SHOCK, Vol. 13, No.5, pp. 361-366, 2000
`
`Clinical lnt·estigations
`
`NEUTROPHIL APOPTOSIS IS DELAYED IN PATIENTS WITH
`INFLAMMATORY BOWEL DISEASE
`
`Ann E. Brannigan,* P. Ronan O'Connell,* Hilary Hurley,* Amanda O'Neill,*
`Hugh R. Brady,t John M. Fitzpatrick,* and R. William G. Watson*
`Department of ·surgery and tMedicine and Therapeutics, University College Dublin, Mater Misericordiae
`Hospital, 47 Eccles Street, Dublin 7, Ireland
`
`Received 30 September 1999; first review completed 25 October 1999; accepted in final form 11 November 1999
`
`ABSTRACT - Delayed neutrophil apoptosis is a feature of persistent acute Inflammation. Neutrophil(cid:173)
`mediated damage has been shown to be associated with the development of inflammatory bowel disease
`(lBO). Persistence of these cells both at the colonic site and circulation may further contribute to lBO. The
`aims of this study were to determine whether neutrophils isolated from lBO patients delay apoptosis and to
`investigate possible mechanisms involved in this delay. We studied 20 patients with lBO, 13 with Crohn's
`disease, and 7 with ulcerative colitis, all of whom were undergoing intestinal resection for symptomatic
`disease. Seventeen patients undergoing elective resection of colon cancer acted as operative controls.
`Systemic, mesenteric arterial, and mesenteric venous blood was harvested. Neutrophlls isolated from pa·
`tients with IBD showed decreased spontaneous apoptosis compared to cancer patients. Mesenteric venous
`serum of lBO patients contribU1ed to this delay, which contained higher concentrations of interteukin-8 (IL-8).
`Pro-caspase 3 express1on was also reduced in lBO neutrophils, which may contribute to decreased spon(cid:173)
`taneous and Fas antibody-induced apoptosis. Neutrophil apoptosis may be altered In Crohn's disease and
`ulcerative colitis through release of anti-apoptotic cytokines and altered caspase expression. The alterations
`in cell death mechanisms may lead to persistence of the inflammatory response associated with lBO.
`
`KEYWORD5-Caspase, IL-8, Fas antibody, reactive oxygen intermediates, hydrocortisone
`
`INTRODUCTION
`
`Apoptosis or programmed cell death is partiall y responsible
`for resolution of a neutrophil-mediated acute inflammatory re(cid:173)
`sponse (1). This process clears potentially LOxic neutrophils
`from the focus of inflammation without induction of an addi(cid:173)
`tional inflammatory stimulus that might otherwise result. Ap(cid:173)
`optosis is a constitutively expressed program, regulated by in(cid:173)
`flammatory mediators (2). Lipopolysaccharide, GM-CSF (3).
`interlekin-8 (1 ~8) (4, 5), and glucocorticoids (6) as well as the
`process of migration (7) all delay apoptosis, whereas ingestion
`of Escherichia coli (8). ligation of the Fas (9. 10), and tumor
`necrosis factor (TNF) receptors ( II) induce this process.
`Caspases are central proteases involved in the induction of
`apopLOsis ( 12). Fas antibody and TNF trigger their activation
`during apoptosis ( 12, 13). Altered ex pre ion of these caspases
`may represent mechanisms for resistance to apoptotic death as
`they would not be available for acti\'ation.
`The term inflammatory bowel di ease (lBD) generally refers
`to Crohn's disease (CD) and ulcerative coliti (UC). Crohn's
`disease is characterized by areas of transmural inflammation of
`the bowel wall interspaced by macroscopically healthy areas.
`Classically. it effects the tenninal ileum, perineum. and colon
`but can effect any area of the enteric tract ( 1-l ). ln contrast, UC
`
`Address reprint request> to R. W. G. Watson. Department of Surgery,
`University College Dublin. Mater Mosericordiac Hospital. 47 Eccles Srreet.
`Dublin 7. Ireland.
`
`361
`
`is characterized by mucosal inflammation confined to the colon
`and rectum. Both C D and UC pursue an indolent course inter(cid:173)
`spaced by acute exacerbation, during which features of an
`acute inflammatory illness are manifest ( 15).
