Identification of the Major Chemokines That Regulate Cell Influxes in Peritoneal Dialysis Patients1

Identification of the Major Chemokines that Regulate Cell Influxes in Peritoneal Dialysis Patients1

Janneke Tekstra,2 Caroline E. Visser, Cornelis W. Tuk, Joke J.E. Brouwer-Steenbergen, Curt W. Burger, Raymond T. Krediet, and Robert H.J. Beelen

(P < 0.05). One of the monocyte-alfracting chemo- J. Tekstra, CE. Visser. C.W. Tuk, J.J.E. Brouwer- kines, RANTES, could not be detected in any of the Steenbergen, R.H.J. Beelen. Department of Cell Biol- effluents, whereas the other, MCP-i , was significantly ogy and Immunology. Faculty of Medicine, Vrije Uni- elevated during peritonitis (P < 0.02). In contrastto the versiteit. Amsterdam, The Netherlands other chemokines measured, MCP-1 concentration C.W. Burger. Department of Gynecology. Academic was relatively high in steady-state peritoneal dialy- Hospital Vrije Universiteit. Amsterdam. The Netherlands sates. An absolute correlation between dialysate MCP-1 concentration and the number of macro- R.T. Krediet. Department of Nephrology. Academic Medical Center. Amsterdam. The Netherlands phages in these effluents was absent. However, in a 48-well chemotaxis assay, monocyte migration to- (J. Am. Soc. Nephrol. 1 996; 7:2379-2384) ward peritonitis, as well as steady-state patient dialy-

sates, could be blocked with antibodies to MCP-i . It ABSTRACT was concluded, therefore, that MCP-1 is the most To investigate which members of the recently discov- important monocyfe chemoaltractant in peritoneal ered family of chemotactic cytokines (chemokines) dialysis steady-state and peritonitis patients; whereas, are important in leukocyte recruitment to a bacterial besides interleukin-8, huGROa was identified as a inflammation site, four different chemokines in the major neutrophil-attracting chemokine in the perito- effluent of peritoneal dialysis patients suffering from nitis situation. acute bacterial peritonitis were measured. The pres- Key Words: Bacterial inflammation, peritonitis. chemotaxis. ence of two neutrophil-alfracting chemokines, inter- neutrophils. monocytes leukin-8 and human melanoma growth-stimulating activity (huGROa), and two monocyte-.altracting B acteriab inflammation is characterized by a neu- members of the chemokine superfamily, monocyfe trophilic cell influx, followed by a monocytic cell chemotactic protein-i (MCP-1) and regulated on ac- infiltrate. Such a pattern of inflammatory events also tivation normal T cell expressed and secreted (RAN- occurs in continuous ambulatory peritoneab dialysis TES), was investigated in patient effluents just before, (CAPD) patients suffering from bacterial peritonitis. The rapid neutrophibic cell influx and the subsequent during, and after a peritonitis episode. This was stud- monocytic cell influx in peritonitis can be observed for ied in seven peritonitis effluents of five patients by a distinct period in the spent dialysis effluents of using chemokine-specific enzyme-linked immunosor- patients (2). bent assays. Cell populations in the dialysates were Although the incidence of peritonitis has been re- differentiated on cytocentrifuge preparations. The duced over the years to approximately 0.8 episodes contribution of the detected chemokines to neutro- per patient per year ( 1 ), the complication still leads to philic and monocytic cell influxes in the inflamed patient dropout from this renal replacement therapy. peritoneal cavity was analyzed by correlating con- It is now generally accepted that cell mnfluxes in the centrations of chemokines to the relevant cell num- area of inflammation are regulated by mediators bers present in the dialysates of these patients. The which are induced by promnflammatory cytokines and detection of the neutrophil-aifracting chemokine in- have specific chemotactic properties. These so-cabled terleukin-8 during peritonitis was in accordance with chemokines constitute a recently discovered group of structurally and functionally related cytokmnes con- other studies. Moreover, a second neutrophil che- taming highly conserved cystemne residues (3). The moattractant, huGROa, was identified in vivo. Both chemokines can be divided in the a- and -subfamiby, were elevated during inflammation (P < 0.02) and in which the a-chemokines mainly have chemotactic contributed significantly to the neutrophilic cell influx properties for neutrophils and the -chemokmnes have chemotactic properties for monocytes (4,5). In the

