Podocytes Express the Endothelial Form of Interleukin 8 in Human Glomerulonephritis*

ORIGINAL PAPERS Adv Clin Exp Med 2004, 13, 2, 245 254 ISSN 1230 025X ZOFIA I. NIEMIR 1, PAWEŁ OLEJNICZAK 1, GRZEGORZ DWORACKI 2 Podocytes Express the Endothelial Form of Interleukin 8 in Human Glomerulonephritis* Ekspresja śródbłonkowej formy interleukiny 8 w nerkach osób chorych na kłębuszkowe zapalenia nerek 1 Department of Nephrology, University of Medical Sciences, Poznan, Poland 2 Department of Clinical Immunology, University of Medical Sciences, Poznan, Poland Abstract Objectives. Interleukin 8 (IL 8) exists in the two major forms, as a 77 amino acid (aa) peptide or as a 72 aa pro tein, known as endothelial (IL 8E) and monocyte (IL 8M) forms of this chemokine, respectively. Recent data sug gest that the biological activities of these peptides might be different. Since podocytes are able to produce IL 8 in vitro and they express IL 1α and IL 1β, i.e. the principal IL 8 inducing factors, in glomerulonephritis (GN), the authors decided to look for the expression of IL 8 in GN. Material and Methods. Fifty patients with different morphological forms of GN were included into the study. Nine normal kidneys served as controls. The alkaline phosphatase anti alkaline phosphatase (APAAP) method was used to analyze the expression of IL 8E, IL 1α, and IL 1β in tissue specimens. Antibodies to synaptopodin and CD31 were employed to confine the expression of the examined peptides to podocytes or endothelial cells, respec tively. The specificity of an antibody to IL 8E was verified by polyacrylamide gel electrophoresis of protein extracts isolated from kidneys and subsequent immunoblotting. Results. The major sources of IL 8E in control kidneys were glomerular podocytes and endothelial cells of the interstitial vessels. Substantial increase in the podocytes expression of IL 8E was noted in minimal change dis ease, early stages of idiopathic membranous GN and IgA GN. The glomerular expression of IL 8E in GN reflect ed the ones, which were observed for IL 1α and IL 1β. The immunoreactivity for all these peptides was reduced in acute endocapillary GN, IgA GN with marked glomerular cell proliferation, as well as in advanced phases of membranous nephropathy and focal segmental glomerulosclerosis. Conclusions. IL 8E is expressed by the normal podocytes and the increase in its expression in GN, related with that of IL 1α and IL 1β, seems to be associated with the preservation of the glomerular architecture (Adv Clin Exp Med 2004, 13, 2, 245 254). Key words: IL 8, IL 1, glomerulonephritis, podocytes, maintaining of the glomerular architecture. Streszczenie Cel pracy. W świetle wyników badań wskazujących, że podocyty kłębuszka nerkowego są istotnym źródłem in terleukiny 1 (IL 1) u chorych na kłębuszkowe zapalenia nerek (k.z.n.) oraz faktu, że ta cytokina należy do głów nych czynników stymulujących wytwarzanie IL 8, celem badań było porównanie ekspresji IL 8, IL 1α i IL 1β w nerkach niezmienionych chorobowo oraz w przebiegu k.z.n. Podjęto próbę oceny ekspresji, tzw. formy śródbłon kowej IL 8 (IL 8E), której działanie ma się różnić od wykazywanych dla formy leukocytarnej IL 8, uwalnianej przede wszystkim przez monocyty. Materiał i metody. Badaniami objęto 50 chorych z różnymi formami morfologicznymi k.z.n.. Materiałem kontrol nym było 9 fragmentów nerek z otoczenia usuniętego nowotworu nerki. Ekspresję IL 8E, IL 1α i IL 1β oraz an tygenów własnych komórek kłębuszka nerkowego (CD31 i synaptopodyny) oceniano za pomocą immunohistoche mii wykonywanej metodą APAAP (alkaliczna fosfataza antyalkaliczna fosfataza). Swoistość przeciwciała dla IL 8E potwierdzono techniką Western blot. Wyniki. W nerkach kontrolnych ekspresję IL 8E wykazywały podocyty w kłębuszkach nerkowych, komórki śród błonka naczyń śródmiąższowych oraz niektóre komórki zlokalizowane w śródmiąższu nerek. Znaczny wzrost eks * Supported by the KBN grant No. 4PO5A 076 14.