`During the acute phase of ffiD in response to putative im(cid:173)
`mune challenge, inflammatory cells are present in the colonic
`wall ( 16). Both systemic and localized neutrophils release re(cid:173)
`active oxygen intennediates and proteolytic enzymes that con(cid:173)
`tribute to tissue damage ( 17). Neutrophil-mediated bystander
`tissue injury is an inevitable feature of the acute local inflam(cid:173)
`matory response in a number of clinical and experimental mod(cid:173)
`els ( 14. 17-19). The extended neutrophil life span can thereby
`resull in persistent inflammation and extensive tissue damage
`at the site of inflammation.
`We hypothesized that rBD might be associated with de(cid:173)
`creased neutrophil apoptosis and that mesenteric factors may
`contribute to this delay. which mediate their effects by altering
`the signaling mechanisms of apoptosi .
`This study demonstrates that neutrophils isolated from pa(cid:173)
`tients with LBO have decreased apoptosis compared to opera(cid:173)
`tive control cancer patients and healthy volunteers. This delay
`is as ociated with the production of survival factors in the
`serum as well as alterations in the cell death pathway in the
`neutrophil. Serum factors specifically from the mesenteric vein
`decrease apoptosis of normal neutrophils. lL-8 is increased in
`the serum but monoclonal antibodies to fL-8 do not inhibit the
`anti-apoptotic effects of serum. IBD neutrophlls also have de-
`
`

`

`362 SHOCK VoL. 13, No. 5
`
`BRANNIGAN ET AL.
`
`creased pro-caspase 3 expression, which may explain their de(cid:173)
`lay in spontaneous apoptosis. IL-8 decreases both spontaneous
`and Fas anti-body induced apoptosis that is also associated
`witJ1 decreased pro-caspase 3 expression. AJtered caspase ex(cid:173)
`pression may represent a central mechanism for altered apop(cid:173)
`totic responses.
`
`MATERIALS AND METHODS
`
`Patients
`Patients undergoing inteStinal resection for symptomatic CD or UC were
`assigned a disease activity index score (20). Patients with inlnlperitoneal sepsis
`or a pre-existing immune or haematological disorder were excluded. No patient
`received pre or peri-operative blood t:ransfusioo. The control group comprised
`healthy volunteers. Pati.:nlS undergoing colonic resection for adenocarcinoma
`acted as operative controls. The study protocol was reviewed and approved by
`the Mater Misericordiae Hospital Ethics Committee and written consent was
`obtained from all patients or a surrogate.
`We studied 20 patients with ffiD, 13 with CD, and 7 with UC, mean age 40
`years (range 1&-77) with a male:female ratio 7:12. All patients selected were
`only on long-term, low-dose oral glucocortiCQids. At lhe time of operation,
`hydrocortisone (100 mg, i.v.) as administered Seventeen patients undergoing
`elective resection of colon cancer acted as operative controls. mean age 78
`years (range 54-83) with a male:female ratio of 12:5. Disease severity was
`graded according to lhe Crohn's disease activity index of Harvey and Brad(cid:173)
`shaw (20). All patients had moderate or severe disease. Teo cont:rol healthy
`volunteers with a mean age of 34 years (range 21-44) wilh a male: female ratio
`of 5:5 were used. Peri-operatively the mesenteric vessels of lhe intestinal
`segment to be resected were isolated. Mesenteric venous and arterial blood (I 0
`mL) was withdrawn prior to vascular clamping. specifically from lhe capil(cid:173)
`laries adjacent to the bowel. Simultaneously, 10 mL of systemic blood was
`withdrawn via a peripheral arterial line. All samples were collected into hep(cid:173)
`arin-coated rubes (I U heparin per mL blood). Samples were transported
`immediately to the laboratory where serum and neutrophi1s were isolated.
`
`Reagents and antibodies
`Dulbecco's Modified Elagle Medium (DMEM), penicillin and streptomycin
`solutio n, L-glutamine, and fetal calf serum (FCS) were purchased from
`GIBCO Life Technologies Ltd .. Paisley, UK. Dextran T 500 and Ficoll were
`purchased from Pharmacia. Buclcinghamshire, UK. E-Lysis was purchased
`from Cardinal Associates (Santa Fe, NM). Murine anti-human CD II b (Bear-1)
`PE-conjugated antibody was purchased from Becton Dickinson, Cambridge
`UK. Pro~Caspase 3 antibody (mouse anti-human) was purchased from Trans(cid:173)
`duction laboratories (Lexington, UK); this anti-body is specific for the pro(cid:173)
`(32kDa) protein and does not cross react wilh lhe active p17 and p12 proteins.