1 Received March 26, 1996. Accepted June 25, 1996. course of inflammation, the chemokines contribute to

2 Correspondence to Dr. J. Tekstra, Department of Cell Biology & Immunology, the fine tuning of cell infiltrates that differ in cell type Faculty of Medicine, Vrije Universiteit, van der Boechorststraat 7, 1081 BT Amster- and time of occurrence by exerting their effects on dam, The Netherlands. different target cell populations. 1046-6673/071 1-2379$03.00/0 Earlier in vitro studies revealed that the mesothebial Journal of the American Society of Nephrology Copyright © 1996 by the American Society of Nephrology cells lining the peritoneal cavity can be an important

Journal of the American Society of Nephrology 2379 Chemokines in Peritoneal Dialysis

source of chemokmne production (6-8), just as me- TABLE 1 . Clinical data of the CAPD patient group sotheliab cells lining the pleural cavity (9), when stim- . Duration ubated with proinflammatory cytokines. Also, an in- Peritonitis ,, . Patient Age/Sex of CAPD Microorganism, verse correlation has been demonstrated between the E isode p (Months) mesothebial cell number in the effluent and the pen- tonitis incidence (10). Evidence has already been pre- 1 A 50/male 19 Staphylococcus sented that the a-chemokine lnterleukmn-8 (IL-8) is epidermidis involved in attracting the neutrophils into the penito- 2 A 50/male 20 5. epidermidis neal cavity during peritonitis (2). However, IL-8 could 3 B 55/male 41 S. epidermidis not account for all the chemotactic activity causing 4 C 61/male 16 5. epidermidis neutrophil influx, as the chemotactic activity of the 5 D 53/male 10 Staphylococcus dialysate could not be blocked completely with neu- aureus tralizing anti-IL-8 antibody ( 1 1 ). Therefore, the aim of 6 E 37/male 13 5. epidermidis this study was to analyze other chemokines in the 7 E 37/male 17 5. epidermidis peritoneal dialysates that could account for the additional, non-IL-8-dependent neutrophil chemotaxis. In addition, we investigated the presence of monocyte- tial cell counts were performed on May Grunwald Giemsa- attracting chemokines in dialysate to explain the stained cytocentnifuge preparations. monocytic cell Influx seen during peritonitis. Consti- Control experiments to validate cell counts in prepenitoni- tis effluents that were refrigerated for up to 48 h were tutive expression of monocyte-attracting chemokines performed earlier ( 13) and showed no significant influence on was also expected in the uninfected steady-state the yield on viability of the peritoneal cell population. To CAPD situation, because a continuous influx of reba- assess the stability of chemokmnes in the stored cell contain- tively immature macrophages in the peritoneal cavity ing effluents, a known amount of recombinant IL-8, hu- of stable patients has been described that can be GROa, RANTES, and MCP- 1 was added to a freshly obtained characterized as a sterile inflammation (12). CAPD effluent. This effluent was refrigerated and the chemo- In this study, protein bevels of the chemokmnes IL-8 kmne concentration was measured after 0, 1 , and 2 days of and human melanoma growth-stimulating activity storage at 4#{176}C,centnifugation. and subsequent freezing at (huGROa), which are important neutrophil chemoat- -80#{176}C. In the case of a peritonitis, effluents were always tractants (3), were analyzed in spent dialysis effluents. processed and frozen immediately after arrival. Also, monocyte