J Am Soc Nephrol 11: 1426–1438, 2000 Strong and Selective Glomerular Localization of CD134 Ligand and TNF Receptor-1 in Proliferative Lupus Nephritis

JAN ATEN,* ANJA ROOS,* NIKE CLAESSEN,* ESTHER J. M. SCHILDER-TOL,* INEKE J. M. TEN BERGE,† and JAN J. WEENING* Departments of *Pathology and †Internal Medicine (Renal Transplant Unit), Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands.

Abstract. CD134 (OX40) is a member of the tumor necrosis microscopy indicated colocalization with subepithelial im- factor (TNF) receptor (TNFR) family that can be expressed on mune deposits. In none of the other renal disorders examined, activated T . Interaction between CD134 and its including nonproliferative forms of lupus nephritis, was glo- ligand (CD134L) is involved in costimulation of T and B merular staining for CD134L detected in a similar pattern. activation, and in adhesion to . Endothelial CD134L expression was frequently observed in To examine the possible role of this interaction in the patho- different types of vasculitis. CD134 was detected on perivas- genesis of systemic lupus erythematosus (SLE), expression of cular infiltrating leukocytes and on part of the tubular epithe- CD134 and CD134L on peripheral blood leukocytes was stud- lium, but not on glomerular resident cells. Immunohistology ied, and no significant differences between SLE patients and for several other TNF(R) family members revealed in prolif- control individuals were found. Immunohistology on renal erative lupus nephritis a similar distribution for TNFR1 as was biopsies from patients with lupus nephritis or other renal dis- observed for CD134L. In contrast, glomerular expression of orders, using a recombinant human CD134-containing chi- TNFR2 was similar in all cases examined. The glomerular meric molecule to detect CD134L, demonstrated the abundant presence of CD134L and TNFR1 in proliferative lupus nephri- presence of CD134L in all cases of proliferative lupus nephritis tis in association with subepithelial immune deposits may be of in a granular distribution predominantly along the epithelial pathogenetic significance and have diagnostic value. side of the glomerular capillary wall. Confocal laser scanning