`Fas-antibody (CH-11) was purchased from lmmunotech, Bedfordshire, UK.
`All remaining chemicals were purchased from Sigma-Aldrich Company Ltd.,
`Dorset. UK if not otherwise stated.
`
`Neutrophil isolation
`l".feutrvphils ~·ere isolated by dext:-..n (6%) sed!mentati{)n !..fld cerurifuga•-inn
`through a discontinuous Ficoll gradient as described previously (7). Neutro(cid:173)
`phil-rich peiiCLS were subjected to hypotonic lysis of the remaining erythro(cid:173)
`cytes with E-Lysis. Cell pellets were resuspended in Dulbecco's modified
`Eagle's medium (DMEM) supplemented with 10% FCS (heat inactivated). 1%
`glutamine, and I% penicillio/streptomycin solution at a concentration of l x
`106 cells /mL. Cells were incubated in polypropylene tubes (Falcoo!Becton
`Dickonson, Cambridge, UK) to prevent adherence. Neut:rophil purity was 95%
`as assessed by s ize and granularity on now cytometry and viability 98% as
`assessed by trypin blue and propidium iodide excll!Sion.
`
`Quantification of apoptosis
`Apoptosis was quantitated by flow cytomeu:y as lhe percentage of cells with
`hypodiploid DNA. Cell buspensions were centrifuged at 200 g for 10 min. The
`cell pellets were resuspended in 500 j.i.L of hypotonic fluorochrome solution
`(50 j.l. g/mL PI, 3.4 mM sodium cirrate, I mM EDTA. 0.1 % Triton X-100) and
`stored in the dark at 4°C before they were analyzed using a Coulter Elite
`cytofluorometer (Coulter, Bedfordshlre. UK).
`
`Respiratory burst activity
`Generation of oxygen intermediates was assessed using the technique of
`Smith (2 1). Neutrophils ( I x IW/mL) were pre-incubated wilh dibydrorhoda(cid:173)
`mine123 for 5 min at 37°C and then stimulated with PMA (5 nM) for 10 min.
`Fluorescence intensity was assessed by spectroscopy (Perkin-Elmer LC-30.
`London, IJK.). Dihydrorhodamine123 bas been shown to detect mainly 1{20 2
`and OH radicals.
`CD11 b expression
`Surface expression was assessed by flow cytometry. Ten thousand neut:ro(cid:173)
`phils in 100 j.j.L of medium were incubated with 10 ILL of antibody at4°C for
`30 min, washed with DMEM, and analyzed.
`Western blot analysis
`Total protein was isolated from 2 x l<f human neutrophils using NP-40
`isolation solution (0.5% NP-40; Tris IOmM. pH 8.0: 60mM KCI; lrnM EDTA,
`pH 8.0; I mM OTT; 10 llll\1 PMSF; and I j.j.M Leupeptin and Aprotinin).
`Isolated protein was measured by lhe Bradford assay Protein Detection Kit
`(Bio-Rad) and loaded at 50 j.l.g per well. Samples were then run on 12% SDS
`polyacrylamide gradient gel ( 140 V for 60 min) and electropboretically trans(cid:173)
`ferred to lmmobilon-P (Millipore. Bedford. MA) (100 V, 45 min). The re(cid:173)
`maining gel was stained after lnlnsfer with ponceau S solution (2%) to confirm
`equal loading. Blots were incubated with mouse anri-pro-caspase 3 primary
`antibody (1:1000) in 1% BSA TBS and 0.1% Tween 20 for I hat room
`temperature and lhen incubated with horseradish peroxidase-conjugated anti(cid:173)
`mouse .lgG at 1:5000 dilution for 1 b. Blots were developed using an enhanced
`chemiluminescence system ( 10).
`
`Caspase activity assay
`Cell lysates were prepared from 10 x106 neutrophil membrane fractions.
