Published online before print November 3, 2003
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,1
* Institute of Experimental Dermatology and
Department of Pediatrics, University of Münster, Germany; and
Institute of Pathology, Heidelberg, Germany
1 Correspondence: Institute of Experimental Dermatology, University of Münster, Röntgenstr. 21, D-48149, Münster, Germany. E-mail: rothj{at}uni-muenster.de
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Key Words: systemic lupus erythematosus • interstitial nephritis • extracapillary GN • S100 protein • S100A8 • S100A9
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MRP8 and MRP14 are two calcium-binding proteins that belong to the S100 family [20 , 21 ]. MRP8 and MRP14 represent the predominant calcium-binding capacity in early differentiation stages of monocytes and neutrophils [22 ], but both proteins cannot be detected in resting tissue macrophages or in lymphocytes [21 , 23 ]. Noncovalently associated complexes of both molecules play a role in calcium-dependent modulation of cytoskeletal-membrane interactions [24 25 26 ]. Calcium-dependent translocation of MRP8 and MRP14 from the cytoplasm to the plasma membrane correlates with the inflammatory activation of these cells as shown by an elevated secretion of cytokines, e.g., tumor necrosis factor (TNF) or interleukin (IL)-1ß, and activation of the respiratory burst [27 , 28 ]. In various experimental models of inflammation, the expression of MRP8 and MRP14 by infiltrating cells showed a strong correlation with the inflammatory process [29 30 31 ]. Accordingly, MRP8 and MRP14 have been detected in monocytes and neutrophils in the infiltrate of various human diseases, e.g., chronic bronchitis, rheumatoid arthritis, Crohn’s disease, or colitis ulcerosa [21 , 32 33 34 35 ]. During renal allograft rejection, determination of a distinct expression pattern of these two calcium-binding proteins and their complex formation in monocytes in vivo allowed differentiation between acute and chronic courses of this disease [36 ]. Furthermore, in a prospective study, it has been shown that the intensity of the MRP8/MRP14-positive infiltrate is a prognostic factor for the course and outcome of acute renal allograft rejection [37 ]. No reliable marker has so far been identified to analyze inflammatory activity of the monocyte/macrophage lineage in GN. In the present study, we now demonstrate that expression of MRP8 and MRP14, concomitant with complex formation in infiltrating monocytes, is an indicator for a severe and acute inflammatory process in GN, whereas monocytic expression of these two proteins without concomitant complex formation rather indicates a chronic interstitial inflammation in GN.
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Control tissue comprised five renal biopsies from the unaffected pole of kidneys removed for renal cell carcinoma from patients with no history of inflammatory disorders. The investigations were conducted in accordance with the guidelines proposed in the Declaration of Helsinki; informed consent was obtained.
Antibodies
Rabbit antisera against recombinant MRP8 (aMRP8) and MRP14 (aMRP14) were produced as described earlier [25
]. Monospecificity of antibodies was analyzed by immunoreactivity against recombinant MRP8 and MRP14, Western blot analysis of lysates of monocytes and granulocytes, as well as by immunoreactivity against MRP8- and/or MRP14-transfected fibroblastic cell lines as described earlier [25
]. In addition, the mouse monoclonal antibody (mAb) 27E10 was used, which detects exclusively the MRP8/MRP14 heterodimer but not single monomers [27
]. Mouse mAb KP1 against the CD68 antigen, a 110-kDa transmembrane glycoprotein highly expressed by human monocytes and tissue macrophages, was used for single- and double-labeling experiments (Dako Diagnostika, Hamburg, Germany) [38
, 39
]. Peroxidase or alkaline phosphatase-conjugated second-stage antibodies were obtained from Dianova (Hamburg, Germany).
Cell culture
Human peripheral blood monocytes were isolated from buffy coats by Ficoll-Paque and Percoll (Pharmacia, Freiburg, Germany) density-gradient centrifugation. Purity of monocytes was >90%, as demonstrated by flow cytometry using mAb against CD14, CD15, and CD16 (Dianova). Monocytes were cultured for 1 day in Teflon bags using McCoy’s 5A medium supplemented with 2 mM calcium chloride and 15% fetal calf serum as previously reported [25
].