The receptor (TNFR) family consists of , as a consequence of either alternative mRNA splicing a number of type I transmembrane glycoproteins characterized or proteolytic cleavage of the transmembrane (3,4). The by homologous cysteine-rich domains in their extracellular expression and function of members of the TNFR and TNF region (1). The intracellular parts of these proteins vary in size families have been mainly studied with respect to the immune and structure, corresponding to the wide array of functions of system. More evidence now points to an important role of TNFR proteins, ranging from regulation of cell activation and TNFR- and TNF-related proteins in regulating the function of differentiation to induction of cell death (1,2). Twenty-two nonlymphoid cells as well (5,6). TNFR family members have been identified, including Qualitative and quantitative alterations in the interactions TNFR1, TNFR2, the low-affinity nerve growth factor receptor, between several members of the TNF and TNFR families have CD27, CD30, CD40, CD95 (Fas), and CD134 (OX40). Except been implicated in the pathogenesis of different forms of for the ligands of the low-affinity nerve growth factor receptor, autoimmune disease, including systemic lupus erythematosus the ligands of TNFR proteins form the family of TNF-related (SLE) (7–16). Nephritis is commonly part of the spectrum of proteins. Most TNF family members can be expressed as type disorders associated with SLE (17). The severity of lupus II transmembrane receptors, and some of these proteins medi- nephritis is an important prognostic factor for SLE (17–19). ate signal transduction via their intracellular domain (1). Sev- Staging of the histologic abnormalities observed in renal biop- eral members of the TNFR family (e.g., CD95, TNFR1, and sies from patients with lupus nephritis according to the World ␣ TNFR2) and the TNF family (e.g., TNF- , CD40 ligand Health Organization (WHO) classification (20) provides guide- [CD40L], and CD95L) can be produced as functional soluble lines for therapy (17,18). WHO class III and class IV prolif- erative lesions represent the most severe forms of glomerular involvement in patients with lupus nephritis. The deposition of Received May 6, 1999. Accepted December 11, 1999. immune complexes at the endothelial side of the glomerular Correspondence to Dr. Jan Aten, Department of Pathology, Academic Medical basement membrane (GBM) in these classes causes activation Center, Meibergdreef 9, L2-256, 1105 AZ Amsterdam, The Netherlands. of the complement cascade, production of inflammatory me- Phone: ϩ31 20 566 4935/5635; Fax: ϩ31 20 696 0389; E-mail: [email protected] diators, and infiltration by inflammatory cells. In turn, disrup- 1046-6673/1108-1426 tion of the capillary wall leads to extracapillary proliferation Journal of the American Society of Nephrology and extensive scarring (18). Copyright © 2000 by the American Society of Nephrology Information on the expression and distribution of members J Am Soc Nephrol 11: 1426–1438, 2000 CD134L and TNFR1 in Proliferative Lupus Nephritis 1427 of the TNFR and TNF families in lupus nephritis is limited. Table 1. Tissue specimens of kidney and skin examined by Yellin et al. recently demonstrated increased glomerular and immunohistology for the presence and distribution tubular expression of CD40 in several types of renal inflam- of TNF and TNFR family membersa mation, including WHO class III and IV lupus nephritis, but Total No. not in the membranous type of lupus nephritis, i.e., WHO class Diagnosis n of Cases V (21). Interestingly, TNF-␣ was shown to be strongly ex- pressed by glomerular visceral epithelial cells (GVEC) in Lupus nephritis 40 WHO class V lupus nephritis and in membranous glomeru- WHO class II 4 lopathy, but not by GVEC in lupus nephritis of the proliferative WHO class III 1 types (22). WHO class IV 24 In an experimental model for drug-induced SLE-like auto- WHO class V 11 immunity, we have shown enhanced expression of CD134 on Postinfectious glomerulonephritis 7 a subset of activated T lymphocytes (23,24). A functional role Membranoproliferative 6 for the interaction of CD134 with its ligand (CD134L) has been glomerulonephritis demonstrated in maturation of dendritic cells (25), in costimu- type I 5 lation of T lymphocyte proliferation and production type II 1 (26–30), in B lymphocyte proliferation and Ig production IgA nephropathy 6 (31,32), and in T lymphocyte adhesion to endothelial cells Membranous glomerulopathy 12 (33,34). Here, we hypothesize that the interaction between Vasculitis 7 CD134 and CD134L is of relevance in the pathogenesis of ANCA-associated 6 SLE. Therefore, we studied expression of CD134 and CD134L anti-cardiolipin -associated 1 on peripheral blood leukocytes from patients with SLE and in Cryoglobulinemia 1 a series of renal biopsies representing the various classes of Diabetes mellitus nephropathy 6 lupus nephritis. Several other proliferative and nonproliferative Amyloidosis 2 renal disorders were studied for comparison. The expression of Minimal change nephropathy 9 other members of the TNFR and TNF families in renal tissue Allograft rejection 9 was investigated to assess the specificity of our observations. acute, grade I 1 The main result of this study is the strong and specific local- acute, grade II 5 ization of CD134L and TNFR1 in the glomerular capillary wall chronic 3 in patients with proliferative types of lupus nephritis. Renal cell 7 No histologic abnormalities 1 Total tissue specimens of kidney 113 Materials and Methods Cutaneous discoid lupus 3 Study Subjects and Samples erythematosus Peripheral blood samples were obtained from eight patients with Systemic lupus erythematosus 3 SLE (seven women, 1 man; average age 40 yr; range, 30 to 56). Five Healthy skin 2 of these patients were treated with prednisolone and . Total tissue specimens of skin 8 Nine healthy volunteers served as control subjects (four women, five a men; average age 32 yr; range, 25 to 45). In a second set of in vitro TNF, tumor necrosis factor; TNFR, tumor necrosis factor activation experiments, peripheral blood was studied from four pa- receptor; WHO, World Health Organization; ANCA, antineutrophil tients with a recently diagnosed active phase of SLE (four women; cytoplasmic antibody. average age 27 yr; range, 20 to 37) and, in parallel, from five healthy volunteers (five women; average age 28 yr; range, 20 to 37). Prolif- erative lupus nephritis was diagnosed in these four patients (one with or with SLE-associated skin lesions were studied and compared with type III and three with type IV lupus nephritis) based on renal biopsies biopsies from healthy skin (Table 1). taken during the same period of hospitalization. One of these patients was treated with prednisone and one with azathioprine at the time Generation of Chimeric Molecules when blood was sampled; for the other two patients examined, therapy To detect CD134L, CD134-containing molecules were used, which was started after blood sampling. Blood samples were obtained with were a kind gift from Dr. J. Shields (Cantab Pharmaceuticals, Cam- informed consent of the donors. bridge, United Kingdom). These constructs were generated as out- Renal tissue from 113 patients was selected for the present study lined below. from the files of the Department of Pathology, Academic Medical A cDNA construct encoding the extracellular region of human Center, Amsterdam (n ϭ 110), and the Department of Pathology, CD134 and the Fc part of human IgG1 was kindly provided by Dr. University of Utrecht, Utrecht (n ϭ 3), The Netherlands. These W. R. Godfrey (Department of Pathology, Stanford University School specimens comprised the diagnostic groups listed in Table 1. As of Medicine, Stanford, CA) (27). The part encoding human IgG1 was control tissue, histologically normal parts of kidneys that had been substituted by cDNA encoding the hinge region and the CH2-CH3 resected because of renal cell carcinoma and a renal biopsy without domains of mouse IgG2a. The resulting construct was transfected into histologic abnormalities were used. In addition, skin biopsies from the Chinese hamster ovary cells. Positive clones were selected by G418; lesional area of patients with cutaneous discoid lupus erythematosus secretion was assessed by incubation of culture super- 1428 Journal of the American Society of Nephrology J Am Soc Nephrol 11: 1426–1438, 2000 natants with CD134L-transfected Sp2/0 mouse myeloma cells, and Immunohistology detection of binding was done by flow cytometry. Fusion proteins Immunoperoxidase histology was performed on acetone-fixed were purified from supernatants of secreting cells using protein G- 4-␮m-thick cryostat sections. Nonspecific binding sites were blocked Sepharose; purity of the eluted material was confirmed by sodium by preincubation with 10% normal goat serum in PBS. All incubations dodecyl sulfate-polyacrylamide gel electrophoresis. with first-step reagents were performed in PBS for 16 h at 4°C and The resulting protein, named hCD134-mFcIgG2a, was used to were followed by inhibition of endogenous peroxidase activity using examine expression of CD134L on cell suspensions by flow cytom- 0.1% NaN3 and 0.3% H2O2 in PBS for 15 min at room temperature. etry and in tissue sections by immunohistology. The chimeric protein, Additional antibody incubations were performed in PBS containing which binds specifically to Sp2/0 cells that had been transfected with 10% normal human serum for 30 min at room temperature. In all full-length human CD134L, could precipitate CD134L from these cases, enzyme activity of horseradish peroxidase (HRP) was finally cells. No binding of hCD134-mFcIgG2a was observed either to detected using 3-amino-9-ethyl-carbazole. Sections were counter- hCD134-transfected SP2/0 cells or to untransfected, or to human stained with hematoxylin. CD40L-transfected mouse 3T3 fibroblasts (not shown). Expression of CD134, CD40L, and Fas was studied using the mAb ACT35 (anti-CD134, mouse IgG1; Pharmingen) (36), 24-31 (anti- Cell Isolation and Cell Culture CD40L, mouse IgG1; Ancell Corp.), and UB2 (anti-Fas, mouse IgG1; Immunotech, Marseille, France), respectively, in the first step. As a Peripheral blood mononuclear cells (PBMC) were isolated from TM second step, HRP-conjugated goat anti-mouse IgG1 was used. For heparinized blood using Ficoll-Paque (Pharmacia Biotech, Upp- signal amplification, fluorescein-tyramide (DuPont, Boston, MA) was sala, Sweden). Culture of PBMC was performed in 24-well plates 6 applied according to the instructions of the manufacturer, followed by (2.10 /ml per well), using RPMI culture medium (RPMI supple- HRP-conjugated rabbit anti-FITC (Dako). mented with 10% heat-inactivated fetal calf serum, 100 IU/ml peni- To examine expression of CD40, TNF-␣ cillin, 100 ␮g/ml streptomycin, and 2 mM glutamine), for 18 h at , and TNFR1, the mAb 37°C. Cell culture was performed in the presence or absence of a CLB-14G7 (anti-CD40, mouse IgM; from the Central Laboratory of (mAb) anti-human CD3 (1XA, purified mouse The Netherlands Red Cross blood transfusion service (CLB), Amster- ␣ IgA) (35), which was previously coated on the culture wells during 2 h dam, The Netherlands), 4C6-H6 (anti-TNF- , mouse IgM; Instruche- at 37°C. Nonbound mAb were washed away before addition of the mie, Hilversum, The Netherlands), and H398 (anti-TNFR1, mouse cells. In some cases, cells were stimulated by addition of phorbol IgG2a; Instruchemie), respectively, were used. Furthermore, hCD134- 12-myristate 13-acetate (10 ng/ml; Sigma, St. Louis, MO) and iono- mFcIgG2a was used to examine expression of CD134L. Binding of mycin (100 ng/ml; Calbiochem, La Jolla, CA). these reagents was detected using HRP-conjugated isotype-specific goat anti-mouse (SBA). For analysis of TNFR2 expression, the mAb M1 was used (anti- Flow Cytometry TNFR2, rat IgG2b; Instruchemie), followed by a mouse mAb anti-rat All cell incubations for flow cytometry were performed in phos- IgG2b (Zymed, San Francisco, CA) and HRP-conjugated goat anti- phate-buffered saline (PBS) containing 1% bovine serum albumin and mouse Ig (SBA). FasL expression was analyzed using an affinity-