246 Z. I., NIEMIR, P. OLEJNICZAK, G. DWORACKI presji IL 8E, który wykazywały podocyty i któremu towarzyszył równoległy wzrost ekspresji IL 1α i IL 1β, ob serwowano w kłębuszkach nerkowych chorych na submikroskopowe k.z.n., błoniaste k.z.n. w jego wczesnych okresach oraz nefropatię IgA z niewielkim rozplemem mezangium. Istotnie statystycznie mniejszą ekspresję po wyższych białek obserwowano w kłębuszkach nerkowych chorych na ostre wewnątrzwłośniczkowe k.z.n., nefro patię IgA ze znacznym rozplemem komórek mezangialnych oraz w zaawansowanych stadiach błoniastego k.z.n. i ogniskowego stwardnienia kłębuszków nerkowych. Wnioski. Podocyty niezmienionych chorobowo kłębuszków nerkowych wykazują ekspresję śródbłonkowej formy IL 8. Wzrost ekspresji tej chemokiny, który towarzyszy równoległemu wzrostowi ekspresji IL 1α i IL 1β, kojarzy się z zachowaniem struktury kłębuszków nerkowych (Adv Clin Exp Med 2004, 13, 2, 245 254). Słowa kluczowe: IL 8, IL 1, podocyty, kłębuszkowe zapalenia nerek, zachowanie struktury kłębuszka nerkowego. Interleukin (IL 8/CXCL8) is the prototypic CXC chemokine, which was purified as a mono cyte derived factor that attracts neutrophils, but not monocytes in Boyden chamber assays. The further studies have revealed that a variety of cells including cultured fibroblasts, endothelial cells, keratinocytes, epithelial cells, as well as lymphocytes and neu trophils, are capable of producing IL 8 [1]. With respect to the kidney, tubular epithelial cells [2 3], fibroblasts [4], mesangial cells [5 6] and podocytes [7] have been shown to produce IL 8 in vitro. The N terminal amino acid (aa) sequence of IL 8 displays heterogeneity that results in length of the secreted protein varying from 79 aa through 77, 72, 71, 70 aa forms to a 69 aa variant [8 10]. The 77 aa and 72 aa forms have been reported to repre sent the majority of the protein released [9]. The 77 aa IL 8 form, originally purified from human cytokine activated umbilical vein endothelial cells and shown to protect these cells from neutrophil mediated damage [8], is also known as endothelial form of IL 8 (IL 8E). Human dermal and gingival fibroblasts stimulated with lipopolysaccharide, IL 1 or tumor necrosis factor α (TNF α) also produce this form of IL 8 [8, 11 12]. Human monocytes and lymphocytes stimulated with the above factors release both the IL 8E and 72 aa forms of IL 8, the latter known as a monocyte derived IL 8 form (IL 8M) [10 11, 13]. In vitro, IL 8M has been shown to be 10 fold more effective than IL 8E in a receptor binding assay, as well as in inducing neutrophil adherence to laminin coated plates and in elevation of cytoplasmic levels of Ca 2+ in neu trophils [14]. Though, both forms of IL 8 have been shown to be equipotent in inducing neutrophil accumulation and edema formation in vivo [14]. A suggested explanation of the latter observation is that the extended IL 8E form of IL 8 is proteolyti cally cleaved in vivo to the more biologically active IL 8M form of this chemokine [12, 14, 15]. Yet, cardioprotective and endothelial protective effects of IL 8E have been shown in a rabbit model of myocardial ischemia and reperfusion. These bene ficial effects of IL 8E have been related to inhibi tion of neutrophil adherence to the myocardium and preserved vasorelaxant responses of coronary arterioles [16]. The novel findings show that IL 8E suppresses cell growth and induces apoptosis in most leukemic cell lines [17]. In addition, this form of IL 8 has been proven as indispensable factor in wound healing [18]. Among the most potent IL 8 inducing factors are IL 1 and TNF α [1 6]. The authors as well as others have previously shown that intrinsic glomerular cells, podocytes and mesangial cells, respectively, express these cytokines in GN [19 22]. Thus, it seemed reasonable to address the question of IL 8 