`Aliquots ofi.he lysates (250 j.j.L) were diluted in assay buffer (100 mM HEPES,
`pH 7.5; 10% sucrose; 0.1% CHAPS) containing 20 JJ.M CBZ-Asp-Giu-Val(cid:173)
`Asp-AFC (caspase 3 substrate) (Enzyme Systems. Livermore. CA), and incu(cid:173)
`bated for 45 min at room temperature. The release of AFC was detected by
`continuous measurement in a luminescence spect:rometer (Perkin-Elmer LS30,
`London. UK) with an excitation of 400 nm and an emission slit at 505 nm.
`Specific activicy is measured as pmoVs·mg protein.
`Serum IL-8 and glucocorticoid concentration
`Standard ELISA technique (R&D Quantikine"~'M, R&D Systems, Oxan, UK)
`was used to measure lL-8 in serum isolated from the systemic. mesenteric
`arterial, and mesenteric venous ci rculation of control patients and those with
`IBD. Blood was collected and rransponed on ice. Serum was separated within
`I h of collection and stores at -80°C. All samples were analyzed within 3
`months of collection. Serum glucoconicoid concentration was assessed by
`immunoassay (lmmunol, Bayer Technicon).
`Statistical analysis
`Statistical analysis was carried out by ANOVA with Bonferroni's correc(cid:173)
`tion. All resultS are expressed as mean ± standard deviation (SO).
`
`RESULTS
`IBD effects on neutrophil apoptosis
`Neutrophils isolated from lBD patients decreased apoptotic
`rates compared to cancer controls and health volunteers. This
`delay occurred in both mesenteric arterial and venous blood as
`well as in the systemic circulation. There was no significant
`difference in the rate of neutrophil apoptosis between the three
`sources of blood (Fig. Ia). Serum from lBD patients signjfi(cid:173)
`cantly delayed apoptosis of control normal neutrophils after 24
`h of incubation. Serum isolated from the mesenteric venous
`circulation induced significant decreases in apoptosis com(cid:173)
`pared with that from the mesenteric arterial or systemic circu(cid:173)
`lation of the same patients (Fig. lb).
`Functional activity in IBD neutrophils
`There was a trend towards an increase in respiratory burst
`activity [generation of reactive oxygen intermediates (ROls)]
`in neutrophils isolated from patients with mo. but this differ-
`
`

`

`SHOCK MAY'2000
`
`NEUTROPHIL APOPTOSIS IN 180 363
`
`a Systemic
`• Artery
`• Veln
`
`(a) 40
`35
`·130
`iz5
`8.zo <
`~15
`10
`s 0 ,__....._,__..,.....-..~
`
`(b)J5
`
`~ ~10
`
`s
`0
`
`mo
`Normal
`Cancer
`FIG. 1 Effects of lBO on neutrophil epoptosls. (a) Neutrophils
`were isolated from systemiC, mesenteriC artenal, and mesenteric ve(cid:173)
`nous blood of carK:er patients (n = 17) and those With lBO (n = 20).
`SystemiC neutroph1ls were isolated from healthy controls (n = 10). Iso(cid:173)
`lated neuttophlls were irK:Ubated for 24 h and then assessed for percent
`apoptosis by propid1um iodide DNA staining using flow cytometry. (b)
`Healthy neutrophils (n = 3) were irK:Ubated With the serum of cancer (n
`= 17) and lBO (n "'20) patients for 24 hand then assessed for percent
`apoptosis by propid1um iodide DNA S1a1ning us1ng flow cytometry. This
`experiment waa carried out wtth blood from three healthy volunteers on
`2 separate days. Results represent data from the first healthy volunteer
`W1th all study serum. Control normal serum was collected from five
`volunteers ·p < 0.05 vs. cancer and control, P < 0.05 vs. lBO anerial
`and systemiC serum.
`
`ence was not stati tically ignificant. There was a significaot
`increase in surface expression or CD II b isolated from patients
`with LBO compared with the cancer control (Table 1). Serum
`from patients with lBO significantly increased normal neutro(cid:173)
`phil rcspirntory burst activity. In particular. erum from the
`mesenteric venous circulation induced a significantly greater
`result than either that of the mesenteric arterial or systemic
`circulation (Table I). Normal neutrophil CD ll b expression
`was increa..sed by lBD erum but there was no greater increase
`induced by mesenteric venow serum.
`
`Detection of /nter/eukln-8 In 180 serum
`IL-8 was JOcreac;cd in patient with lBO compared with
`cancer controls. Specifically, the concentration of lL-8 in the
`mesenteric venou<, serum of patient! with I BD was greater than
`that in either the mesenteric arterial or y temic serum (Fig. 2).