0.01% NaN3, for 30 min on ice. Flow cytometry on cultured cells was purified rabbit antiserum directed against FasL (Santa Cruz Biotech- performed using biotin-conjugated mAb L106 (anti-CD134, mouse nology, Santa Cruz, CA), followed by HRP-conjugated goat anti- IgG1; kindly provided by Dr. V. C. Maino, Becton Dickinson Immu- rabbit Ig (Dako). nocytometry Systems, San Jose, CA) (27), the chimeric molecule Negative controls were performed by replacement of the first-step hCD134-mFcIgG2a, or nonbinding mAb (biotinylated mouse IgG1, antibody with incubation buffer only or with isotype- and species- mouse IgG2a; Pharmingen, San Diego, CA) in the first step. Subse- matched mAb, which do not bind to human tissue. In addition, to quently, either phycoerythrin (PE)-conjugated streptavidin (Dako, control for specificity of binding of the hCD134-mFcIgG2a construct, Glostrup, Denmark) or PE-conjugated goat anti-mouse IgG2a anti- immunohistology was performed with OKT3 (anti-human CD3, bodies (Southern Biotechnology Associates, Birmingham, AL) in the mouse IgG2a; purified from culture supernatant of the hybridoma presence of 1% normal human serum were applied. After blocking obtained from American Type Culture Collection, Manassas, VA). with 2% normal mouse serum, FITC-conjugated mAb anti-CD4 (SK3, The staining distribution was analyzed, and the glomerular staining mouse IgG1; Becton Dickinson), anti-CD8 (DK25, mouse IgG1; intensity was scored by two pairs of the authors (J.A. and N.C.; A.R. Dako), or anti-CD19 (HD37, mouse IgG1; Dako) was added. The and N.C.). Negative staining was expressed as 0, and positive staining cells were analyzed in the presence of propidium iodide (Molecular was semiquantitatively classified from 1 (weak or sparse, but unequiv- Probes, Leiden, The Netherlands) for identification of dead cells. ocal staining) to 5 (diffuse and global, intense staining). Flow cytometry on freshly isolated PBMC was performed as fol- For all cases of lupus nephritis examined by immunohistology, lows. In the first step, biotinylated ACT35 was applied (mAb anti- 4-␮m-thick sections of renal biopsies that were fixed in buffered CD134, mouse IgG1; Ancell Corp., Bayport, MN) (36), followed by formalin and embedded in paraffin had been stained with hematoxylin streptavidin-CyChrome (Pharmingen). Subsequently, FITC-conju- and eosin (HE), periodic acid-Schiff reagent, and silver, according to gated mAb anti-CD4 (Becton Dickinson), anti-CD8 (Dako), or anti- Jones, for routine diagnostic assessment. These sections were ana- CD3 (SK7, mouse IgG1; Becton Dickinson) was added, together with lyzed to determine indices for activity and chronicity of the lesions, anti-CD45RO conjugated to PE (UCHL-1, mouse IgG2a; Becton using the scoring system of Austin and coworkers (37). Dickinson). Finally, cells were fixed using 1% paraformaldehyde in PBS. Appropriately conjugated nonbinding isotype-matched mAb served as negative controls. Confocal Laser Scanning Microscopy Data acquisition was performed using FACScan or FACSCalibur Two-color immunofluorescence histology was performed on ace- flow cytometers (Becton Dickinson). During analysis, cell popula- tone-fixed 4-␮m-thick cryostat sections that were preincubated with tions were gated based on scatter parameters and, when appropriate, 10% normal goat serum. To compare localization of CD134L with negative staining for propidium iodide. that of human Ig, sections were incubated with hCD134-mFcIgG2a J Am Soc Nephrol 11: 1426–1438, 2000 CD134L and TNFR1 in Proliferative Lupus Nephritis 1429

