specific dialysis fluid peritoneal dialysis pathol 1995;48:115-119 specific increase in...

I Clin Pathol 1995;48:115-119

Specific increase in interleukin-8 concentrationsin dialysis fluid of patients with peritonitisreceiving continuous ambulatory peritonealdialysis

Y C Ko, N Mukaida, T Kasahara, S Muto,Y Itoh, Y Yamagishi, T Kawai

K Matsushima, E Kusano, Y Asano,

Departments ofClinical Pathology,Medical Biology andParasitology, andNephrology, JichiMedical School,Tochigi, JapanY C KoT KasaharaS MutoE KusanoY AsanoY ItohY YamagishiT Kawai

Department ofPharmacology, CancerResearch Institute,Kanazawa University,Kanazawa, JapanN MukaidaK Matsushima

Correspondence to:Dr T Kasahara, Departmentof Medical Biology &Parasitology, Jichi MedicalSchool, Minamikawachi-machi, Tochigi-ken 329-04,Japan.Accepted for publication1 July 1994

AbstractAims-To evaluate the influence of in-terleukin-8 (IL-8) and other inflammatorycytokines (IL-6, IL- 1 P and tumour necrosisfactor a (TNFa)) on the occurrence ofperi-tonitis in patients receiving continuousambulatory peritoneal dialysis (CAPD).Methods-The study population com-prised 12 patients with peritonitis, 33 with-out peritonitis, all undergoing CAPD,and five patients undergoing peritonealcatheter implantation. Cytokine con-centrations in dialysis fluid were de-termined by immunoassay and theirvalues compared.Results-Concentrations of both IL-8(median 147 pg/ml, range 20-2273 pg/ml;n= 12) and IL-6 (median 1120 pg/ml, range96-10 600 pglml) were substantially el-evated, while the IL-lIp concentration waslower and TNFa was not detectable inpatients at diagnosis. The IL-6 con-centration was also elevated in patientsundergoing catheter implantation as wellas in those with peritonitis. The IL-8 con-centration, however, was elevated onlyupon infection. Intraperitoneal pro-duction of IL-8 was evident on de-termination of paired serum and dialysisfluid cytokine concentrations, and im-munostaining of peritoneal cells withmonoclonal anti-IL-8 antibody.Conclusions-These results suggest thatdetermination ofthe IL-8 concentration indialysis fluid maybe useful as a specificmarker for following patients with peri-tonitis receiving CAPD.(J Clin Pathol 1995;48:115-119)

Keywords: Interleukin-8, peritonitis, peritoneal dialysis.

Peritonitis, characterised by neutrophil in-filtration in the peritoneal cavity, is one of themajor complications in patients with end stagerenal failure undergoing continuous am-bulatory peritoneal dialysis (CAPD). We havepreviously demonstrated that interleukin-8(IL-8), a potent chemotactic cytokine for neu-trophils," is associated with pyuria in patientswith urinary tract infections.3 Therefore, it isreasonable to assume that IL-8 may contributeto peritonitis in patients undergoing CAPD.

Elevated IL-6 and IL-8 concentrations haverecently been reported in the peritoneal dialysis

fluid of patients with peritonitis.45 The presentstudy was designed to investigate whether dia-lysis fluid IL-8 or IL-6 concentrations are el-evated in patients with and without peritonitisand in patients undergoing peritoneal catheterimplantation. The contribution of IL-1p andtumour necrosis factor ct (TNFoc) and evidenceof the intraperitoneal production of IL-8 inpatients with peritonitis receiving CAPD werealso evaluated.

MethodsTwelve patients with peritonitis (10 men andtwo women; mean age 616 years, range 33-94years) undergoing CAPD were studied. Themicro-organisms isolated included Streptococcusviridans (three cases), Staphylococcus epidermidis(two cases), S aureus (two cases), methicillinresistant S aureus (two cases), Candida albicans(one case), and culture negative (four cases).Thirty three patients without peritonitis (25men and eight women; mean age 47-7 years,range 23-70 years) undergoing CAPD for atleast one month served as controls. Fivepatients undergoing peritoneal catheter im-plantation, three of whom subsequently de-veloped peritonitis, were also studied. Samplesfrom the latter patients were taken within24 hours of catheter implantation. Evidence ofperitonitis included abdominal symptoms orcloudy dialysis fluid, or both, and the presence100 white blood cells (WBC)/mm3 in the dia-lysis fluid or positive culture, or both.