`
`Effect of lnterleukln.IJ on neutrophil spoptosls
`IL-8 delayed normal neutrophil apoptosis in a do~ depen(cid:173)
`dent manner (Table 2). Thill effect wa-; loo;t at higher concen(cid:173)
`trauons and re\ulted in necrosis. A octated with delayed ap(cid:173)
`opto ts, IL-8 (5 pglmL) treated neutrophih were resistant to
`Fas-antibody induced cell death (Fig. 3). To determine whether
`IL·8 was the only survival factor in the serum of IBD patients
`a blocking monoclonal antibody to IL-8 was used to inhibit the
`effects or lL-8. The IL-8 blocking monoclonaJ antibody bad no
`
`effect on mhibiting the erum delay in normal neutrophil ap(cid:173)
`opto is (data not shown). This re ult would indicai.e that other
`serum factors contribute to the delay.
`Neutrophil pro-caspsse 3 expression In 180
`Fas-antibody induce neutrophil apopto j
`through the acti(cid:173)
`vation of the caspase cascade. A\ these cells are resistant to thi
`ligation, it was hypothe iled that inhibition may be at Lbe level
`of ca pase e>.pre
`ion. We. tern bloning demon trated that neu(cid:173)
`trophil i olated from the \'ein of patients with IBD have re(cid:173)
`duced pro-caspase 3 expre ion (Fig. 4). A caspase 3 plays an
`important role in the induction or neutrophil apoptosis. total
`pro-caspase 3 expres ion was al. o asses ed in IL-8-treated neu(cid:173)
`lrophils. There was a :.ignificant reduction (approximately
`20%) in the overall expression or intracellular pro-caspase 3 in
`normal neutrophils treated with IL-8 (5 pglrnL) compared with
`control cells (Fig. 5). This was further confirmed by assessing
`caspase 3 activity. Neutrophils incubated for 18 h in vitro have
`a basal caspase 3 activity of 24 ± 8 pmoVs·mg protein. Caspasc
`3 activity decreases to 13 ± 3 pmoVs·mg protein after 18 h
`incubation with LL-8 (5 pglmL).
`
`Hydrocortisone and neutrophil spoptosls
`Serum hydrocorti one concentration was increased from
`control ( 162 nmoVL. range 106-270 nmoVL) to IBD patients
`(1210 nmoVL, range 1150-1320 nmoVL). This was due to the
`fact that the e patients were on -.teroid treatment due to the
`stage of their disease. However. there was no difference in the
`concentration of hydrocorti one belween the three sources of
`blood in each patient (data not <;hown).
`Healthy neutrophils (n = 3) "'ere incubated with increasing
`concentrations of hyd.rocorttsone, which was demonstrated to
`have no effect on neutrophil apopto is. Concentrations of 0, 25,
`50, 100, 250. 750, 1000. and 1250 (nmoVL) yielded apoptotic
`rates of 23 ± 8, 20 ± 10, 16 ± 8, 16 ± 6, 22 ± 5, II± 7, 16 ±
`4, 18 ± 6. and 2 1 ± II % apopto i , respectively.
`
`DISCUSSION
`Neutrophil apoptosis is an important step in the resolution of
`an innanunatory re pon. e (I). Delayed neutrophil apoptosis is
`as ociated with persisting innammation and resulting host tis-
`ue injury (21 ). Neutrophil are concentrated in the gastroin(cid:173)
`t~tinal mucosa during acute exacerbations of mo and are
`involved inttmately in mucosal inOammation during thi phase
`of the disease proces ( 15). In thic; study, we demonstrate a
`delay in neutrophil apopto i in lB O patients. Serum factors
`contribute to this delay. specifically mesenteric venous erum
`in patients with lBD. Previous studie. clearly have associated
`delayed neutrophil apoptosi. with pathological persistence or
`the inOammatory respon'-C tn condiuon ucb as the acute re-
`plratory distre!. -;yndrome (22). the '>Y temic inflammatory
`re pon e yndrome (23), and thermal injuries (24). In these
`condition'>. delayed neutrophtl apoptost~ i believed to contrib(cid:173)
`ute igntficantJy to the end organ damage characteristic of the
`individual disea e process. Thi\ <.,tudy confirms this concept in
`patient with CD and UC but alo,o establishes that Lbe major
`factors that contribute lO the delay are released from the in(cid:173)
`named bowel.