Ј for 16 h at 4°C, followed by FITC-conjugated rabbit F(ab )2 antibod- ies specific for human Ig ␬ chain, human Ig ␭ light chain, or human IgA (all from Dako). Subsequently, Texas Red-conjugated goat anti-mouse IgG antibodies (Rockland, Gilberstville, PA) were applied. In addition, localization of CD134L was compared with that of collagen type IV. Sections were incubated with hCD134-mFcIgG2a and with rabbit anti-human collagen type IV antibodies (ICN, Zoe- termeer, The Netherlands) in the first step. In the second step, Texas Red-conjugated goat anti-mouse IgG antibodies (Rockland) and FITC-conjugated goat anti-rabbit IgG antibodies (Jackson, West Grove, PA) were used. Negative controls were performed as detailed above. Sections were mounted in Vectashield (Vector Laboratories, Figure 1. CD134 is expressed on CD4ϩCD45ROϩ T lymphocytes. Burlingame, CA) to inhibit fluorescence fading. Confocal laser scan- Freshly isolated peripheral blood mononuclear cells (PBMC) from a ning microscopy was performed using a Leitz CLSM (Leica, Heidel- healthy donor were used. The percentage of positive cells within gated berg, Germany), applying double excitation with the 488 and 563 nm CD4ϩ (left panel) and CD8ϩ lymphocyte populations (right panel) is lines of an Argon/Krypton laser and double detection with a 530 nm indicated in the quadrants. Isotype-matched negative control antibod- bandpass filter for FITC emission, and a 610 nm longpass filter for ies were used to determine the position of the markers. Texas Red emission. Both images were adjusted to the full dynamic range (8 bit). Subsequently, FITC- and Texas Red-derived images were corrected for cross-talk and merged using the Multicolor Anal- ysis Software (Leica) and a look-up table to convert FITC signals to PBMC from SLE patients and from healthy donors were green, Texas Red to red, and overlapping areas to white. compared for their expression of CD134. Also in SLE patients, CD134 expression was predominantly restricted to lympho- cytes expressing CD3, CD4, and CD45RO. The number of Statistical Analyses ϩ ϩ Differences between patients with SLE and control individuals CD134 cells within the CD4 T lymphocyte population was with respect to fractions of CD134ϩ lymphocytes were evaluated more variable in the group of SLE patients than in the control using the Mann–Whitney sum test. Scores for glomerular binding group, and reached high levels in some patients with SLE of hCD134-mFcIgG2a, anti-TNF-␣, anti-TNFR1, and anti-TNFR2 (Figure 2). However, the difference between SLE patients and were analyzed for possible differences between groups, the latter control subjects did not reach statistical significance, and a consisting of control renal tissue specimens as defined above (group clear relationship between T cell CD134 expression and clin- 1); renal biopsies from patients with lupus nephritides of WHO class ical disease parameters could not be detected. Similar results II (group 2), classes III or IV (group 3), and class V (group 4); and were obtained when the fractions of CD134ϩ T lymphocytes renal biopsies from patients with membranous glomerulopathy (group ϩ ϩ were calculated within the population of CD4 CD45RO 5). To determine whether an overall difference exists between the ϩ groups with respect to the variable considered, the Kruskal–Wallis cells, or within the CD3 cell population (not shown). rank sum test was applied with correction for ties. For subsequent Flow cytometry did not reveal CD134L expression on any comparison of specific groups, differences in rank sum were analyzed population in peripheral blood at a significant level, either in using the Dunn procedure, applying correction for ties, for variable SLE patients or in healthy control subjects (results not shown). group size, and for multiple comparison of groups (38). Relations between indices for activity and chronicity and scores for glomerular binding of hCD134-mFcIgG2a, anti-TNF-␣, anti-TNFR1, and anti- TNFR2 were analyzed for all cases of lupus nephritis by calculating the nonparametric Spearman rank correlation coefficient ␳. Differ- ences or correlations were considered statistically significant when P values were Ͻ0.05.