Immunoperoxidase technique
Cryostate sections of renal biopsies of GN obtained during diagnostic needle biopsies at the University of Heidelberg (Germany) were processed for immunoperoxidase staining as described previously. Briefly, acetone-fixed serial sections (5 µm) were placed in 0.1% hydrogen peroxide (v/v) and 0.12 M sodium azide (Merck, Darmstadt, Germany) in phosphate-buffered saline (PBS) to destroy endogenous peroxidase activity. Nonspecific protein binding was blocked with 1% (w/v) bovine serum albumin (BSA; Sigma Chemical Co., St. Louis, MO) in PBS, and sections were subsequently incubated with 1 µg/ml antibodies against MRP8, MRP14, or the mAb 27E10, respectively, followed by peroxidase-conjugated second-stage antibodies (Dianova). Peroxidase activity was detected with 3-amino-9-ethylcarbazole (Sigma Chemical Co.). Sections were counterstained with Mayer’s haemalaun (Merck). For negative controls, isotype-matched antibodies of irrelevant specificity were used. For double-labeling experiments, tissue samples were incubated successively for 1 h with 1% BSA, 10% normal goat serum in PBS, aMRP14 rabbit antiserum, peroxidase-conjugated goat anti-rabbit IgG F(ab')2, 1.5 mM 3-amino-9-ethylcarbazole (Sigma Chemical Co.), 0.02 mM H2O2 in acetate buffer (pH 5.2, 10 min) for color reaction, mAb KP1 against CD68, phosphatase-conjugated goat anti-mouse IgG F(ab')2, 0.02% (w/v) naphtol AS-MX phosphate, 0.1% (w/v) levamisole, and 0.1% (w/v) fast blue RR salt (all Sigma Chemical Co.) in 0.1 M Tris buffer, pH 8.2, as substrate (30 min). Slides were not counterstained after double-labeling procedures. Two independent observers separately analyzed the expression patterns of leukocyte-specific molecules within glomeruli as well as in the interstitium. Glomerular expression of different leukocyte antigens is presented as positively stained cells per glomerulus. At least five cross-sections of glomeruli per biopsy were counted. For quantification of interstitial expression, at least 250 cells per biopsy were counted at the site of maximal inflammatory reaction. Under some conditions, MRP8 and MRP14 show a slightly diffuse staining pattern, which may be a result of extracellularily secreted proteins. Only cytoplasmatic stainings of MRP8 or MRP14 with a clear nuclear association were counted as positive cells. Two observers (M. Frosch and J. Roth) independently did all quantifications. The overall inter-observer variability was less than 10%.
Flow cytometry
For detection of surface expression of the MRP8/MRP14 heterodimer on monocytes, mAb 27E10 was used, which detects specifically the MRP8/MRP14 heterodimer [27
]. Monocytes cultured for 1 day were treated with different anti-inflammatory drugs for 4 or 16 h as indicated. To analyze stimulatory as well as inhibitory effects on the surface expression of MRP8/MRP14, 0.1 mM arsenite was added to the medium in parallel sets of experiments during the last 2 h of the incubation period to up-regulate MRP8/MRP14 surface expression. After incubation, cells were harvested, washed, and resuspended in 20% normal goat serum for 30 min to block nonspecific binding. Primary antibodies (1 µg/ml) against the MRP8/MRP14 heterodimer (27E10) and against CD14 were allowed to react for 1 h at 4°C. After additional washing, cells were incubated with fluorescein isothiocyanate-conjugated goat anti-mouse F(ab')2 fragments. Thereafter, propidium iodide (Sigma Chemical Co.) was added to permit determination of cell viability and exclusion of nonviable cells. Cell viability was found to be greater than 95% in all experiments presented. Expression of surface molecules on monocytes was analyzed using a FACScan equipped with Lysis-II software (Becton Dickinson, San Jose, CA). Purity of monocyte populations was controlled by high expression of CD14.
Northern blot analysis
Monocytes were treated with different immunosuppressive agents for 4 h as indicated. Total RNA of monocytes was prepared using a two-step method. Cells were lysed with sodium dodecyl sulfate in a citric acid-containing buffer. This procedure was succeeded by salt precipitation to remove contaminating DNA and protein and by a final alcohol precipitation of RNA [40
]. Aliquots of the RNA (20 µg) were separated on a denaturing 1.5% agarose gel, blotted onto a nylon membrane, and probed with 32P-labeled human MRP8 and MRP14 cDNA probes as described earlier [41
]. Finally, the membranes were washed twice with 0.1% saline sodium citrate at 65°C and exposed to an X-ray film for 2 days. Expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) served as control.