`It is well established that neULrophil functional activity is
`
`

`

`364 SHOCK VOL. 13, No. 5
`
`BRANNIGAN ET AL.
`
`Source of
`neutrophils
`
`Patient neutrophils
`
`Normal neutrophils
`
`Functional
`activity
`
`AOI
`(LnMCF)
`C011b
`(LnMCF)
`ROI
`(LnMCF)
`CD11b
`(LnMCF)
`
`TABLE 1. Neutrophil functional activity
`Cancer (n = 17)
`Artery
`
`Vein
`
`Systemic
`
`Systemic
`
`lBO (n = 20)
`Artery
`
`Vein
`
`127 ± 22
`
`109± 28
`
`114 :t: 30
`
`259 ± 184
`
`207 :t: 95
`
`237 :t: 114
`
`16.8 :t:6.7
`
`16.5 ± 7.3
`
`16.1 ± 6.6
`
`29.5 :t: 14.2'
`
`29.7 :t: 14.0'
`
`28.1 ± 12.0 '
`
`142 :t: 2
`
`159:t:5
`
`148 :t: 1
`
`152 ± 5'
`
`158 :t:.,.
`
`172 :t:8'11
`
`10.2:!; 3.4
`
`11.4±2.1
`
`9.9 ± 4.1
`
`30.4 ± 15'
`
`34.9% 1'
`
`33.1:!; 9.2'
`
`*P < 0.05 vs. cancer serum; "Ap < 0.05 vs. IBD Systemic and IBD Arterial serum.
`Neutophils were isolated from cancer (n = 17), lBO (n = 20), or normal healthy controls (n = 2). Patient neutrophils were assessed for ROI production
`and CD11 b expression directly on isolation by flow cytometry. Normal neutrophils (n = 2) were pre-incubated with cancer (n = 17) or lBO (n = 20)
`serum and after 24 h assessed for ROI and CD11 b expression. Data In this table represent neutrophils isolated from one normal volunteer incubated
`with serum for all study patients. Data = mean ± SO and expressed as Ln Mean Channel Fluorescence (LnMCF), the intensity of fluorescence per
`cell. ROI, reactive oxygen Intermediates.
`
`a Systemic
`II Artery
`• Vein
`
`*
`
`150
`
`0
`
`Cancer
`FtG. 2. Serum IL-8 concentrations. Serum was collected from the
`systemic circulation, mesenteric arterial and venous circulation of all
`patients with IBD (n = 20) and cancer controls (n = 17). IL-8 concentra(cid:173)
`tions were then assessed in all serum samples by standard ELISA. • P <
`0.05 vs. cancer; '-P < 0.05 vs. IBD arterial and systemic serum.
`
`mn
`
`TABLE 2. Effects of IL-8 o n neut rophil apo ptosls
`
`Concentration of
`IL-8 (pg/mL)
`
`Percent
`necrosis
`
`Percent
`apoptosis
`22:7
`13± 4
`8± 3'
`6:2'
`10 ± 6'
`12 ± 3'
`21 ± 9
`
`0
`1
`2
`5
`7
`10
`20
`Data= t/eean ± SD. •p...:. 0.05 vs. 0 p.g/mL !L·8.
`Healthy neutrophils (n = 3) were incubated with increasing concentra·
`lions of IL-8 (1 pg-20 pg). After 24 h incubation in vitro, apoptosis was
`assessed by propidium iodide DNA staining using now cytometry. Ne·
`crosis was assessed by the ability of the cell to exclude propidium io·
`dide, non-viable cells had increase intenslity of fluorescence.