chemotactic protein- 1 (MCP- 1 ) and Enzyme-Linked Immunosorbent Assay regulated on activation normal T cell expressed and secreted (RANTES), known as monocyte chemoattrac- IL-8 and huGROa. Chemokine concentrations in the spent tants (3) were measured in the spent effluents of effluents were determined by using commercially available patients during peritonitis and steady-state periods. human IL-8 enzyme-linked immunosorbent assay (ELISA) kits (Central Laboratory of Blood Transfusion, Amsterdam, Of these four chemokines, IL-8, huGROa, and MCP- 1 The Netherlands) with a detection limit of 50 pg/mL, and were present in the dialysates, whereas RANTES could huGROa ELISA kits (Quantikmne, R&D Systems, Mmnneapo- not be detected. To further characterize the role of bis. MN) with a detection limit of 9 pg/mL. MCP- 1 , blocking studies with neutralizing anti-MCP- 1 MCP-1 and RANTES. To measure these chemokmnes, ELISA antibody in an in vitro chemotaxis assay were per- were developed in our laboratory. For the human MCP- 1 formed to investigate whether MCP- 1 is the major ELISA, maxisorb 96-wells plate were coated overnight at 4#{176}C monocyte chemoattractant In the dialysis effluents. with monocbonal mouse-antihuman MCP- 1 antibody ascites (14) (dilution i04 in phosphate-buffered saline IPBSI). The METHODS plates were blocked with 0.3% gelatin in PBS/0.05% Tween 20, 1 h at room temperature. After incubation with the Patients and Material samples ( 1 h at room temperature), plates were washed with A randomly selected group of patients (Table 1) stoned their PBS/0.05% Tween 20 and incubated with polycbonal goat- overnight effluent for three consecutive days at 4#{176}C.The antihuman MCP- 1 antibody (2.5 jg/mL obtained from R&D overnight dialysis effluent of 2 days before (Day -2) was Systems). After 1 h at room temperature and washing, discarded if the patient showed no clinical signs of peritoni- incubation with penoxidase-conjugated swine-antigoat mm- tis. When peritonitis occurred, the patient was seen in the munoglobubmn (bg) followed. After washing, substrate solution outpatient clinic and the overnight fluids of Day -2, - 1 , and (freshly made of 0. 1 1 M NaAc buffer with 0. 1 mg/mL 0 (peritonitis) were obtained for analysis. The overnight dwell (3,3’,S,S’-tetramethyb benzidmne) and 0.0375 mg/mL H202) time was 8 to 10 h. One month after peritonitis, control was added and the substrate was allowed to be converted for effluent from the same patient was collected. All patients 15 mm. The reaction was stopped by adding H2S04 and the used commercial dialysis fluid Dianeal (Baxter, Maanssen, optical density was measured at 450 nm. The human RAN- the Netherlands) with various glucose concentrations. Seven TES ELISA is based on the same protocol, with the use of a peritonitis episodes in five diffenent patients could be moni- monocbonal mouse-antihuman RANTES antibody (R&D Sys- toned in this way. tems, 0.5 g/mL) as catching antibody and a pobyclonab The collected effluents were centrifuged for 10 mm at goat-antihuman RANTES (R&D Systems, 1 pg/mL) as detec- 300 X 9 to separate the cells from the diabysate. Cell-free tion antibody. On every 96-well plate, the optical density effluents were stored at -80#{176}Cuntil chemokine analysis. The values of a standard dilution range of the relevant chemokine total peritoneal cell numbers were determined and differen- (recombinant human IL-8, huGROa, MCP-1, and RANTES,