Determination of MRP8/MRP14 concentrations by sandwich enzyme-linked immunosorbent assay (ELISA)
Monocytes were incubated with different immunosuppressive drugs as indicated. Monocytes were harvested and lysed in PBS containing 1% Nonidet P-40. Cellular debris was separated by centrifugation for 10 min at 10,000 rpm in an Eppendorf desk centrifuge, and a specific sandwich ELISA, as described earlier, determined concentrations of MRP8 and MRP14 in the fraction of soluble cellular proteins [42
]. For calibration, different amounts (0.25–250 ng/ml) of the native complex of human MRP8 and MRP14 were solubilized in dilution buffer and applied to the system. MRP8 and MRP14 form noncovalently associated complexes, which are detected by our ELISA system [35
]. We therefore calibrate our ELISA with the native MRP8/MRP14 complex and present our data as amount of MRP8/MRP14 complex per total cellular protein (µg/mg).
Statistical analysis
The U-test, according to Mann and Whitney (for values without normal distribution), was performed to determine significant differences in the number of MRP8- and MRP14-expressing cells in biopsies of GN. Values of P > 0.05 were considered not to be significant. Correlations of different disease parameters and MRP8/MRP14 expression are presented as Pearson’s correlation coefficients (r).
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Table 1. Clinical and Demographic Data of the Study Population
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Figure 1. Expression and complex formation of MRP8 and MRP14 in glomeruli of different GN. Serial sections of different GN were stained with antibodies specific for MRP8, MRP14, for the heterodimer MRP8/MRP14 (mAb 27E10), and for the macrophage-specific surface antigen CD68. Primary antibodies were detected by immunoperoxidase-conjugated second-stage antibodies. Data are presented as mean ± SEM of positively stained cells per glomerulus. *, Significant differences to controls (P 0.05). Healthy controls, Contr.
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Figure 2. Immunohistochemical stainings and double-labeling experiments. Serial sections of GN were stained with antibodies specific for MRP8, MRP14, the heterodimer MRP8/MRP14 (mAb 27E10), and CD68 (A–E). Primary antibodies were detected by immunoperoxidase-conjugated second-stage antibodies (red color), and slides were subsequently counterstained with hemalaun. Staining of MCD with mAb 27E10 (A) shows only a few MRP8/MRP8-expressing cells within the glomerulus. SLE-GN of World Health Organization class IV (B–H) shows a high abundance of MRP8 (B), MRP14 (C), and MRP8/MRP14 complex formation (D; mAb 27E10). CD68 shows an identical expression pattern in glomeruli of SLE-GN as MRP8 and MRP14 (E). Double-labeling experiments with aMRP14 (phosphatase staining, blue color) and aCD68 (peroxidase staining, red color) revealed that almost all CD68+ cells within the glomeruli express MRP14 (dark brown color, F). Double-labeling with aMRP14 (phosphatase staining, blue color) and 27E10 (peroxidase staining, red color) showed an almost complete double-labeling of positive cells within the glomeruli of SLE (dark brown color, G), whereas infiltrating cells in the interstitium of the identical section does not form the 27E10 epitope despite staining for MRP14 (H, exclusively blue-color reaction). (F–H) Not counterstained after immunohistochemical double-labeling. Original bars represent 50 µM.
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Glomerular MRP8 and MRP14 expression and disease activity
In patients with SLE-GN, IgA-GN, or ECGN, there is a striking negative correlation between the number of MRP8- and MRP14-expressing cells in the glomeruli and the duration of acute flares after which the biopsy was obtained (r =-0.6 for SLE-GN, -0.7 for IgA-GN, and -0.7 for ECGN). Furthermore, there is a negative correlation between the number of MRP8- and MRP14-expressing cells and general duration of disease in IgA-GN (r=–0.8) and ECGN (r=–0.8). The observed relation of MRP8 and MRP14 expression with earlier stages of disease in these distinct subgroups of GN supports the assumption that a high abundance of MRP8 and MRP14 in the infiltrate is an indicator for an active type of inflammation in the glomerulus. This is in accordance with the finding that those GN that are known to present weaker inflammatory reactions in glomeruli, e.g., MCD, also show significantly lower numbers and lower percentages of MRP8- and MRP14-expressing cells. No correlation was observed between MRP8 or MRP14 expression in the glomeruli and long-term parameters of renal functions in the whole patient population, i.e., blood pressure, serum concentrations of creatinine, or daily urinary protein excretion.