Results Expression of CD134 and CD134L on Peripheral Blood Leukocytes from SLE Patients and Control Subjects Expression of CD134 could be detected on T lymphocytes in unstimulated PBMC from healthy donors (Figure 1). Among the CD4ϩ T lymphocytes, CD134 was expressed only on the subset expressing CD45RO. In most donors tested, CD134 expression could not be detected on resting CD8ϩ T lympho- cytes. Flow cytometry performed on unseparated blood sam- ples revealed that granulocytes and monocytes, which were Figure 2. Expression of CD134 on CD4ϩ lymphocytes from control distinguished on basis of their scatter characteristics, did not donors and from systemic lupus erythematosus (SLE) patients. Flow express detectable levels of CD134 (not shown). Furthermore, cytometry was performed on freshly isolated PBMC. CD4ϩ lympho- all lymphocytes expressing CD134 coexpressed CD3. cytes were gated for analysis. 1430 Journal of the American Society of Nephrology J Am Soc Nephrol 11: 1426–1438, 2000

However, using the same technique, clear expression of vasculitis (50%), and renal allograft rejection (75%). In the CD134L was detected on human umbilical vein endothelial tubulointerstitial area, moderate to high numbers of scattered cells (results not shown), as reported previously by others (33). CD134ϩ leukocytes were observed in cases of renal allograft In vitro stimulation of PBMC from either patients with rejection (60%). In control renal tissue, sporadic CD134ϩ active SLE or control individuals by immobilized anti-CD3 or leukocytes were observed in three of eight cases. a combination of phorbol 12-myristate 13-acetate and ionomy- CD134 was not found to be expressed by any glomerular cin showed similar upregulation in the numbers of CD134ϩ resident cell type in any condition studied. However, strong cells, with a tendency of CD8ϩ cells from SLE patients to be CD134 expression was detected at the apical and lateral mem- higher in CD134 expression than those from control subjects brane of epithelial cells in a distinct segment of the tubules, (Figure 3). Similar results were obtained for the mean fluores- presumably the duct of Henle, in all renal biopsies examined cence intensity values (not shown). Anti-CD3 did not induce (Figure 4A). Because CD134 has not been described to be significant expression of CD134L on T or B lymphocytes, expressed by nonlymphoid cells earlier, we further examined either in cells obtained from SLE patients or in cells from the specificity of this finding. Binding of the ACT35 anti- control donors. CD134 mAb to this type of tubular was inhibited by preincubation of the renal tissue with a chimeric molecule Expression of CD134 in Lupus Nephritis and other consisting of human CD134L linked to the Fc portion of Renal Disorders human IgG1, as well as by preincubation of the ACT35 mAb Few CD134-expressing leukocytes were detected in the glo- with the chimeric hCD134-mFcIgG2a molecule (results not meruli in 50% of patients with WHO class III or class IV lupus shown). nephritis. In these cases, at most five CD134ϩ leukocytes were present per glomerular section in the minority of the glomeruli. Distribution of CD134L in Lupus Nephritis and other In only one patient with WHO class II lupus nephritis and in Renal Disorders one patient with WHO class V lupus nephritis were glomeruli CD134L was abundantly present in glomeruli in almost all observed to contain CD134ϩ leukocytes. Renal biopsies con- cases of proliferative lupus nephritis, as detected by binding of tained several CD134ϩ leukocytes per glomerular section in 20 the hCD134-mFcIgG2a construct (Figure 4, B and C). The to 30% of patients with postinfectious glomerulonephritis, type localization of CD134L was predominantly along the glomer- I membranoproliferative glomerulonephritis, or antineutrophil ular capillary wall (Figure 4C and Figure 5A) and in most cases cytoplasmic antibody (ANCA)-associated vasculitis. In all confined to the epithelial side of the basement membrane other biopsies studied, CD134ϩ leukocytes were only occa- (Figure 6). Glomerular CD134L colocalized with (sub)epithe- sionally detected in glomeruli. lial human Ig deposits, as indicated by the double-positive CD134ϩ leukocytes, amounting to 5% of the total number of white staining with anti-human ␬ light chain (Figure 6A, leukocytes, were found in perivascular infiltrates in 50% of arrow) or anti-human ␭ light chain (Figure 6B). In addition, patients with proliferative types of lupus nephritis, as well as in single-positive red staining for CD134L was observed at the 25% of patients with nonproliferative forms of lupus nephritis. epithelial side of the GBM (Figure 6A, long arrow, and Figure Perivascular infiltrates also contained CD134ϩ leukocytes in 6B). Occasionally, focal colocalization with (sub)endothelial cases of postinfectious glomerulonephritis (40%), membrano- immune deposits was present, as demonstrated by double- proliferative glomerulonephritis (50%), IgA nephropathy positive white staining with anti-human ␬ light chain (Figure (65%), membranous glomerulopathy (25%), ANCA-associated 6A, arrowhead) or anti-human IgA (Figure 6C, arrowhead), in

Figure 3. Anti-CD3 stimulation induces CD134 expression on CD4ϩ and CD8ϩ lymphocytes. PBMC from a control donor and from an SLE patient were stimulated in parallel with immobilized anti-CD3 at the indicated coating concentrations. CD4ϩ and CD8ϩ lymphocytes were gated. Cells permeable for propidium iodide were excluded from analysis. Results represent one of at least three experiments. J Am Soc Nephrol 11: 1426–1438, 2000 CD134L and TNFR1 in Proliferative Lupus Nephritis 1431

