0192507.5010 © 1995 Plenum Publishing Corporation

NEUTROPHIL DYSFUNCTION IN EXPERIMENTAL COLITIS

some condit ions. For example, impaired neutrophil chemotaxis and phagocytosis associated with glycogen storage disease lb has been suggested to predispose these patients to the development of Crohn ' s disease (9). In addition, while impaired neutrophil superoxide product ion is characterist ic of chronic granulomatous disease, it is also possible that such an impairment of neutrophil funct ion could contribute to the pathogenesis of Crohn ' s disease (7). Indeed, when chronic granulomatous disease involves the intestine, the histopathological features are indistinguishable f rom Crohn ' s disease (10).

One of the difficulties in interpreting the collec- tive data of the many studies of peripheral neutrophil function in IBD is determining whether the observed changes preceded the disease or were purely a consequence of it. Another difficulty in interpreting these studies relates to the variability in a number of factors from one study to the next, including variability in: (1) disease activity and/or duration; (2) patient characterist ics (eg, sex, age, smoking); (3) presence or absence of extragastrointestinal manifestat ions; (4) use of drugs that can alter neutrophil function; and (5) methods em- ployed to assess neutrophil function. There also appear to be significant differences in various neutrophil functions between Crohn ' s disease and ul- cerat ive colitis (1, 2).

We have therefore at tempted to determine if peripheral neutrophil function is altered as a consequence of colitis and to characterize these changes in an experimental model of colitis, which has previously been shown to exhibit many of the histopathological character is t ics of human IBD (11). While animal models do not provide a complete representat ion of human IBD, they do provide a useful tool for s tudying neutrophil function before and at var ious times after induction of colitis, in the absence o f the confounding influence of medication and in a homogeneous " 'pa t ient" population.

MATERIALS AND METHODS

Induction of Colitis. Male Wistar rats (175-200 g) obtained from Charles River Canada Inc. (St. Constant, Quebec, Canada) were housed in the Animal Resources Centre of the University of Calgary. The rats were given access to water and standard laboratory chow throughout the experimental period. All experimental protocols were approved by the Animal Care Committee of the Univer- sity of Calgary.

Colitis was induced by the intracotonic administration of 0.5 ml TNBS (60 mg/ml in 50% ethanol), as previously

described in detail (11). Groups of five to six rats each were studied 12 hr and one, seven, and 14 days after induction of colitis. Untreated rats served as controls.

The severity of colitis was assessed after blood had been withdrawn for isolation of peripheral blood neutrophils (see below). The distal 10 cm of colon was excised, opened by a longitudinal incision, and the luminal con- tents were removed in warm tap water. The colon was pinned onto a wax mount and microscopically visible damage was assessed using previously described criteria (12). Segments (-100-200 mg) of colonic tissue were then excised for subsequent assay of myeloperoxidase (MPO) activity (12), as well as for histological assessment.

Total and Differential Leukocyte Counts. Rats were anesthetized with ether and approximately 5 ml of blood was removed by cardiac puncture and placed into a tubes containing 0.1 M sodium citrate. Cell smears were performed for differential analysis and 100-lxl samples of blood were treated with an equal volume of 3% acetic acid to lyse erythrocytes. Total leukocyte numbers were determined using a Coulter counter.

Neutrophil Function Assays. The phagocytic ability of the peripheral blood neutrophils, in the absence and presence of endotoxin, was determined using a commercially available nitroblue tetrazolium (NBT) kit, as previously described (13).

The generation of LTB 4 by neutrophils was determined following activation of cells with calcium ionophore (A2.3187). In these experiments, 1-ml samples of heparin- ized blood were placed into a 37°C shaking water bath and A23187 (10 ~M) was added. The samples were incubated for 30 min. The blood samples were then centrifuged for 3 min at 9000g in an Eppendorf microfuge, and superna- tants were removed and frozen at -70°C for subsequent determination of LTB 4 concentration using a specific radioimmunoassay (14).