Cytokine concentrations in the dialysis fluidor serum were determined by immunoassay asdescribed in detail elsewhere.36 The detectionlimits were 16, 10, 5, and 4 pg/ml for IL-8, IL-1 , TNFct, and IL-6, respectively.

Centrifuged sediment from dialysis fluid ofpatients with peritonitis was fixed on the glassslides and endogenous peroxidase activityblocked. Slides were stained using the avidinbiotin complex immunoperoxidase techniqueand colour developed using diaminobenzidine.

Results were expressed as medians andranges. The values of each parameter at diag-nosis of each episode of peritonitis or duringcatheter implantation were used for com-parison. Specimens with values below the de-tection limit were excluded from comparisonsinvolving that variable. Data analysis was per-formed using Hollander and Wolfe's method;p values less that 0 05 were regarded as sig-nificant.

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C = ControlsCl = Catheter implantationP = Peritonitis

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P < 0.01° B Catheter implantation elicited a substantial0B~ increase in IL-6 concentrations (460 pg/ml,

* range 185-1130pg/ml) and WBC counts (93o-- i .........30cells/mm3,range 71-400 cells/mm3), but not in

IL-8 concentrations (<16 pg/ml), in patientsE without infection compared with controls (figCD 1A). No significant differences were noted0 ° ° +¢ ..........20 M when theWBC counts and IL-6 concentrations

4- z in the dialysis fluid ofpatients undergoing cath-eter implantation were compared with those of

0o 10 patients with peritonitis, although these levelstended to be higher in the latter. Patients un-

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dergoing catheter implantation are capable ofo 0 producing IL-8, as IL-8 concentrations in-c p c p creased in those who subsequently developed

Concentrations of cytokines in dialysis fluid of peritonitis. As shown in fig 2, IL-8 and IL-6with (P; n = 12 for panel A and n = 11 for panelwithout (C; n = 33 for panel A and n = 11 for concentrations and WBC counts increased onperitonitis and five patients undergoing catheter development ofperitonitis; IL-8 concentrationsrtion (CI). The dotted lines denote detection limits were the first to return to normal in responsenoassay. The horizontal lines denote median to antibiotic treatment.J*P<nnn A*P<n.A 6AP=n.V V) tLtetetNSUeUs. (n-osign 4 )-U'Ul,cl-aUnUt VcontrlaI

NS = not significant)

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ResultsAs shown in fig IA, IL-8 concentrations werebelow the detection limit (<16 pg/ml) in thedialysis fluid of all patients without peritonitis(n = 33). These patients also had low IL-6concentrations (median 155 pg/ml, range0-52 pg/ml) and WBC counts (6-8 cells/mm3,range 1 2-65 cells/mm3). In patients withperitonitis (n= 12) IL-8 and IL-6 concen-trations and theWBC counts were significantlyelevated at diagnosis with median values of147 pg/ml (range 20-2273 pg/ml), 1120 pg/ml(96-10 600 pg/ml), and 680 cells/mm3 (125-6500 cells/mm3), respectively.Although IL-1,B and TNFa stimulate the

production of both IL-6 and IL-8, IL-1iconcentrations in patients with peritonitiswere significantly lower (152 pg/ml, range48-256 pg/ml; n= 11) than those in the con-trols (317 pg/ml, range 180-392 pg/ml; n= 1 1)(fig 1 B). TNFa concentrations were below thedetection limit (<5 pg/ml) in patients with (n=1 1) and without (n= 11) peritonitis.

E 100-;

10

10-

Serum Dialysate

Figure 3 Concentrations of IL-8 and IL-6 in paireddialysis fluid and serum specimens from patients withperitonitis undergoing CAPD.