MRP8 and MRP14 in infiltrating interstitial cells in different forms of GN
Immunohistochemical data of the interstitial infiltrate are presented as percentage of positively stained cells in the infiltrate. The expression pattern of MRP8 and MRP14 markedly differed between glomeruli and the interstitium (Fig. 3
). IN, which by definition presented almost no inflammatory infiltrate in glomeruli and only a weak glomerular immunoreactivity against MRP8 and MRP14, showed dense infiltration and high percentage of positive cells for both proteins in the interstitium. In severe forms of GN, such as SLE-GN and ECGN, there was also a significantly raised expression of MRP8 and MRP14 in the interstitium, paralleling the findings in glomeruli, whereas MCD showed normal numbers of MRP8/MRP14-expressing monocytes in the interstitium. It is interesting that in IgA-GN and especially in SLE-GN, there was a lack of MRP8/MRP14 complex formation in infiltrating cells of the interstitium despite expression of the monomers and despite complex formation in the glomeruli (Fig. 2G
and 2H)
. Accordingly, r, between expression of single MRP8 and MRP14 monomers and complex formation, were relatively low in the interstitium of SLE-GN and IgA-GN (r between 0.38 and 0.68). IN also revealed lack of complex formation, i.e., lack of 27E10-positive cells, despite a high number of MRP8/MRP14-expressing cells in the interstitium. This suggests that the phenotype of 27E10-/MRP8+/MRP14+ cells reflects a more chronic, inflammatory reaction in the interstitium (r=0.40 for monomer expression and complex formation). This assumption is supported by the finding that the expression of MRP8 or MRP14 in the interstitium shows no relation to duration of acute flares in disease activity during GN. However, serum concentrations of creatinine as a parameter of chronic renal failure showed a significant correlation to expression of MRP8 and MRP14 in the interstitial infiltrate of all these forms of GN, which lacked complex formation (IgA-GN: r=0.91/0.86; FSGS: r=0.89/0.75; SLE-GN: r=0.57/0.60 for MRP8/MRP14, respectively). The parallel detection of MRP8, MRP14, and 27E10 reactivity in ECGN (r=0.93/0.97) probably points to a more acute type of inflammatory reaction in the interstitium in this special GN, but the number of cases examined is too small for a general statement.
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Figure 3. Expression and complex formation of MRP8 and MRP14 in the interstitium of different GN. Serial sections of different GN were stained as described in Figure 1
. Data are presented as percentage (%, mean±SEM) of positively stained cells in the interstitium. *, Significant differences to controls (P0.05).
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Figure 4. Expression of MRP8 and MRP14 after treatment of monocytes with different anti-inflammatory drugs. Monocytes were incubated with different anti-inflammatory drugs for 4 h on culture day 1 (CON, medium as control). (A) Expression of MRP8 and MRP14 mRNA in monocytes was investigated by Northern blot analysis. Expression of GAPDH served as control for loading of RNA. (B) MRP8 and MRP14 expression at protein level was analyzed by a specific sandwich ELISA. Data are presented as content of MRP8/MRP14 complex/total cellular protein (µg/mg). (C) Monocytes cultured for 1 day were treated with different anti-inflammatory drugs for 4 h. Surface expression of the MRP8/MRP14 heterodimer on monocytes was detected by flow cytometry. Open graphs show the autofluorescence of cells treated with control IgG of nonrelevant specificity. Surface reactivity of mAb 27E10 is presented by the shaded histograms. None of the anti-inflammatory drugs increases basal expression of MRP8/MRP14 on nonstimulated monocytes (left column of histograms). Any anti-inflammatory agent investigated did not inhibit up-regulation of MRP8/MRP14 surface expression by treatment of monocytes with 0.1 mM arsenite for 2 h (histograms in the right column). Almost identical results were obtained after treatment of the monocytes for 16 h with the anti-inflammatory drugs mentioned above (data not shown).
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Thus, differences of MRP8 and MRP14 expression as well as of MRP8/MRP14 complex formation observed in distinct GN are a result of the underlying inflammatory process and are not directly modified by immunosuppressive treatment.