Superoxide anion production and the chemotactic responses of neutrophils were determined using pooled blood samples from five rats in each experiment. Neutro- phils were enriched using a modification of a method previously described (13). Briefly, 6 ml of whole blood was subjected to dextran sedimentation in round-bottom polypropylene tubes. The buffy coats were transferred into a 50-ml centrifuge tube and subjected to two hypo- tonic lysis steps to remove contaminating erythrocytes. After washing the cells in 50 ml of phosphate-buffered saline (PBS; pH 7.4) at 300g for 6 min (4°C), the pellet was resuspended in 2.5 ml of PBS and layered over 3 ml of Histopaque and centrifuged at 350g for 30 min. The pellet was resuspended in PBS. Final cell yields in untreated rats were 9-10 x 107 cells/ml with >85% of the cells determined microscopically to be neutrophils. The generation of superoxide anion was determined using a kinetic spectrophotometric assay previously described (15). The agonists used for generation of superoxide included phorbol myristate acetate (PMA, 2.5 v.M) and n-formyl-leucyl- methionyl-phenylalanine (FMLP, 50 p.M). In the latter case, dihydrocytochalasin B (25 mM) was used as a priming agent. Chemotaxis to LTB 4 (150 nM) and FMLP (10 nM) was determined using an in vitro microchemo- taxis assay (13). Aliquots (2 x 105) of [Xlqn]HCl (2-5 i~Ci/106 cells)-labeled neutrophils were placed in each

Digestive Diseases and Sciences, Vol. 40, No. 1 (January 1995) 193

CARTER AND WALLACE

upper well. The chemotactic index of migration of the neutrophils was determined after removal of background values (13).

Migratory responses of neutrophils were also assessed using an in vivo model. These studies were performed using groups of five to six rats each on days 1, 7, or 14 after intracolonic administration of TNBS. The rats were lightly anesthetized with ether, the back was shaved, and 0.1 ml of LTB 4 (150 riM), IL-8 (100 nM), and sterile Ringer's solution were injected intradermally at discrete sites. After 2 hr, the injection sites were excised and stored at -70°C until assay for MPO activity as an index of neutrophil infiltration (16). As it was possible that neutrophil MPO content might change as a consequence of colitis, and therefore influence the results obtained in the experiments described above, a series of experiments were performed in which the MPO activity in 75-~1 samples of peripheral blood taken at 1-14 days after induction of colitis was measured. The MPO activity was then expressed on a per 10 6 neutrophils basis, using neutrophil counts performed on separate blood samples from the same animals.

Statistical Analyses. All data are presented as the mean SEM. Comparisons between groups of data were made

using a one-way analysis of variance followed by a Bon- ferroni test. An associated probability (P value) of 5% or less was considered significant.

Materials. TNBS was obtained from J.T. Baker Chem- ical Co. (Toronto, Ontario, Canada), recombinant human IL-8 from R&D Systems (Minneapolis, M!nnesota), LTB 4 from Cayman Chemical Co. (Ann Arbor, Michi- gan), [tllln]Cl and [~H]LTB4 from Amersham Canada (Oakville, Ontario, Canada). The antibody to LTB4 was generously supplied by Dr. A. Ford-Hutchinson of Merck-Frosst (Dorval, Qu6bec, Canada). All other re- agents were obtained from Sigma Chemical Company (St. Louis, Missouri).

RESULTS

A s descr ibed in detai l p rev ious ly (11), in t raco- lonic adminis t ra t ion of T N B S resul ted in the devel- opmen t o f ex tens ive u lcera t ion and inf lammation in the distal co lon , which pers i s ted throughout the two-week s tudy per iod . As shown in Figure 1, both the colonic damage score and t issue mye lope rox i - dase ac t iv i ty were s ignif icant ly (P < 0.01) e l eva ted above that of un t rea ted con t ro l s at all t imes examined.

Induct ion of coli t is resu l ted in an app rox ima te ly twofold increase in total numbers of b lood leuko- cy t e s wi th in 12 hr (controls : 5.7 _+ 1.5 x 10 s cel ls / ml; T N B S : 10.7 -+ 2.2 × 108 cel ls /ml; P < 0.05), wi th a d i s p r o p o r t i o n a t e i nc rease (ca. fourfold) in the n u m b e r of neu t roph i l s (F igure 2). The number s of pe r iphe ra l neu t roph i l s s u b s e q u e n t l y d e c r e a s e d to con t ro l l eve l s wi th in s even days . In c o n t r a s t to the enhanc ing effects of induc t ion of col i t is on

194

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o3

E ¢3 6 o

F, 8 3

0 0 , II ,

(Control] Days after TNBS

o

lO0

a0 '~.