production in the renal tissue with various morphological forms of GN. The results show that IL 8E is expressed by the normal podocytes and its expression, related to that IL 1α and IL 1β, varies in different types of GN. Material and Methods Patients Fifty patients with biopsy proven GN were included in the study: 5 with acute endocapillary (AGN), 25 with IgA GN, 10 with idiopathic mem branous GN (IMGN), 5 with minimal change dis ease (MCD), and 5 with focal segmental glomeru losclerosis (FSGS). Morphological, clinical and laboratory data of the patients are summarized in Table 1. All the patients presented with variable degrees of proteinuria and erythrocyturia, and nor mal or impaired renal function. In the Table 1 iden tified were patients biopsied for a revision of the diagnosis due to ineffectiveness of the adminis tered steroid and/or steroid cytotoxic treatment. In the AGN patients, a detailed clinical histo ry revealed an upper respiratory tract infection 2 4 weeks prior to the occurrence of urinary abnor malities. In three of the patients from this group, a renal biopsy was performed during an early acute phase of GN with renal function impairment. In the other two patients, a biopsy was prescribed due to the persistence of urinary abnormalities 6 and 12 months after the onset of the disease. One

Podocytes Express the Endothelial Form at IL 8 in Human Glomerulonephritis 247 Table 1. Histological and clinical data of patients at the time of renal biopsy Tabela 1. Histologiczne i kliniczne dane pacjentów podczas biopsji nerki Morphology No. of patients, Age, yr Serum creatinine Renal insufficiency a, Urinary protein, Nephrotic syndrome b, Gross hematuria, Patients undergoing (Rozpoznanie male/female (Wiek, lata) (Stężenie kreaty no. of patients mean ± SD no. of patients no. of patients biopsy/repeat biopsy morfologiczne) (Liczba chorych, mean range niny w surowicy) (Niewydolność (Białkomocz, (Zespół nerczy (Makroskopowy during therapy, kobiety/ (średnia) (zakres) mean ± SD, nerek, liczba zakres ± SD) cowy, liczba krwiomocz, no. of patients mężczyźni) mg/dl chorych) g/24 h chorych) liczba chorych) (Liczba chorych, u których biopsję nerki wykonano w czasie leczenia) AGN 5 (4/1) 46 33 71 1.92 ± 1.00 3 2.58 ± 2.97 2 3 IgA GN MGA 4 (4/0) 18 12 22 0.83 ± 0.17 0 1.10 ± 0.58 0 3 MesPGN 10 (4/6) 40 14 71 1.43 ± 1.10 2 2.46 ± 2.67 3 4 CrGN 7 (6/1) 33 8 71 1.54 ± 1.30 2 7.04 ± 4.91 5 4 ScGN 4 (4/0) 45 37 57 3.67 ± 4.25 3 3.82 ± 3.24 1 2 IMGN Stage I/II 4 (4/0) 43 7 61 0.90 ± 0.25 0 8.55 ± 3.07 4 0 Stage II/III 4 (3/1) 58 42 71 1.05 ± 0.38 1 5.57 ± 3.09 3 0 2 c Stage III 2 (2/0) 38 26 49 1.15 ± 0.49 1 6.25 ± 2.47 2 0 2 e MCD 5 (1/4) 21 7 47 0.74 ± 0.18 0 4.48 ± 3.92 3 0 1 e FSGS 5 (4/1) 43 33 48 3.02 ± 3.15 2 6.32 ± 7.82 2 0 2 c, 2 d AGN acute endocapillary GN, MGA minimal glomerular abnormalities, MesPGN mesangial proliferative GN, CrGN mesangial proliferative GN with crescent formation, ScGN scleros ing GN, IMGN idiopathic membranous GN, MCD minimal change disease, FSGS focal segmental glomerulosclerosis. a Serum creatinine concentration above 1.5 mg/dl. b Proteinuria > 3.5 g/1.73 m 2 /24 h, accompanied by hypoalbuminemia and lipid abnormalities. c Steroids. d Steroid cyclophosphamide treatment. e Steroid chlorambucil therapy. AGN ostre wewnątrzwłośniczkowe k.z.n., MGA zmiany minimalne, MesPGN mezangialne rozplemowe k.z.n., CrGN mezangialne rozplemowe k.z.n. z formowaniem półksiężyców, ScGN stwardniające k.z.n., IMGN idiopatyczne błoniaste k.z.n., MCD zmiany minimalne, FSGS ogniskowe stwardnienie kłębuszków nerkowych. a Stężenie kreatyniny w surowicy powyżej 1.5 mg/dl. b Białkomocz > 3.5 g/1.73 m 2 /24 h z towarzyszącą hipoalbuminemią i zaburzeniami gospodarki lipidowej. c Steroidy. d Leczenie skojarzone steroidami i cyklofosfamidem. e Leczenie skojarzone steroidami i chlorambucylem.