Figure 4. Immunohistology for CD134 (A), CD134L (B through D), tumor necrosis factor receptor type 1 (TNFR1) (E and F) and TNFR2 (G and H) in renal biopsies from patients with World Health Organization (WHO) class IV lupus nephritis (A, B, C, E, and G) or WHO class V lupus nephritis (D, F, and H). Magnification: ϫ75 in A and B; ϫ240 in C through H. 1432 Journal of the American Society of Nephrology J Am Soc Nephrol 11: 1426–1438, 2000

Figure 5. Immunohistologic staining for CD134L in a renal biopsy from a patient with WHO class IV lupus nephritis. Staining for CD134L is present along the glomerular capillary wall and focally on parietal epithelial cells (A). In addition, staining for CD134L is found on endothelial cells in inflamed extraglomerular vasculature (B). Magnification: ϫ405.

the latter case on a section of a renal biopsy in which IgA was mFcIgG2a present was bound to the glomerular visceral epi- only deposited at the endothelial side of the GBM (Figure 6C). thelium (not shown). The prevalence of CD134L at the epithelial side of the GBM is In contrast to the strong binding of hCD134-mFcIgG2a emphasized by the granular single-positive red staining for along the glomerular capillary wall observed in almost all CD134L adjacent to the urinary space (arrows in Figure 6, C patients with proliferative lupus nephritis (Figure 7), glomeruli and D) when double staining was performed with anti-human of patients with nonproliferative types of lupus nephritis IgA (Figure 6C) or with anti-collagen type IV (Figure 6D). showed no or only weak binding of hCD134-mFcIgG2a, Double-staining experiments with mAb anti-factor VIII con- mainly in the mesangial area (Figure 4D and Figure 7). Re- firmed that part of the glomerular endothelium was positively markably, in all other renal disorders characterized by the stained by hCD134-mFcIgG2a and that most of the hCD134- presence of subendothelial and/or subepithelial immune depos- J Am Soc Nephrol 11: 1426–1438, 2000 CD134L and TNFR1 in Proliferative Lupus Nephritis 1433

Figure 6. Confocal laser scanning microscopy of immunofluorescence staining for CD134L (A through E: red) and human Ig ␬ light chain (A and E: green), human Ig ␭ light chain (B: green), human IgA (C: green) or human collagen type IV (D: green) in renal biopsies from patients with WHO class IV lupus nephritis. Double-positive areas are depicted in white. CL, capillary lumen; US, urinary space; CB, capsule of Bowman. Magnification: ϫ485 in A; ϫ430 in B; ϫ1000 in C; ϫ965 in D; and ϫ685 in E. its, such as postinfectious glomerulonephritis, membranopro- rank correlation coefficient ␳ is 0.753 (P Ͻ 0.0001) for all liferative glomerulonephritis, and membranous glomerulopa- cases of lupus nephritis and 0.435 (P ϭ 0.0371) for cases of thy (Figure 7), the at most weak staining for CD134L was not proliferative lupus nephritis only (Figure 8A). associated with immune deposits. Endothelial staining for CD134L in the extraglomerular The extent of glomerular staining for CD134L was shown to vasculature was also observed (Figure 5B and Figure 6E) and be positively correlated with the histologic activity index of was increased in frequency and intensity in several renal dis- lupus nephritis, as determined according to the scoring system orders, i.e., in proliferative lupus nephritis (15 of 24); in of Austin and coworkers (37). The nonparametric Spearman membranoproliferative glomerulonephritis (5 of 6); in allograft 1434 Journal of the American Society of Nephrology J Am Soc Nephrol 11: 1426–1438, 2000

indicated the presence of double-positive immune deposits in the vessel wall (Figure 6E, arrow), as well as CD134L single- positive endothelial cells (Figure 6E, arrowhead), in prolifer- ative lupus nephritis. In control renal tissue, in two of eight cases weak staining for CD134L was observed on some ves- sels. CD134 on activated T cells was reported to mediate adhe- sion to CD134L-expressing endothelial cells in vitro (33,34). Interestingly, perivascular infiltrates often contained CD134ϩ leukocytes in cases of vasculitis in which endothelial cells were observed to express CD134L. Perivascular infiltrates fre- quently contained CD134ϩ leukocytes, whereas only low num- bers of large CD134Lϩ leukocytes, presumably foam cells, were detected around the large vessels in seven of eight cases of renal allograft rejection.

Expression of CD134 and CD134L in Lupus- Associated Skin Lesions In all biopsies from lupus-associated skin lesions, CD134L was clearly expressed on the endothelial cells of almost all vessels (not shown). Leukocyte infiltrates contained moderate numbers of CD134ϩ cells. Importantly, in one of four cases, staining for CD134L was detected at the dermal-epidermal junction in association with granular staining for IgG and C3. In healthy skin, CD134L-expressing endothelial cells were detected less abundantly and with lower staining intensity, and CD134ϩ leukocytes were not observed.