40 ~ ~

~t 0

2o ~

Fig 1. Colonic damage score and tissue myeloperoxidase activity at various times after induction of colitis with trinitrobenzene sulfonic acid (TNBS). Both parameters were significantly elevated (P < 0.01) above control levels at all time points shown. Each point represents the mean _+ SEM o f at least five rats.

p e r i phe ra l neu t roph i l n u m b e r s , the ab i l i ty o f the neu t roph i l s to r e l ease L T B 4 in r e s p o n s e to s t imu- la t ion wi th ca lc ium i o n o p h o r e was p r o f o u n d l y de- p r e s s e d in i t ia l ly and r e t u r n e d to no rma l leve ls t he r e a f t e r (F igure 2). A s p e r i p h e r a l ne u t roph i l n u m b e r s r e t u r n e d to con t ro l leve ls (by s e v e n d a y s p o s t - T N B S ) , so too did the i r ab i l i ty to r e l ease L T B 4.

R e s p o n s i v e n e s s of neu t roph i l s to c h e m o t a c t i c s t imuli in vitro was also p ro found ly a l te red subsequent to induct ion of coli t is (Figure 3). Neu t roph i l migrat ion in r e sponse to L T B 4 was r educed b y 89% at 12 hr after T N B S admin is t ra t ion and remained

5 E

== ,

a

i=

j 2 z

z 0

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100

80 .*~ ..o i ' 2 0

' 0

Fig 2. Circulating neutrophil number and leukotriene B 4 synthesis by peripheral neutrophils at various times after induction of colitis with trinitrobenzene sulfonic acid (TNBS). Neutrophil numbers were significantly (P < 0.05) elevated at 12 hr post- TNBS, but not significantly different from controls thereafter. LTB, synthesis was significantly (P < 0.05) depressed at 12 and 24 hr post-TNBS, but not significantly different from control levels thereafter. Each point represents the mean _+ SEM for at least 5 rats.

Digestive Diseases and Sciences, Vol. 40, No. 1 (January 1995)

NEUTROPHIL DYSFUNCTION IN EXPERIMENTAL COLITIS

g

I t

m¢ 6 qII

m

U

o Q

0

0 t i i ~ t i

0 3 6 9 12 15 (Control)

Days after TNBS Fig 3. Peripheral neutrophil chemotaxis in vitro in response to FMLP (10 nM) and LTB 4 (150 nM) at various times after induction of colitis with trinitrobenzene sulfonic acid (TNBS). Aster- isks denote significant differences from the control group with LTB4 used as an agonist (**P < 0.001, ***P < 0.001), while stars indicate significant differences from the control group with F M L P used as an agonist ( * * P < 0.01, * * * P < 0.0001).

suppressed throughout the study period. Chemotaxis in response to FMLP was also markedly suppressed, particularly at seven and 14 days after TNBS administration. When migration of neutrophils was assessed in vivo, in response to intradermal injections of LTB 4 or IL-8, once again, the responses were depressed in rats with colitis. As shown in Figure 4, in which the degree of neutrophil recruitment into the skin injection sites has been normalized for changes in circulating neutrophil numbers, the re- sponsiveness of the neutrophils was significantly depressed at all times examined. Neutrophil infiltration in these experiments was determined by measuring tissue myeloperoxidase activity. These changes in recruitment of neutrophils after induction of colitis were not attributable to changes in cellular content of MPO, as these levels remained unchanged throughout the study period (eg, 63 4- 21 units/106 neutrophils in controls; 72 4- 30 units/106 neutrophils in the two week TNBS group).

Phagocytosis of NBT was examined under resting conditions and following stimulation with endotoxin. Under resting conditions, 8 4- 3% of neutrophils from controls rats were NBT-positive. This was dramatically increased following induction of colitis. At 12 hr after TNBS administration, 28 --- 4% of neutrophils were NBT-positive. The percent of NBT-positive cells remained markedly elevated throughout the study period (22 ___ 5% at day 14). When stimulated with endotoxin, the number of

Digestive Diseases and Sciences, VoL 40, No. 1 (January 1995)

o 4

.

o Control 1 7 14

Days After TNBS

Fig 4. Recruitment of granulocytes into skin in response to intraderma] injections of 100 p.] of LTB 4 (150 nM) and [L-8 (]00 nM) at various times after induction of colitis with trinitrobenzene sulfonic acid (TNBS). Granulocyte infiltration was assessed 90 min after injection of the chemotaxins, by measuring tissue myeloperoxidase (MPO) activity. Each bar represents the mean ± SEM of at least five rats. Asterisks denote significant differences from the corresponding control (day 0) group (*P < 0.05, **P < 0.01., ***P < 0.001).