2380 Volume 7 - Number 1 1 ‘ 1996 Tekstra et al

obtained from PeproTech Inc. , London, England) were deter- RESULTS mined to create a standard curve. The optical density values of patient samples were compared with this curve to calcu- Chemokine Production During Bacterial bate the chemokmne concentrations in the samples. By creat- ing a standard curve for every plate, interplate variation was Peritonitis overruled. The lower detection limit of the MCP- 1 ELISA is 30 Results on chemokine concentrations in spent efflu- pg/mL and 50 pg/mL for the RANTES ELISA. Cross-reaction ents of five patients suffering seven peritonitis epi- with other chemokmnes was not detected in the above-men- sodes are depicted in Figure 1 . Every line represents tioned ELISA (data not shown). one of seven peritonitis episodes; every symbol represents one of five patients. Analogous to IL-8 (data not shown), huGROa and MCP- 1 could be detected in the Monocyte Chemotaxis effluents and these chemokines obviously peaked during peritonitis compared with preceding days (P < Peripheral blood monocytes were isolated from healthy 0.02 in Wilcoxon signed rank test), whereas they volunteers as described earlier by Fneundbich and Avdabovic ( 1 5). Mononuclear cells were isolated by Lymphoprep (Nyco- decreased to control values (stable CAPD) when in- prep, Oslo, Sweden) density gnadient centrifugatmon (30 mm flammation had passed. The chemokine RANTES at 900 X g) of hepanmnized blood. The cells were washed and could not be detected in any of the CAPD effluents suspended at a concentration of 2 to 4 106 cells/mI. In RPMI (data not shown). All four chemokines remained stable 1640 (Gibco BRL Life Technologies, Bneda, the Netherlands) in CAPD effluents, because the added fixed concen- containing 20% newborn calf serum. This cell suspension trations were recovered unchanged on Days 0, 1 , and was allowed to adhere to plasma-treated, gelatin-coated 2 of storage at 4#{176}C.These experiments additionally culture flasks for 45 mm at 37#{176}C, then washed to deplete showed that there was no cross-reaction between the nonadhenent cells, and incubated with 10 mM ethylenedma- four mentioned chemokines (results not shown). minetetraacetic acid in PBS to recover adherent monocytes (>95% purity). Monocytes were suspended in RPMI supple- mented with 1 % newborn calf serum in the concentration of 4 X i05 cells/mL. In a standard 48-webb chemotaxis chamber A (Neuro Probe Inc., Cabin John, MD) (1 6), 25 L of dialysis effluent with anti-MCP- 1 (polycbonal goat antihuman anti- 10000 body, R&D Systems, 75 j.tg/mL) or with a nonnelevant antibody (pobycbonal goat antimouse IgG, Cappel, Onganon Teknika, Belgium, 75 ig/mL) was administered to the lower 1000 wells. As a positive control, 30 ng/mL human recombinant MCP- 1 with goat antimouse IgG was used. MCP- 1 with anti-MCP-1 showed the blocking capacity of this specific I antibody. All samples were tested In triplicate. The bower 100 wells of the chemotaxis chamber were covered with a 5-.tm pore polycarbonate filter (pobyvinylpynrolidone-free, Costar I Europe, Badhoevedorp, the Netherlands) and the chamben was assembled. In the upper wells, 50 L of the monocyte suspension (2 x i04 cells/well) was loaded. The chamber -2 -1 0 28 was incubated for 1 h at 37#{176}C,5% CO2 to allow monocyte time, days chemotaxis through the filter. Theneaften, the nonmigrated cells were scraped off and the filters were dried. The cells on B the filters were fixed and stained in Coomassie Brilliant Blue. Migrated cells were counted by light microscopy with a scored eyepiece. Chemotaxis was expressed as the percent- age of total cells that had migrated through the filter. .

Statistical Analysis 0 a The chemokmne concentrations in effluents on the day of peritonitis (Day 0) were compared with the concentration in control effluents (Day 28) by the Wibcoxon signed rank test. Peritonitis effluent concentrations of chemokmnes were con- sidened significantly increased compared with controls when -2 -1 0 28 P < 0.05. The Spearman’s rank coefficient was calculated to time, days correlate neutnophib numbers to IL-8 and huGROa concentrations in the effluents, and macrophage numbers to MCP- 1 Figure 1 . Chemokine concentration in dialysis effluents. Con- concentrations in control, Day -2, Day - 1 , and peritonitis centrations of huGROa (A) and MCP-1 (B) in the dialysates of situations. A P value lower than 0.05 was considered a five patients during seven peritonitis episodes are given. statistically significant positive correlation between chemo- Every line represents a peritonitis period; every symbol rep- kmne concentration and cell number. resents a patient.