Renal Expression of other Members of the TNF and TNFR Families in Lupus Nephritis CD134L was present in large amounts in proliferative lupus nephritis and was detected only at low levels in nonprolifera- tive lupus nephritis, membranous glomerulopathy, and other renal disorders examined. In contrast, TNF-␣ was found to be present in similar quantity and distribution in proliferative lupus nephritis and membranous lupus nephropathy. In idio- pathic membranous glomerulopathy, TNF-␣ tended to be ex- pressed at even higher levels. Also, in histologically normal tissue from kidneys that were resected because of urinary tract carcinoma, glomerular TNF-␣ expression was clearly detected (Figure 7). Interestingly, the presence of TNFR1 in glomeruli was found to be strongly increased in proliferative lupus nephritis compared with its near absence in the other classes examined (Figure 4, E and F, and Figure 7). The glomerular staining for Figure 7. Scatter diagrams of glomerular scores of staining for TNFR1 is correlated with the histologic activity index of lupus ␳ CD134L, TNF-␣, TNFR1, and TNFR2 performed on control renal nephritis (Spearman rank correlation coefficient is 0.660; tissue (Ctrl) and on renal biopsies from patients with SLE-associated P Ͻ 0.0001) (Figure 8B). Both the intensity and the pattern of nephritides of WHO classes II, III/IV, and V, and with membranous glomerular staining for TNFR1 showed a clear positive corre- glomerulopathy (MGP). Stars indicate the calculated P values for lation with those for CD134L, as can be observed in two differences between groups, according to Dunn rank sum analysis: adjacent sections (Figure 4, C and E) and as confirmed by ૺ ૺૺ ૺૺૺ P Ͻ 0.05; P Ͻ 0.01; P Ͻ 0.001. Spearman’s ␳, which is equal to 0.866 (P Ͻ 0.0001). Glomer- ular TNFR1 staining was not detected in control renal tissue, in WHO class II lupus nephritis, and in most cases of membra- rejection (6 of 8); and in ANCA- and anti-cardiolipin-associ- nous glomerulopathy and WHO class V lupus nephritis (Figure ated vasculitis (6 of 7). Confocal laser scanning microscopy 7). In contrast, glomerular staining for TNFR2 was readily analysis of double staining with anti-human ␬ light chain observed in each renal biopsy examined, and no apparent J Am Soc Nephrol 11: 1426–1438, 2000 CD134L and TNFR1 in Proliferative Lupus Nephritis 1435

Figure 8. Relation of glomerular scores of staining for CD134L (A) and for TNFR1 (B) with the histologic activity index of lupus nephritis. Renal biopsies from patients with SLE-associated nephritides of WHO classes II (Œ, n ϭ 4), III and IV (F, n ϭ 24), and V (f, n ϭ 11) were analyzed. differences between the various diagnostic groups were ob- ecule. This method of detection does not identify the exact served (Figure 4, G and H, and Figure 7). Neither glomerular nature of the local ligand to which hCD134-mFcIgG2a binds. staining for CD134L, nor for TNF-␣, TNFR1, and TNFR2, In this respect, it is noteworthy that the TNFR family member were correlated with the histologic chronicity index of lupus CDw137, previously known as 4-1BB, was reported to bind to nephritis. several extracellular matrix proteins, including laminin and Glomerular expression of CD40, Fas, and FasL was detected collagen type IV (42,43). However, in view of the restricted to a variable degree in all diagnostic groups examined. None of staining patterns obtained in immunohistology, specificity of these TNFR and TNF family members, however, was observed CD134 for matrix proteins is unlikely. Western blotting with in a glomerular expression pattern as described here for the hCD134-mFcIgG2a molecule on lysates from human um- CD134L and TNFR1. The various diagnostic groups could not bilical vein endothelial cells revealed a protein band at 31 kD be discerned on the basis of glomerular expression of CD40L, after reduction (O. J. de Boer et al., submitted for publication), CD40, FasL, or Fas (not shown). corresponding to the molecular weight of the CD134L mono- mer (26). The limited size of human renal biopsies has thus far Discussion hampered further characterization of the ligand in glomeruli In this study, we demonstrate the abundant presence of the from patients with proliferative lupus nephritis by immunopre- TNF family member CD134L and of TNFR1 in association cipitation. with the glomerular capillary wall in proliferative lupus ne- The observation that in proliferative lupus nephritis CD134L phritis. The high amount of CD134L and TNFR1, in combi- and TNFR1 were localized in an immune complex-like pattern nation with its glomerular immune complex-like distribution may suggest that deposition and accumulation of these proteins pattern, is a highly specific characteristic of proliferative lupus into the capillary wall takes place from the circulation. Staining nephritis compared with many other inflammatory as well as for CD134L and also for TNFR1 was also observed in asso- noninflammatory renal disorders. We have obtained analogous ciation with immune deposits along the dermal-epidermal junc- results in experimental models for immune-mediated nephritis. tion in one patient with lupus-associated skin lesions. Soluble Glomerulonephritis in mice undergoing a chronic semi-alloge- forms of CD134L have not yet been demonstrated in vivo, but neic graft-versus-host reaction, which shares many features are likely to occur in view of the existence of soluble forms of with lupus nephritis (39,40), was associated with binding of a most members of the TNF family, such as TNF-␣ and CD95L. human CD134-human FcIgG1 chimeric molecule to the glo- Circulating levels of soluble TNF receptors, including TNFR1, meruli. In contrast, glomeruli of proteinuric mice that had have been described to be elevated in SLE (10,11,44). There- received anti-aminopeptidase A (41) or anti-dipeptidyl pepti- fore, at least part of the TNFR1 detected in the glomeruli in dase IV mAb did not show staining for CD134L (unpublished proliferative lupus nephritis may have been derived from the observations in collaboration with Dr. K. J. M. Assmann, circulation. Department of Pathology, St. Radboud Hospital, Nijmegen, Apart from deposition from the circulation, glomerular The Netherlands). CD134L and TNFR1 in proliferative lupus nephritis may also The presence of CD134L was defined by detection of bind- have been locally produced by glomerular resident cells. Dou- ing of a recombinant human CD134-containing chimeric mol- ble staining with mAb anti-factor VIII demonstrated that 1436 Journal of the American Society of Nephrology J Am Soc Nephrol 11: 1426–1438, 2000