`
`5:1
`3±2
`4±2
`6±5
`5:3
`11 :!; 3'
`20±9'
`
`altered in mo (16, 25-27). [n this study, we have confirmed
`these observations when patient serum was cultured with nor(cid:173)
`mal neutrophils, resulting in an increase in both respiratory
`burst activity and CDll b expression. This data also specifi(cid:173)
`caJly showed that mesenteric venous serum increased signifi·
`cantly these markers of functional activity above the artery and
`systemic :.aroph:::.. There was no change in respiratory burst
`activity between cancer controls and IBD patients, but this
`could be attributed to the power of the study. Lncreased respi-
`
`50
`
`* • Medium
`~ Fasantibody
`
`IL-8 (Spglml)
`Control
`FIG. 3. Effects of IL·8 on Fas-antlbody-induoed apoptosls. Nor(cid:173)
`mal healthy neutrophils were pretreated with IL-8 (5 pg/mL) for 4 h and
`then incubated with Fas-antibody (CH-11 , 100 ng/mL) for 24 h. Apop(cid:173)
`tosis was assessed by propidium iodide DNA staining using flow cytom(cid:173)
`etry. 'P< 0.05 vs. control unstimulated medium.
`
`ratory burst activity and CD 11 b expression are associated with
`enhanced neutrophil functional activity and migration to the
`site of inflammation.
`Inflammatory mediators play important roles in delaying
`neutrophil apoptosis. G-CSF is increased in colonic mucosal
`specimens of mo patient; these supernatants also significantly
`delay normal neutrophil apoptosis (28). In addition, increased
`concentrations of lL-8, a potent chemoattractant and anti(cid:173)
`apoptotic factor for neutrophiJs (29), are found in the systemic
`circulation of !BD patients :wd within homogen<~te.~ of in(cid:173)
`flamed mucosa (30, 31). This study demonstrates an increase in
`both mesenteric and systemic serum 1L-8 in patients with IBD.
`Of particular interest was that the IL-8 detected in the mesen(cid:173)
`teric venous circulation was significantly greater than in the
`systemic or mesenteric arterial circulation. fL-8 was also
`shown to delay spontaneous apoptosis in vitro, albeit at I 0-fold
`lower concentrations compared to that found in the serum. This
`could be explained by the fact that serum may contain anti(cid:173)
`inflammatory proteins such as lL-10, which has been shown to
`block the delays in apoptosis. Identification of serum factors
`that contribute to delayed apoptosis may not have clinical rel(cid:173)
`evance, .as blocking these factors in the context of inflamma(cid:173)
`tion may and has proven unrealistic. Blocking IL-8 in these(cid:173)
`rum had no effect on delayed normal neutrophil apoptosis, this
`
`

`

`SHOCK MAY 2000
`
`Control
`,.............
`
`IDDPatients
`
`........... .-.....
`I .
`U~ll i
`
`JlkDa #1
`
`#2
`
`#3
`
`#9. #~ #14 . #15
`
`~
`
`~=too
`
`'tJ?,
`lBD
`Control Cancer
`FIG. 4. Caspase 3 protein expression In lBO patients. Protein was
`extracted from neutrophils isolated from all mesenteric venous samples
`of blood of lBO patients (n = 20). Pro-caspase 3 expression was as(cid:173)
`sessed by western blotting. Gel is a representative of 2 controls and 5
`experimental patients that were selected from the groups and run on a
`single gel. Equal protein (50 ~g) was added to each well and gel stained
`after transfer with ponceau S solution (2%) to confirm equal loading.
`Densitometery was carried out on all samples run by western blotting.
`Gel images were digitized using a Nikon CCD video camera and density
`of image using the imaging device software (Imaging Research Inc,
`Ontario, CA) was calculated as an arbitrary unit, control (n = 6). cancer
`(n = 10), and lBO (n = 10). Results are expressed as a percent of control
`due to the variation in exposure times giving different intensities for the
`bands between different westerns run. • P < 0.05 vs. control and cancer.
`
`Control
`FIG. 5. Effect of IL-8 on caspase 3 protein expression. Total pro·
`tein was extracted from neutrophils incubated for 4 h with IL-8 (5 pg/mL).
`Equal protein (50 l)g) was added to each well and run on a standard
`SDS-Page western blot. Gels were stained after transfer with ponceau
`S solution {2%) to confirm equal loading. Pro-caspase 3 antibody was
`used to stain the membrane and band intensity analyzed by densrtom·
`etry. Gel represents one of five experiments carried out. Densitometry
`was carried out on these five experiments and results expressed as
`percentage pro-caspase 3 expression with control levels set at 1 00%.
`
`would strongly indicate the presence of other survival factors
`in the serum. We beJjeve that understanding the central intra(cid:173)
`cellular mechanisms resulting in delayed apoptosis may be
`more beneficial, than identifying the inflammatory mediators
`that cause these delays. If a central mechanism is identified.