248 Z. I., NIEMIR, P. OLEJNICZAK, G. DWORACKI Table 2. Antibodies used in the immunohistochemical procedure Tabela 2. Przeciwciała stosowane w badaniu immunohistochemicznym Antibody Dilution Source (Przeciwciało) (Rozcieńczenie) (Producent) Primary antibodies Mouse anti human endothelial form of IL 8 1: 200 American Bio Technologies Inc., Cambridge, MA, USA Mouse anti human IL 1α 1: 50 Immunotech, Marseille, France Mouse anti human IL 1β 1: 200 Immunotech Mouse anti rat synaptopodin 1: 2 Kindly supplied by Dr. Peter Mundel (Department of Anatomy and Cell Biology, University of Heidel berg, Germany) [23] Mouse anti human CD31 (PECAM 1) 1: 200 Novocastra Laboratories, Newcastle, UK Secondary antibodies Rabbit anti mouse IgG 1: 40 DAKO A/S, Glostrup, Denmark Mouse anti rabbit IgG 1: 40 DAKO APAAP complex 1: 40 DAKO patient displayed microscopic hematuria, while the other presented with microscopic hematuria and mild proteinuria. Patients with IgA GN presented morphologi cally with varied degrees of mesangial expan sion/proliferation and/or sclerosis along with vari able severity of tubulointerstitial lesions. They were categorized as minimal glomerular abnor malities (MGA; n = 4), mesangial proliferative GN (MesPGN; n = 10), mesangial proliferative GN with segmental crescents (CrGN; n = 7), and sclerosing GN (ScGN; n = 4) [20, 22]. Histological classification of IMGN was made according to the extent of glomerular membrane alterations revealed by silver staining. Immunohistochemistry The immunohistochemical evaluation of IL 8E, IL 1α and IL 1β was performed on acetone fixed cryostat sections, using the APAAP (alkaline phosphatase anti alkaline phosphatase) method as previously described [20, 22]. The primary and secondary antibodies applied in the staining proce dure are listed in Table 2. Antibodies to CD31 and synaptopodin were used as markers of endothelial cells and podocytes, respectively [20, 23]. Control experiments were conducted by omit ting the incubation with the primary antibody and by substituting a non immune murine serum for the primary antibody. SDS PAGE Western Blot Verification of the Detected IL 8 Protein Kidney samples from 3 patients (2 with MesPGN in the course of IgA GN and 1 with MCD) were homogenized in an extraction buffer (20 mm Tris HCl, 500 mm NaCl, ph 7.5) supple mented with 0.5% CHAPS (Sigma), containing phenylmethylsulfonylfluoride (1 mm, Sigma), as well as trasylol (200 IU/ml, Sigma) as protease inhibitors [23]. Isolated proteins were diluted 1: 4 (v/v) with sample buffer containing 20% SDS and 100 mm 2 mercaptoethanol, and analyzed on 5 15% SDS PAGE gradient gels [24]. Due to small amounts of tissue available, only 10 mg of protein, as determined by the method of Bradford [25], were applied per lane. The 77 aa and 72 aa forms of human IL 8 (500 ng, Antigenix America Inc., Franklin Square, NY, USA) were used as pos itive controls. After SDS PAGE, proteins were transferred onto Immobilon P membrane (Millipore, Eschborn, Germany). The sheets were preincubated in Tris HCl buffer, ph 7.6, supple mented with 1.5% dry milk and 2% BSA for 1 hour at room temperature. Subsequently, an overnight incubation with anti IL 8 antibody at 4 C was performed. The antigen antibody reaction was detected using the APAAP method and the BCIP/NBT system (Boehringer Mannheim) for signal visualization [20, 22]. Statistical Analysis The mean glomerular expression score (MES) for IL 8 was calculated basing on the staining intensity (I) and percentage of glomerular area stained (A) by the applied antibody. For calcula tion of MES, the formula used was MES = + I(1) A(1)/100% +... + I(n) A(n)/100%/(n), where N was the number of glomeruli in the renal specimen. The staining intensity was graded from