Journal of the American Society of Nephrology 2381 Chemokines in Peritoneal Dialysis

Correlation of Cell Numbers to Chemokine Monocyte Chemotaxis Toward Peritonitis and Concentrations Control Effluent

The Spearman’s rank correlation data are visually As is demonstrated in Figure 3A, monocytes of represented in scatter plots (Figure 2). A similar con- healthy donors were chemotacticably attracted by six relation that was present between the number of of seven peritonitis effluents. When a neutralizing neutrophibs in the dialysates (peritonitis and preced- anti-MCP- 1 antibody was administered to the chemo- ing days) and the diabysate IL-8 concentration (r = taxis assay, monocyte migration toward peritonitis 0.59, P < 0.05, data not shown) could be demon- effluents was totally abolished in six of the seven strated even more strongly for huGROa (r = 0.91, P < episodes, although this was not the case when an 0.05, Figure 2A). No distinct relationship was found irrelevant antibody was used (Figure 3A).This mdi- between the number of dialysate macrophages and cates that MCP- 1 is the only important monocyte the MCP- 1 concentration regarding the whole popuba- chemoattractant in these six cases. One distinct case tion (r = 0. 1 7, Figure 2B). A few effluents are observed of peritonitis (Episode 1 , Patient A), however, showed containing low MCP-1 concentration (below 100 pg/ high chemotactic activity which could not be blocked mL), although they contain high numbers of macro- by anti-MCP- 1 antibody nor by another chemokine phages. antibody, anti-RANTES (not shown). Episode 2 of the same patient also showed high chemotactic activity which could be completely inhibited by anti-MCP- 1 A 10000 (Figure 3A) but not by anti-RANTES (not shown). Episode 5 (Patient D) showed little chemotaxis, but 1000 0 0 0 this was abolished when the specific anti-MCP- 1 an- 0 100 tibody was added.

U) 0 Monocyte chemotaxis was also found, however to a

0. 10 00 lesser extent, toward effluents of the same patients 0 . under stable CAPD conditions. This chemotactic ac- #{149}. S C 0 #{149}1 (U 0,1 U) . A (U ... 0 0,01

0,001 .9) 10 100 1000 10000 E

dialysate HuGRO, pg/mi a 0 E a C B 1000 a

0 0 100 0 irrelevant anti-MCP-1 antibody 0 0 U) 10 U) S 03 S #{149}0 (U 0 10 .C S B 0. S 2 S a .55 SS (U 21 E . S U) 0,1 (U .9) E . (U 0,01 5 0

x a 0,001 0 E a. a 10 100 1000 1000C C . a : : #{149} . . a dialysate McP-1, pg/mi . -. #{149}a 0 Figure 2. Correlation of cell numbers to chemokine concen- irrelevant anti-McP-1 trations in dialysis effluents. The concentrations of huGROa antibody (A) and the neutrophil numbers are given and concentrations of MCP-1 (B) in relation to macrophage numbers in the Figure 3. Monocyte chemotaxis toward CAPD effluents: dialysates of five patients during seven peritonitis episodes blocking with anti-MCP-1 antibody. Chemotaxis of blood

are shown. Closed symbols represent stable, Day - 1 , and monocytes toward peritonitis (A) and stable CAPD (B) efflu- Day -2 effluents; open symbols represent Day 0 (peritonitis) ents in the presence of neutralizing anti-MCP-1 antibody and effluents. an irrelevant antibody (75 g/mL) is shown.

