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 addi- tional, 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