NBT-positive neutrophils from control and TNBS- treated rats increased significantly (69 -- 9% in controls; 71 4- 9% in the 12-hr TNBS group), with no significant differences among the various groups examined.

Superoxide production stimulated by FMLP or PMA increased significantly (more than fivefold) within 24 hr of TNBS administration and returned to control levels thereafter (Figure 5).

DISCUSSION

Numerous clinical studies have documented significant abnormalities in neutrophil function in patients with inflammatory bowel disease, although the nature of the abnormalities reported varies con- siderably from one study to the next. For example, neutrophil migration in IBD patients has been described as increased, unchanged, and decreased, depending on the population of IBD patients studied (ulcerative colitis versus Crohn's disease) and the methods and agonists used (1, 4-8). While some of these studies have correlated the neutrophil func- tional alteration with disease activity, it is not possible to draw conclusions regarding the question of whether the alteration preceded or was a consequence of the intestinal disease. The present study was performed to address this question in an animal model of colitis. The TNBS model of colitis is characterized by inflammation with both an acute

195

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~ ' ~ PMA

0

0 7 {Control)

Days after TNBS

Fig 5. Superoxide generation by peripheral neutrophils at various times after induction of colitis with trinitrobenzene sulfonic acid (TNBS). Superoxide production was stimulated with phorbol myristate acetate (PMA, 2.5 ~M) or FMLP (50 ~M). Superoxide production was significantly increased (***P < 0.0001) with both agonists at 24 hr post-TNBS, but was not different from control levels thereafter. Each point represents the mean -+ SEM for four to six experiments.

and chronic phase, the latter persisting for several weeks after administration of the hapten (11). While not representing all aspects of human IBD, this model does bear numerous similarities to Crohn's colitis, including transmural, granuiomatous inflammation of the distal colon (11). The results presented herein document significant changes in neutrophil function, both in vitro and in vivo (Table 1), although most of these changes were transient in nature. While some of the parameters measured

TABLE 1. SUMMARY OF EFFECTS OF COLITIS ON NEUTROPHIL FUNCTION*

Acute Chronic Function Stimulus change change

Superoxide production PMA '~ ~, FMLP 1' ,-~

Phagocytosis none t 1' endotoxin ~ *-,

LTB 4 synthesis A23187 ,L ~-,

Chemotaxis LTB 4 (in vitro) ~ FMLP (in vitro) ~-, LTB 4 (in vivo) ~ J, ILl-8 (in vivo) ~

* " A c u t e " refers to the first 12-24 h after induction of colitis, while "chron ic" refers to 7-14 days after induction of colitis. 1', indicates a significant increase; $ indicates a significant

decrease, ,-% indicates no significant effect (P < 0.05 versus controls).

CARTER AND WALLACE

(phagocytosis, superoxide production) were enhanced, chemotaxis was profoundly suppressed throughout the two-week study period. This obser- vation is consistent with some of the clinical studies of Crohn's disease and most of the clinical studies of ulcerative colitis (1). Changes in chemotactic activity could be attributable to changes in the numbers of receptors for the chemotaxin expressed on the neutrophil , as has been demons t ra ted in Crohn's disease for FMLP (2). However, as the depressed chemotaxis was observed with three different agonists (FMLP, LTB 4, and IL-8), this seems unlikely. The decreased chemotactic properties of neutrophils from animals with colitis do not neces- sarily indicate neutrophil dysfunction. It is possible that the suppression of chemotactic function is a physiological reaction to the colonic inflammation, acting to down-regulate the inflammatory response.