`then this could be targeted. To study tills. two systems were
`used: the neutrophils isolated from the IDD patients, and the
`IL-8 that was increased in the serum of these patients, which
`was used as a model to stimulate delayed apoptosis and ro
`investigate the intracellular effects leading to this response.
`Caspases are now considered to play a pivotal role in the
`process of programmed cell death. They are proteolytic en(cid:173)
`zymes that cleave specific intracellular proteins, resulting in
`
`NEUTROPHIL APOPTOSIS IN 180 365
`
`the characteristic features of apoptosis (12, 13). Caspase 3 is
`present intracelluJarly as an inactive pro-enzyme that is cleaved
`to yield the active moiety. Neutropbils express high levels of
`the pro-caspase 3. demonstrating a particular ability to undergo
`apoptosis (32). Ligation of the Fas receptor activates the
`caspase cascade specifically caspase 3 and 8, and results in the
`induction of neutrophil apoptosis (J 0). As this process is de(cid:173)
`pendent on the activation of the caspase cascade, inhibition of
`this cascade either at the level of expression or activity may
`explain delaye.d apoptosis. Neutrophils from healthy controls
`incubated with IL-8 have been shown to be resistant to Pas(cid:173)
`antibody-induced apoptosis (5). lL-8 was confurned in this
`study to delay spontaneous apoptosis and also induces resis(cid:173)
`tance to rhe effects of Fas-antibody. This resistance to apop(cid:173)
`tosis is not associated with altered expression of the Fas re(cid:173)
`ceptor (5, 9), but was demonstrated to reduce pro-caspase 3
`protein expression. This suggests that altered caspase expres(cid:173)
`sion is the mechanism of apoptotic delay. Altered pro-caspase
`expression has been associated previously with decreased ap(cid:173)
`optosis (33, 34). Neutrophils isolated from the venous circula(cid:173)
`tion of the IBD patients also demonstrated a significant de(cid:173)
`crease in pro-caspase 3 expression. As the neutrophils have a
`delay in apoptosis, it is unlikely that the reason for decreased
`expression is due to the conversion to the active protein. This
`alteration in the expression of pro-caspase 3 may well contrib(cid:173)
`ute to the delay in apoptosis and resistance to apoptotic(cid:173)
`inducing agents.
`Conicosteroids are known to have anti-apoptotic effects that
`might have influenced our observations in patients with mo
`(6). Although we did find increased concentrations of gluco(cid:173)
`corticoids in the serum of patients with ffiD that may have
`contributed towards a global reduction in neutrophil apoptosis,
`it does not explain the specific changes in mesenteric venous
`serum of patients with ffiD . This would indicate that gluco(cid:173)
`corticoids alone are not responsible for the potent anti(cid:173)
`apoptotic effects of this serum.ln addition, it was demonstrated
`that i11 vitro incubation of hydrocortisone up to patient serum
`concentrations bad no effect on neutrophil apoptosis. Sul(cid:173)
`fasalazine has been shown to increase the rates of apoptosis
`(35). Despite all patients being on sulfasalazine, there were still
`significant delays in neutrophil apoptosis. In the article by
`Akaho hi et al. (35), they did demonstrate that GM-CSF does
`inhibit sulfasalarine-induced apoptosis. This ability of inflam(cid:173)
`matory survival factors to dominate over the induction of ap(cid:173)
`optosis by sulfasalazine is similar to that found with Fas(cid:173)
`antibody-ioduced apoptosis. Neutrophil apoptosis is delayed in
`patients with both CD and UC. This may contribute towards a
`persistent inflammation within the gastrointestinal tract. Regu(cid:173)
`lation of neutrophil apoptosis in patients with IDD may prevent
`persistence of the inflammatory response and represent a site
`for therapeutic manipulation. Extracellular cytokine manipula(cid:173)
`tion alone may prove difficult due to the adverse inflammatory
`signaling cascade, but targeting the intracellular final common
`pathway of apoptosis may prove fruitful.
`
`ACKNOWLEDGMENTS
`Supported by a grant from the Wellcome Trust (053761 ) and in part from
`the Ma1er College.
`
`

`

`366 SHOCK Voc. 13, No. 5
`
`BRANNIGAN ET AI..
`
`ID
`
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