2382 Volume 7 ‘ Number 1 1 - 1996 Tekstra et al

tivity was also reduced when anti-MCP- 1 antibody statistically significant positive correlation between was administered compared with administration of an MCP- 1 concentrations and macrophage numbers in irrelevant antibody (Figure 3B). the dialysates could be found. However, a trend is The anti-MCP- 1 antibody was not toxic for mono- visible in which high MCP- 1 concentrations come- cytes because chemotaxis toward N-formyl methio- spond to high macrophage numbers in the peritonitis nine-beucine-phenylalanine in the presence of the an- effluents (Figure 2B, open symbols). An unexplainable tibody remained unchanged (between 8 and 10%). population of seven effluents with bow MCP- 1 values Anti-MCP- 1 was neutralizing in a concentration of 75 (below 100 pg/mL) and yet high macrophage numbers g/mL because chemotaxis toward MCP-1 in the was observed In our study as well as in others (19). presence of this concentration of the antibody was This suggests another important monocyte attractant completely abolished (from 5 to 8% to 1 %). Specificity In some distinct cases where MCP- 1 seems to be of was assessed by control experiments with use of minor importance. Excluding the separate popula- irrelevant antibodies (goat-antimouse IgG and goat- tion, the correlation between the number of macro- antihuman RANTES) in the same concentrations, phages and the MCP- 1 concentrations was significant which did not affect monocyte chemotaxis toward (r = 0.58, P < 0.05). MCP- 1 or formyl-methionyl-leucyl-phenylalanine. The In general, high steady-state levels of MCP- 1 were negative control (chemokinesis) ranged from 0.3 to 1 % found which varied considerably per patient. These in the chemotaxis assay. high background levels of MCP- 1 in dialysis fluids may be a phenomenon that is typical for CAPD pa- DISCUSSION tients because every time the dialysis fluid is changed, macrophages are washed away from the penitoneab In the study presented here, we identified besides cavity ( 1 2) and new monocytes have to be recruited IL-8 an additional neutrophil chemokine in CAPD from the blood. An indication that this hypothesis can peritonitis and we demonstrated the importance of be true comes from data (not shown) obtained with MCP- 1 as a monocyte chemoattractant in peritonitis peritoneal fluids from a group of non-CAPD patients. as well as in stable CAPD patients. The involvement of This group consisted of women undergoing general IL-8 in regulating neutrophil cell influx in bacterial gynecologic surgery, of whom the peritoneab cavity peritonitis was mentioned earlier (2,6,7). However, it was rinsed with saline directly after opening the pen- was clear that at least one other neutrophil-attracting toneum and the fluid was recovered. In contrast to chemokmne must play a role in the in vtvo inflamma- CAPD patient effluents, MCP- 1 concentrations were tion. Because IL-8 and huGROct are both potent neu- below detection limit in the peritoneal fluid of non- trophil chemoattractants (3-5) and bevels are raised CAPD patients. Monocyte migration toward the pen- during peritonitis, it can be concluded that these two toneal fluid of these non-CAPD patients was never chemokines contribute significantly to the neutro- measurable, whereas high chemotaxis was observed philic cell influx seen in bacterial peritonitis in CAPD toward CAPD peritonitis effluents and, although to a patients. This conclusion is substantiated by the sig- lesser extent but above control bevel, toward stable nificant correlation between neutrophil numbers and CAPD effluents (Figure 3). The chemotactic activity huGROa concentrations found In the dialysates (Fig- that was found toward the peritonitis effluents could ure 2A). be neutralized completely by a specific anti-MCP- 1 In this investigation, we also addressed the question antibody, suggesting that in the majority of CAPD about which of the members of the chemokine family patients MCP- 1 is the only important monocyte che- would regulate the monocytic cell influx that occurs moattractant In peritonitis. It is tempting to speculate shortly after the neutrophilic cell influx in peritonitis. that high levels of the chemokine are also maintained Therefore, the monocyte chemoattractants RANTES in stable CAPD patients to constantly repopulate the ( 1 7) and MCP- 1 ( 1 8) were measured in effluents as peritoneal cavity with macrophages. possible candidates. It became clear from our results The observation of one peritonitis episode in which that RANTES could not be detected in peritonitis or high chemotactic movement could not be blocked by stable effluents, rendering It improbable that It Is anti-MCP- 1 antibody can not readily be explained, just important as a monocyte chemoattractant in this like the episode in which only very bow monocyte che- inflammatory state. Only in 10 times concentrated motaxis was found (Figure 3A). The only difference peritonitis effluents could a very low concentration of between these two aberrant cases and the others is that the chemokine be detected (5 to 10 pg/mL), which is peritonitis in these two patients occurred during the probably not physiologically relevant for monocyte daytime instead of during the night, which Implies a chemotaxis. Because MCP- 1 could be detected In shorter dwell time (4 instead of 8 h). One possibility every effluent in physiologically relevant amounts, It could be that, at the very early onset, neutrophils are was concluded that this chemokine is involved in the attracted exclusively and only after several hours are the regulation of the monocytic cell influx that follows monocytes attracted. In the early phase of chemotactical neutrophib cell influx. Effluent levels of the chemokmne recruitment of monocytes, one or several other proteins are significantly raised during the inflammation (Fig- may play a role before MCP- 1 becomes active; this may ure 1B, P < 0.02 in the Wilcoxon signed rank test). No be another chemokine, products from neutrophil granu-