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Parallel expression of MRP8, MRP14, and CD68 represents a proinflammatory macrophage, which is found in highly active GN. The question of whether expression of MRP8 and MRP14 reflects infiltration of blood-derived monocytes or re-expression in resident tissue macrophages, as shown for peritoneal macrophages after lipopolysaccharide stimulation [45 ], cannot be answered completely by this phenotypical study. However, the high abundance of these molecules in SLE-GN and ECGN presents strong evidence for the recruitment of blood monocytes. The MRP8-/MRP14-/CD68+ phenotype is compatible with a mature tissue macrophage, which is present in normal kidneys and in higher numbers in GN with less inflammatory activity such as MCD or FSGS. The phenotype of MRP8+/MRP14+/CD68- cells encompasses at least partially infiltrating granulocytes. The assumption that expression of MRP8 and MRP14 characterizes an active inflammatory macrophage phenotype is supported by the findings that MRP8 and MRP14 have been shown to be involved in calcium-dependent activation of macrophages and that translocation of these proteins to the plasma membrane correlates with release of inflammatory active substances such as TNF or IL-1ß and with an increase in respiratory burst [27 , 28 ]. Furthermore, in inflammatory bowel disease, it has been shown that MRP8- and MRP14-expressing cells are also the main sources for respiratory burst and for IL-1ß and TNF release in vivo [46 ]. Our in vitro data point against a direct influence of different anti-inflammatory drugs on de novo synthesis of MRP8 and MRP14 or on formation of their complexes. Thus, expression patterns reported here for different kinds of GN reflect differences in the underlying inflammatory processes rather than differences induced by anti-inflammatory drugs at a single-cell level.
Our findings are in accordance with the expression pattern of MRP8 and MRP14 in renal allograft rejection. During acute allograft rejection, the majority of infiltrating cells express MRP8 and MRP14, and both proteins have been shown to be a sensitive marker for the inflammatory activity of the rejection process [37 ]. In a previous paper, we have shown that absence of the MRP8/MRP14 complex formation, despite expression of both subunits in infiltrating macrophages, is in contrast to acute renal allograft rejections, characteristic for chronic rejections [36 ]. We now present evidence that there are similar differences in the complex pattern of MRP8 and MRP14 in distinct, inflammatory renal diseases. Macrophages in the interstitial infiltrate of IN show a phenotype identical to that found in chronic allograft rejections. It is interesting that this phenotype is also present in biopsies of patients with SLE-GN, IgA-GN, and FSGS, supporting the assumption that a chronic inflammatory process of the renal interstitium is an important pathogenetic factor in some forms of GN and that tubulo-interstitial changes are major determinants in the progression of chronic renal damage [47 , 48 ]. Thus, expression of MRP8 and MRP14 with simultaneous absence of their complex in the interstitial infiltrate of GN indicates an inflammatory pathomechanism distinct from the glomerular inflammation, which may also need specific anti-inflammatory therapies. Therefore, analysis of expression and complex formation of MRP8 and MRP14 provide further information, in addition to the number of infiltrating macrophages, about the quality and activity of the distinct inflammatory processes involved in GN.
Besides this potential diagnostic tool, our data also point to novel aspects about the pathogenesis of GN. Secretion of MRP8 and MRP14 in vivo is induced by contact of monocytes with inflammatory, activated endothelium [42 , 44 ]. A recent report demonstrated that extracellular MRP14 enhances CD11b/CD18 integrin-binding activity on phagocytes [49 ]. High expression of MRP8 and MRP14 during GN may thus point to a positive-feedback mechanism propagating the inflammatory reaction. In this model, contact of monocytes to activated endothelium induced secretion of MRP8 and MRP14, which leads to a higher affinity of integrin-adhesion molecules, thus inducing a switch from selectin-mediated rolling to a tight contact of leukocytes to the endothelium. Subsequently, MRP8/MRP14 mediates binding of leukocytes to specifically carboxylated glycans on endothelial cells and thus promotes leukocyte trafficking and extravasation [50 ]. Analyzing the molecular mechanisms of release and extracellular functions of MRP8 and MRP14 may thus offer molecular targets for novel, immunosuppressive strategies to modulate the important inflammatory response mechanisms of monocytes in GN.
Received February 19, 2003; revised September 4, 2003; accepted September 29, 2003.
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