Podocytes Express the Endothelial Form at IL 8 in Human Glomerulonephritis 249 Fig. 1. Western blot analysis after SDS PAGE of pro tein extracts from kidney samples with GN. An anti IL 8E antibody reacted with a protein of a molecular weight between 6.5 and 14.5 kda isolated from biopsy specimens of patients with MesPGN in the course of IgA GN (lanes 3 and 4) and MCD (lane 5). This anti body also reacted with the 77 aa form of IL 8 (lane 1), whereas no reactivity with the 72 aa recombinant form of IL 8 could be observed (lane 2) Ryc. 1. Analiza Western blot białek izolowanych z fragmentów nerek chorych na k.z.n. Przeciwciało przeciwko IL 8E reagowało z białkami o ciężarze czą steczkowym między 6,5 i 14,5 kda, izolowanymi z fragmentów materiału biopsyjnego chorych na MesPGN w przebiegu nefropatii IgA (ścieżki 2 i 3) i MCD (ścieżka 5). Stosowane przeciwciało reagowało z rekombinowaną formą IL 8 zawierającą 77 amino kwasów (ścieżka 1), nie reagowało natomiast z formą IL 8, zawierającą 72 aminokwasy (ścieżka 2) 0 to 3 points according to the following scale: 0 no immunoreactivity, 1 weak immunoreactivity, 2 moderately intense reactivity, 3 dense, marked immunoreactivity. All values in the text and figures are presented as means ± standard deviations (SD). The ANOVA was used to test differences between the particular groups of patients. A value of p < 0.05 was accept ed as being statistically significant. Results The specificity of an applied anti IL 8 anti body for the 77 aa form of IL 8 was confirmed by SDS PAGE and Western blotting of detergent extracts from kidney samples, which were run under reducing conditions. This anti IL 8 antibody bound a peptide of a molecular weight between 14.5 and 6.5 kda in a used 5 15% polyacrylamide gradient. The molecular weight of the detected peptides corresponded with that of the respective control protein (Fig. 1). In the normal kidney, the reactivity for IL 8E was confined to the outer aspects of glomerular cap illaries indicating that podocytes were the cellular source of this form of IL 8 (Fig. 2A, 2A1). When podocytes were stained with an antibody to synap topodin, i.e. a podocyte marker, the reactivity for synaptopodin largely exceeded that for IL 8E (Fig. 2B). In addition to glomerular podocy tes, endothelial cells of interstitial arterioles, as well as smooth muscle cells of these vessels and some cells located in the interstitium expressed IL 8E. The authors compared the glomerular reactivity for IL 8E, synaptopodin, and an endothelial cell marker, CD31 (PECAM 1; platelet endothelial cell adhesion molecule 1), in the morphological forms of GN examined. In GN, the glomerular expression for IL 8E was, as in the normal kidney, exclusively confined to the outer aspects of glomerular resembling the pattern of reactivity observed for synaptopodin (Fig. 2). However, the glomerular expression for IL 8E in MCD largely exceeded that for synaptopodin (Fig. 2C vs. 2E). In IMGN stage I II, this reactivity was comparable to that of synaptopodin in the normal kidney (not shown). In later stages of IMGN (Fig. 2F vs. 2H) and FSGS, the podocyte reactivity for synap topodin largely surpassed that of IL 8E. The latter trend was more clearly visible in mesangial prolif erative GN. Depending on the extent of mesangial cell proliferation loss of podocyte reactivity for synaptopodin was observed in this group of patients (Fig. 2K and 2N). Nevertheless, even in samples with an advanced disruption of the glomerular architecture, the podocyte staining for synaptopodin exceeded that for IL 8E (Fig. 2L vs. 2N). A semiquantitative evaluation of the glomeru lar expression of IL 8E in renal biopsies with dif ferent forms of primary GN (according to the grad ing system given in the Material and Methods sec tion) is presented in Figure 3. A similar analysis of the expression of both forms of IL 8 in different phases of IgA GN is demonstrated in Figure 4. As it is shown in Figure 5, in the normal glomeruli only trace amounts of IL 1α were detected along the outer aspects of glomerular tufts, whereas no immunoreactivity for IL 1β could be stated in this condition. In GN, however, the glomerular expression of IL 8E exactly resem bled that observed for IL 1α and IL 1β. Clinical Pathological Correlation The most pronounced expression of IL 8E, IL 1α and IL 1β was associated with a well pre served glomerular architecture and only minor interstitial alterations present. Furthermore, no relationship could be stated between the degree of proteinuria and the expression of either cytokine