Paired IL-6 and IL-8 concentrations inserum and dialysis fluid were determined (fig3). Both IL-6 and IL-8 concentrations weresubstantially higher in dialysis fluid than inserum, favouring the local production of IL-8within the peritoneal cavity. To strengthen

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IL-8 dialysate concentrations in patients with peritonitis undergoing CAPD

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Figure 4 Positive immunostaining reaction with monoclonal antibody to IL-8 in peritoneal macrophages (arrow),mesothelial cells (curved arrow), and neutrophils with three or less nuclear segments (arrowheads) of patients withperitonitis undergoing CAPD (original magnification x 1000).

these obversations, peritoneal macrophagesand mesothelial cells, identified by their char-acteristic morphology, together with neu-trophils in the dialysis fluid of patients withperitonitis, were positively stained with mono-clonal anti-IL-8 antibody WS-4 (fig 4). Neu-trophils with four or more nuclear segmentsgenerally did not stain. These findings are spe-cific, as immunostaining with monoclonal anti-CD 15 antibody, which serves as a positivecontrol for granulocytes, did not discriminatebetween these neutrophils with respect to thenuclear segment pattern (data not shown).

DiscussionAccumulAting evidence suggests that in-flammatory cytokines have a vital role in theresponse to infection. The present study dem-onstrates the specific intraperitoneal release ofIL-8 in patients with peritonitis undergoingCAPD, and confirms previous reports onIL-6 and IL-8 in peritoneal dialysis fluid.457

In patients undergoing CAPD for the firsttime exposure to dialysis fluid and foreign ma-terial such as the catheter presumably inducesa local inflammatory response within the peri-toneal cavity,""'3 as indicated by the raisedIL-6 concentrations found in these patients.The failure to detect IL-8 in these patients isnot unexpected as mononuclear, rather thanneutrophil, cell numbers predominate. On ini-tial infection of the peritoneum, IL-8 con-

centrations surge, and provided the infectiondoes not persist, IL-8 concentrations return tonormal. One patient with peritonitis whosedialysis fluid IL-8 concentrations were normalat diagnosis was consequently found to havesignificantly elevated IL-8 concentrations thefollowing day. These characteristic kinetics per-mit the use ofIL-8 as a marker when diagnosingand monitoring the course of patients withperintonitis receiving CAPD. This may alsoapply to other localised infectious diseases suchas urinary tract infections.Both IL-8 and IL-6 are produced by a variety

of cell types, some of which produce bothcytokines."14 Immunostaining for IL-8 iden-tified peritoneal macrophages, mesothelial cellsand neutrophils as the local producing cellswithin the peritoneal cavity during peritonitis.This is consistent with previous in vitrofindings.""'8 Similar findings have been re-ported for IL-6.'920 Other probable sources ofIL-8 production include endothelial cells andfibroblasts within the peritoneum.Another important finding was that neu-

trophils with four or more nuclear segmentsdid not stain for IL-8. The lack of IL-8 pro-duction by these cells may indicate negativeregulation of further neutrophil recruitment.Additional studies are required to confirm thishypothesis.As in cases of urinary tract infection there

were no significant increases in IL-11, andTNFcx concentrations in the dialysis fluid of

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Ko, Mukaida, Kasahara, Muto, Matsushima, Kusano, et al

infected patients.' It is postulated that thesecytokines are produced at a very low level or

that some inhibitory factors which interferewith the immunoassays may be present in thedialysis fluid. It is possible, however, that verylow IL-i1 and TNFa concentrations may act atthe early stage, initiating the cytokine cascade.Alternatively, there may be a bypass pathwayfor the direct stimulation of IL-6 or IL-8 pro-duction. Havell and Shegal demonstrated TNFindependent IL-6 production in murine list-eriosis. Anti-TNFcx and anti-IL-i neutralisingantibodies did not prevent the early phase oflipopolysaccharide (LPS) induced IL-8 syn-thesis in human blood.22 In other animal studiesanti-TNFoc antibody did not protect rats andmice from lethal Escherichia coli peritonitis, sug-gesting that TNF has a minor role in bacterialperitonitis.2324 Fieren et al showed that withoutexogenous stimulation, such as LPS, peritonealmacrophages obtained from infected and un-

infected patients released similar amounts ofIL-1 P and TNFot in vitro.2526 Peritoneal dialysisfluid has been reported to inhibit IL-6 andTNFcx release from mononuclear leucocytes,27while IL-6 suppresses LPS induced productionof IL-i1 and TNFa in peripheral blood.28To confuse the situation further, a recent

report showed that TNFct (median level about340 pg/ml), but not IL-i [P, concentrations wereraised in the ascitic fluid of cirrhotic patientswith spontaneous bacterial peritonitis.29 How-ever, these results must be interpreted cau-

tiously as IL-6 concentrations in the patientswith cirrhosis were very high (about 1-7 x