Journal of the American Society of Nephrology 2383 Chemokines in Peritoneal Dialysis

lae like leukotriene B4 ( 1 9), or early mediators of inflam- 4. Schall TJ: Biology of the RANTES/SIS cytokine family. matlon like complement products or bacterial-derived Cytokmne 1991;3:165-183. 5. Schall TJ, Bacon KB: Chemokmnes, leukocyte trafficking, products (20). The influence of these other mediators on and inflammation. Curn Opmn Immunol 1994;6:856-873. the cell influx in the peritoneal cavity is not investigated 6. Betjes MGH, Tuk CW, Struijk DG, et at.: Interleukmn-8 in this study. However, the contribution of the chemo- production by human peritoneab mesothelial cells in response to tumor necrosis factor-a, intenleukin- 1 and kines IL-8, huGROa, and MCP- 1 to cell Influx in the medium conditioned by macrophages co-cultured with penitoneab cavity could be demonstrated. Staphylococcus epidenmidis. J Infect Dis 1993; 168: Identification of the relevant chemokines in the 1202-1209. clinical situation is of great importance because these 7. Topley N, Brown Z, J#{246}rres A, et at.: Human peritoneal mesothelial cells synthesize interleukin-8. Synergistic mediators are possible subjects of therapeutic inter- induction by interbeukmn- 1 3 and tumor necrosis factor a. vention. In the normal healthy situation, the rapid Am J Pathol 1993: 142:1876-1886. induction of chemokines by promnflammatory media- 8. Topley N, Williams JD: Role of the penitoneal membrane in the control of inflammation in the penitoneal cavity. tors in case of infection will be adequate and the Kidney Int 1994;46lSuppl 481:S71-S78. response will be downregulated by crosstalk with the 9. Goodman RB, Wood RG, Martin TR, Hanson-Painton 0, IL- 1 system (20). A situation is conceivable, in partic- Kinasewitz GT: Cytokine-stimulated human mesothelial cells produce chemotactic activity for neutrophils includ- ubar for a slightly compromised group bike CAPD ing NAP- 1 /IL-8. J Immunol 1 992; 148:457-465. patients, in which intervention with the local immune 10. Betjes MGH, Bos HJ, Krediet RT, Arisz L: The mesothe- system can be beneficial. For instance, patients who hal cells in CAPD effluent and their relation to peritonitis have low mesotheliab cell counts and a higher mci- incidence. Pent Dial mt 1991:11:22-26. 1 1 . Betjes MGH, Visser CE, Zemel D, et at.: Intnaperitoneal dence of peritonitis ( 10) may be helped with autobo- intenleukmn-8 and neutnophil influx in the initial phase of gous mesothelial cell transplantation and specific a CAPD peritonitis. Pent Dial mt 1996:16:135-142. chemokine therapy to compensate for the lack of 12. Bos HJ, Struijk DG, Tuk CW, et at.: Peritoneal dialysis induces a local sterile inflammatory state and the me- chemokmne induction upon infection caused by the sotheliab cells in the effluent are related to the bacterial impaired mesothebial cell layer. It may be advanta- peritonitis incidence. Nephron 