250 Z. I., NIEMIR, P. OLEJNICZAK, G. DWORACKI Fig. 2. A, A1 the immunoreactivity for IL 8E in a normal glomerulus. Outer aspects of glomerular capillaries are positive; B the immunoreactivity for synaptopodin in a normal glomerulus ( 400). Outer aspects of the glomerular capillaries are strongly positive. (C to N) A comparison between the glomerular expression of IL 8E, CD31 and synaptopodin in MCD (C E), IMGN stage II/III (F H), MesPGN with segmental sclerotic lesions in the course of IgA GN (I K), and crescentic IgA GN (L N) Ryc. 2. A, A1 ekspresja IL 8E w kłębuszku nerkowym nerki zdrowej. B ekspresja synaptopodyny w kłębuszku nerkowym nerki zdrowej. C do N porównanie ekspresji IL 8E, CD31 i synaptopodyny w kłębuszkach nerkowych chorych na MCD (C E), IMGN ze zmianami okresu II (F H) oraz nefropatię IgA ze zmianami typu MesPGN (I K) i CrGN (L N)

Podocytes Express the Endothelial Form at IL 8 in Human Glomerulonephritis 251 Fig. 3. Evaluation of the glomerular expression of IL 8E in biopsies with different morphological types of GN (for abbreviations, see the Material and Methods section and Table 1) Ryc. 3. Porównanie ekspresji IL 8E w kłębuszkach nerkowych chorych z różnymi formami morfologicz nymi k.z.n. (znaczenie skrótów patrz Materiał i me tody oraz tab. 1) Fig. 4. Evaluation of the glomerular expression of IL 8E in tissue specimens with IgA GN (for abbrevia tions, see the Material and Methods section and Table 1) Ryc. 4. Porównanie ekspresji IL 8E w kłębuszkach nerkowych chorych z różnym zaawansowaniem zmian morfologicznych w przebiegu nefropatii IgA (znacze nie skrótów patrz Materiał i metody oraz tab. 1) Fig. 5. A comparison between the glomerular expression of IL 1α, IL 1β and IL 8E in a normal glomerulus (A C), advanced stage of FSGS (D F), and during the restitution phase after AGN (G I) (for abbreviations, see the Material and Methods section and Table 1) Ryc. 5. Porównanie ekspresji IL 1α, IL 1β i IL 8E w kłębuszkach nerkowych nerki zdrowej (A C), w zaawansowanym stadium FSGS (D F) oraz w okresie rezolucji po AGN (G I) (znaczenie skrótów patrz Materiał i metody oraz tab. 1)

252 Z. I., NIEMIR, P. OLEJNICZAK, G. DWORACKI in different morphological forms of GN. In this respect, patients exhibiting the higher levels of these peptides expression in glomeruli (MCD, IMGN stage I II or IgA GN with minimal glomerular abnormalities) presented with features of the nephrotic syndrome or only low grade pro teinuria (Tab. 1). Discussion Previous studies on IL 8 expression in GN have related its effects with neutrophil infiltration into glomeruli [26 28]. Consequently, a release of lysosomal enzymes and generation of superoxide anions from neutrophils has been suggested to contribute to the renal damage [26, 27]. Injection of an anti IL 8 antibody into rabbits with immune complex GN reduced glomerular neutrophil infil tration, as well as prevented foot processes fusion and proteinuria [26]. In human GN, the expression of IL 8 has mainly been detected in association with glomerular cell proliferation and confined either to the expanded mesangial areas and crescents [27 29] or spread over the whole glomeruli [27]. In the study, the glomerular expression of IL 8E reflected that for IL 1α and IL 1β. Podocytes were revealed to be the cellular source of the extended 77 aa form of IL 8 in the normal glomeruli, and the expression of this IL 8 form increased in GN. The SDS PAGE of kidneys extracts with subse quent immunoblotting allowed authors to con clude that the used antibody was specific for the IL 8E form of IL 8. Conversely to the expecta tions, the highest