10'ng/ml). Taken together, the involvementof IL-ip and TNFoc in peritonitis cannot becompletely ruled out, and studies using in situhybridisation would have been useful. There-fore, measurement of IL-i [p and TNFot con-

centrations in dialysis fluid for the diagnosis ofperitonitis seems to be of no advantage.The influence of IL-8 on neutrophil

chemotaxis and activation (intraperitonealadministration of human recombinant IL-8causes neutrophil infiltration in mice'0) sug-gests that this cytokine plays an importantrole in the response to bacterial infection ofthe peritoneal cavity. Intraperitoneal ad-ministration of IL-8 may be beneficial inpatients undergoing CAPD as is the case withinterferon-a, which is effective in preventinginfection when administered prophylactically."Nevertheless, a long term study to compare theoccurrence of probable complications, such as

peritoneal fibrosis, in patients with or withoutcytokine treatment is mandatory.

In conclusion, IL-8 concentrations are sig-nificantly raised in the dialysis fluid of patientswith peritonitis undergoing CAPD. Ad-ministration ofrecombinent IL-8 may be usefulfor prophylaxis in patients undergoing CAPD.

The authors are grateful for the assistance of M Hayashi, IShimada and their staff in collecting samples and to K Na-matame for performing the immunostaining. We also thankSRL Inc. and Fuji Rebio Ltd., Tokyo, Japan, for measuring IL-1 and TNFa concentrations and providing the kits for the IL-6 immunoassay, respectively. This study was supported in partby grant from the Ministry of Education, Culture, and Science,Japan.

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2 Baggiolini M, Imboden P,Detmers P. Neutrophil activationand the effects of interleukin-8/neutrophil-activating pep-tide 1 (IL-8/NAP-1). Cytokines 1992;4:1-17.

3 Ko YC, Mukaida N, Ishiyama S, Tokue A, Kawai T,Matsushima K, Kasahara T. Elevated interleukin-8 levelsin the urine of patients with urinary tract infections. InfectImmun 1993;61:1307-14.

4 Lin CY, Lin CC, Huang TP. Serial changes of interleukin-6 and interleukin-8 levels in drain dialysate of uremicpatients with continuous ambulatory peritoneal dialysisduring peritonitis. Nephron 1993;63:404-8.

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18 Betjes MGH, Tuk CW, Struijk DG, Krediet RT, AriszL, Hart M, et al Interleukin-8 production by humanperitoneal mesothelial cells in response to tumour necrosisfactor-a, interleukin-1, and medium conditioned bymacrophages cocultured with Staphylococcus epidermidis. JInfect Dis 1993;168: 1202-10.

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22 DeForge LE, Kenney JS, Jones ML, Warren JS, RemickDG. Biphasic production of IL-8 in lipopolysaccharide(LPS)-stimulated human whole blood. J Immunol 1992-148:2133-41.

23 Bagby GJ, Plessala KJ, Wilson LA, Thompson JJ, NelsonS. Divergent efficacy of antibody to tumor necrosis factor-a in intravascular and peritonitis models of sepsis. J InfectDis 1991;163:83-8.

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25 Fieren MWJA, Van Den Bemd GJCM, Bonta IL. Endo-toxin-stimulated peritoneal macrophages obtained fromcontinuous ambulatory peritoneal dialysis patients showan increased capacity to release interleukin-11 in vitroduring infectious peritonitis. Eur J Clin Invest 1990;20:453-7.

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28 Schindler R, Mancilla J, Endres S, Ghorbani R, ClarkSC, Dinarello CA. Correlations and interactions in theproduction of interleukin-6, IL-1, and TNF in humanblood mononuclear cells: IL-6 suppresses IL- 1 and TNF.Blood 1990;75:40-7.

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31 Carozzi S, Nasini MG, Schelotto C, Caviglia PM, CantaluppiA, Salit M, et al. Intraperitoneal therapy with interferon-a inCAPD patients with relapsing bacterial peritonitis. TransAmSoc ArtifIntern Organs 1989;35:421-3.

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