The ability of neutrophils to phagocytose nitroblue tetrazolium was also altered following induction of colitis, with the alteration persisting throughout the two-week study period. Under resting conditions, a greater proportion of neutrophils in the animals with acute colitis were NBT-positive than those in controls. However, in response to activation with endotoxin, similar proportions of neutrophils in controls and colitis were capable of ingesting NBT. This suggests that the resting neutrophils of colitic animals were "p r imed , " perhaps through exposure to circulating cytokines, inflammatory mediators, or endotoxin. Interestingly, similar results have been reported in clinical studies of neutrophil phagocytosis in IBD. In both ulcerative colitis and Crohn's disease, an increased percent- age of neutrophils were NBT-positive (1, 6, 17, 18).

Whether or not the suppression of chemotaxis is an adaptive defensive response aimed at reducing infiltration of neutrophils into the colon in response to the high levels of chemotaxins being produced remains to be determined. It is noteworthy that one of the mechanisms through which neutrophils can down-regulate their chemotactic response is by in- creasing expression of a neutral endopeptidase on their surface, which can inactivate various peptide chemotaxins. Indeed, it has recently been suggested this adaptive response of neutrophils is impaired in 1BD (19). Further studies are warranted to better characterize the changes in neutrophil function that occur as a consequence of colonic inflammation and the contribution of such changes to the inflammatory process.

196 Digestive Diseases and Sciences, VoL 40, No. 1 (January 1995)

N E U T R O P H I L D Y S F U N C T I O N IN E X P E R I M E N T A L COLITIS

ACKNOWLEDGMENTS

This work was suppor ted by a grant from the C r o h n ' s and Colitis Founda t ion of Canada. Dr. Wal lace is a Med- ical Resea rch Council of Canada Scient is t and an Alber ta Her i tage Founda t ion for Medical Research Scientist . The au thors are grateful to Tai Le for his ass is tance in per- forming these studies.

R E F E R E N C E S

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4. Curran FT, Keighley MRB: Neutrophil motility in Crohn's disease. Digestion 48:104-112, 1991

5. Williams JG, Hughes LE, Hallett MB: Toxic oxygen metab- olite production by circulating phagocytic cells in inflammatory bowel disease. Gut 31:187-193, 1990

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7. Curran FT, Allan RN, Keighley MRB: Superoxide production by Crohn's disease neutrophils. Gut 32:399-402, 1991

8. Elmgreen J, Binder V: The chemotactic function of neutrophils in ulcerative colitis. Scand J Gastroenterol 17:561-564, 1982

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chronic granulomatous disease. N Engl J Med 288:382-387, 1973

11. Morris GP, Beck PL, Herridge MS, Depew W, Szewczuk MR, Wallace JL: Hapten-induced model of chronic inflammation and ulceration in the rat colon. Gastroenterology 96:795-803, 1989

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13. Carter L, Ferrari JK, Davison JS, Befus AD: Inhibition of neutrophil chemotaxis and activation following decentraliza- tion of the superior cervical ganglia. J Leukocyte Biol 51:597-602, 1992

14. Wallace JL, MacNaughton WK, Morris GP, Beck PL: Inhi- bition of leukotriene synthesis markedly accelerates healing in a rat model of inflammatory bowel disease. Gastroenter- ology 96:29-36, 1989

15. Woodman RC, Erickson RW, Rae J, Jaffe HS, Curnutte JT: Prolonged recombinant interferon--y therapy in chronic granulomatous disease: evidence against enhanced neutrophil oxidase. Blood 79:1558-1563, 1992

16. Bradley PP, Priebat DA, Christensen RD, Rothstein G: Measurement of cutaneous inflammation: estimation of neutrophil content with an enzyme marker. J Invest Dermatol 78:206-209, 1982

17. Ward M, Eastwood MA: The nitroblue tetrazolium test in Crohn's disease and ulcerative colitis. Digestion t4:179-193, 1976

18. Kirk AP, Cason J, Fordham N J, Brown KA, Goddard DH, Holbrow E J, Lennard-Jones JE: Polymorphonuclear leukocyte function in ulcerative colitis and Crohn's disease. Dig Dis Sci 28:236-278, 1983

19. Cole AT, Garlick N, Buchanan M, Galvin A, Hawkey CJ, Robins A: Reduced expression of neutrophil neutral endopeptidase in inflammatory bowel disease. Gastroenterol- ogy 106:A666, 1994

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Alterations in rat peripheral blood neutrophil function as a consequence of colitis