expression of IL 8E was observed in MCD and early stages of IMGN, where proliferation of glomerular cells is absent or minimal and a significant number of infiltrating cells, in particular of the PMN cell lineage, is not observed in glomeruli. Intriguingly, an abundant podocyte expression of CXCR1, which is the primary functional recep tor for IL 8, has recently been reported in IMGN [7]. The expression of IL 8 protein has not been examined in these patients. Yet, in vitro, podocytes stimulated with fetal calf serum released IL 8 into the culture medium. Since different culture condi tions did not significantly influence the expression of mrna for CXCR1, the authors suggested that CXCR1 could be down regulated by ligand induced receptor internalization [7]. However, irrespective of the IL 8 binding to its receptors through the N terminal sequences, IL 8 can bind to heparin and heparan sulfate through its C termi nal α helix [30]. We did not find any positive relationship between the clinical activity of GN and the expres sion of IL 8, IL 1α and IL 1β. This is in line with the results of Mezzano et al. who could not observe any relation between the glomerular immunoreac tivity for IL 8 and proteinuria in AGN [28]. In gen eral, the more pronounced distortion of the glomeru lar architecture the lower expression of IL 8E was found. Loss of podocytes and even more deeply visible reduction in the IL 8E expression was stat ed in this condition. Thus, it could be suggested that IL 8 production by glomerular cells seems to be important for the maintaining of the glomerular architecture. The recent study of the group of Svanborg has demonstrated that IL 8 binding to epithelial cells expressing CXCR1 caused IL 8 dependent neu trophil transmigration through these cell lines [31]. In this context, the IL 8E form of IL 8 has been demonstrated to be a potent neutrophil transmigra tion inducing factor [32]. Also IL 8 bound to heparin and glycosaminoglycans has been shown to promote transmigration of neutrophils through the endothelial cell barrier and, simultaneously, protected the tissue from damage through lytic enzymes released by migrating cells [30]. Thus, a plausible hypothesis would be that IL 8 promotes neutrophil transmigration through the glomerular filtration barrier. In addition, the IL 8E induced apoptosis in transmigrating cells [17] can further contribute to the protection of the glomerular filter from neutrophil mediated damage. References [1] Rollins BJ: Chemokines. Blood 1997, 90, 909 928. [2] Schmouder RL, Strieter RM, Wiggins RC, Chensue SW, Kunkel SL: In vitro and in vivo interleukin 8 pro duction in human renal cortical epithelia. Kidney Int 1992, 41, 191 198. [3] Gerritsma JSJ, Hiemstra PS, Gerritsen AF, Prodjosudjadi W, Verweij CL, Van ES, Daha MR: Regulation and production of IL 8 by human proximal tubularepithelial cells in vitro. Clin Exp Immunol 1996, 103, 289 294. [4] Lonnemann G, Engler Blum G, Müller GA, Koch KM, Dinarello CA: Cytokines in human interstitial fibro sis. II. Intrinsic interleukin (IL) 1 synthesis and IL 1 dependent production of IL 6 and IL 8 by cultured kidney fibroblasts. Kidney Int 1995, 47, 845 854.

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254 Z. I., NIEMIR, P. OLEJNICZAK, G. DWORACKI [31] Huber AR, Kunkel SL, Todd RF III, Weiss SJ: Regulation of transendothelial neutrophil migration by endoge nous interleukin 8. Science 1991, 254, 99 102. [32] Godaly G, Hang L, Frendeus B, Svanborg C: Transepithelial neutrophil migration is CXCR1 dependent in vitro and is defective in IL 8 receptor knockout mice. J Immunol 2000, 165, 5287 5294. Address for correspondence: Zofia I. Niemir Department of Nephrology, University of Medical Sciences Przybyszewskiego 49 60 355 Poznań Poland e mail: zniemir@usoms.poznan.pl Received: 16.10.2003 Revised: 17.11.2003 Accepted: 17.11.2003 Praca wpłynęła do Redakcji: 16.10.2003 r. Po recenzji: 17.11.2003 r. Zaakceptowano do druku: 17.11.2003 r.