199159:508-509. geous to intensify the inflammatory response in these 13. Betjes MHG, Tuk CW, Visser CE, et at.: Analysis of the peritoneal cellular immune system during CAPD shortly patients because their situation increases the risk of before a clinical peritonitis. Nephnol Dial Transplant sclerosing peritonitis, which is a severe complication 1994:9:684-692. in peritoneal dialysis. Furthermore, after onset of 14. Pen G, Milanese C, Matteucci C, et at.: A new monocbo- nab antibody (5D3-F7) which recognizes human mono- peritonitis, it may be beneficial for CAPD patients to cyte-chemotactic protein- 1 but not related chemokmnes. accelerate the quenching of the chemokine response Development of a sandwich ELISA and in situ detection by adding antichemokine antibodies to restrict further of producing cells. J bmmunol Methods 1994; 174:249- damage to the mesotheliab layer. 257. 15. Freundlich B, Avdalovic N: Use ofgelatin/plasma coated In conclusion, our data indicate that in the case of flasks for isolating human peripheral blood monocytes. bacterial peritonitis in CAPD patients, IL-8, huGROa, J Immunol Methods 1993:62:31-37. and MCP- 1 are involved in the recruitment of neutro- 16. Martinet Y, Martinet N, Vignaud JM, Plenat F: Blood monocyte chemotaxis. J Immunol Methods 1 994; 174: phibs and monocytes, respectively. Other members of 209-2 14. the chemokine family seem to be less relevant in this 17. Wiedermann CJ, Kowald E, Reinisch N, et at.: Monocyte setting but may be important in other than bacterial haptotaxis induced by the RANTES chemokmne. Curn inflammations, requiring other cell type influxes. It Biol 1993;3:735-739. 18. Leonard EJ, Yoshimura T: Human monocyte chemoat- can be postulated that every kind of inflammation tractant protein- 1 (MCP- 1 ). Immunol Today 1 990; 11:97- may be characterized by a different pattern of chemo- 101. kine production, explaining the redundancy of chemo- 19. Sprenger H, R#{246}sberA, Tonn P, Huffmann G, Gemsa D: Differential recruitment of leukocytes into cerebrospinal kines in the human body (20,2 1 ) and offering a pos- fluid of meningitis patients by chemoattnactant cyto- sibibity for therapeutic intervention. kines (chemokines) (Poster 421. Presented at the Ninth Annual Conference of the European Macnophage Study ACKNOWLEDGMENTS Group, Amsterdam, The Netherlands, October 1995. 20. Kristensen M, Jinquan T, Thomsen MK, et at: ETH615, We thank Dr. A. Mantovani for providing us with the anti-MCP- 1 a synthetic inhibitor of beukotniene biosynthesis and monoclonal antibody 5D3-F7 and Dr. H. Sprenger for suggestions function, also inhibits the production of and biological concerning the MCP- 1 ELISA. This study was partly supported by responses towards mntenleukin-8. Exp Dermatol 1993;2: Grant C92.l234 from the Dutch Kidney Foundation. 165-170.

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2384 Volume 7 . Number 1 1 . 1996