CD134L can be present on, and possibly be synthesized by, is one of the classic characteristics of active, proliferative lupus glomerular endothelial cells in proliferative lupus nephritis. nephritis. CD134L expression by endothelial cells in vitro has been In contrast to TNF-␣, transmembrane CD134L can trans- described previously (33). Recently, we detected upregulation duce signals and activate the cell on which it is expressed, as of CD134L membrane expression on endothelial cells by in- was shown for murine B lymphocytes (31), human dendritic cubation with interleukin-4 or TNF-␣ (O. J. de Boer et al., cells (25), and human endothelial cells (54). Activated ϩ submitted for publication). Preliminary experiments indicated CD134 leukocytes have been demonstrated to adhere specif- CD134L and TNFR1 mRNA expression in a human glomeru- ically to CD134L expressed on endothelial cells in vitro lar visceral epithelial cell line, transformed by SV-40; thus far, (33,34). Whether this interaction may play a role in in vivo we did not detect CD134L or TNFR1 protein expression in this adhesion and infiltration is not known at present, but is sug- ϩ cell line. Additional experiments are under way to establish gested by the increased number of CD134 leukocytes found whether CD134L and TNFR1 are synthesized in situ by glo- in glomeruli and in perivascular infiltrates in proliferative lupus nephritis and in skin lesions of SLE patients, as well as merular resident cells in proliferative lupus nephritis. ϩ In view of the absence of CD134 on resident glomerular in vasculitis. Indeed, these CD134 leukocytes were fre- cells, it is unlikely that CD134L will affect glomerular cell quently found around vessels where the endothelial cells stained positive for CD134L. Although some SLE patients function through direct binding to a specific receptor in the ϩ ϩ glomerulus. Whether CD134L from the glomerular epithelium showed high expression of CD134 on CD4 CD45RO T is secreted into the urinary space and whether it may subse- lymphocytes in peripheral blood, this was not significantly quently affect epithelial cell function in the CD134ϩ segment different from the control individuals. of the tubules is at present unknown. This is the first report of Finally, ligation of transmembrane CD134L on the glomer- CD134 expression on a nonlymphoid cell type. The high ular visceral epithelial cell may affect its function. However, as constitutive expression of CD134 on a distinctive part of the discussed above, we did not detect CD134 in the glomerulus. tubule also suggests a role in physiologic epithelial cell func- Another important possibility would be the presence of auto- antibodies against CD134L in SLE. Autoantibodies against tion for this TNFR family member, as has been hypothesized TNFR2 have been reported to occur in patients with SLE for CD40 (5,6). previously (10). Such autoantibodies to CD134L and possibly TNF-␣ is expressed by glomerular visceral epithelial cells in also to TNFR1 may not only trigger the cells expressing these membranous glomerulopathy, as was first described by Neale signaling proteins, but may in addition cause in situ immune et al. (22) and is supported by the present study. Interestingly, complex formation, possibly explaining the striking distribu- similar to CD134L in proliferative lupus nephritis, TNF-␣ in tion patterns of CD134L and TNFR1 in proliferative lupus membranous glomerulopathy was localized mainly along the nephritis. capillary wall in association with the immune deposits. TNF-␣ is likely to be secreted as a soluble factor by the GVEC, since its presence in the urine of patients with membranous glomeru- Acknowledgments lopathy was demonstrated (22). In addition, TNF-␣ may be The authors thank Dr. K. J. M. Assmann (Department of Pathology, present in its transmembrane form on podocytes. St. Radboud Hospital, Nijmegen); Dr. J. H. M. Berden (Department of Reverse signaling through transmembrane TNF-␣ is not Nephrology, St. Radboud Hospital, Nijmegen); Dr. S. Florquin (De- partment of Pathology, Academic Medical Center, Amsterdam); Dr. known to occur, and to have a biologic effect, TNF-␣ has to W. R. Godfrey (Department of Pathology, Stanford University School bind one of its specific receptors, i.e., TNFR1 or TNFR2. of Medicine, Stanford, CA); Dr. R. Goldschmeding (Department of Signaling via the high-affinity receptor TNFR1 (45) by soluble Pathology, Academic Hospital Utrecht); Dr. G. Ligtenberg (Depart- TNF-␣ has in several cases been shown to require the coordi- ment of Nephrology, Academic Hospital Utrecht); Dr. V. C. Maino nate expression of TNFR2 (46–49) and may induce (Becton Dickinson Immunocytometry Systems, San Jose, CA); Dr. J. (47–49). In contrast, TNFR2 can be triggered by transmem- van Marle and Mr. H. van Veen (Center for Microscopical Research, brane TNF-␣ in the absence of TNFR1 (50,51) and may cause, Academic Medical Center, Amsterdam); Dr. J. Shields (Cantab Phar- among other effects, cell proliferation and cytokine production maceuticals, Cambridge, United Kingdom); and Dr. P. C. Wever (50,52). When TNFR1 and TNFR2 are coexpressed, trans- (Department of Medical Microbiology, Academic Medical Center, Amsterdam) for their advice and kind help in providing reagents and membrane TNF-␣ can efficiently induce apoptosis as well samples for analysis. (53). In the present study, we report constitutive expression of TNFR2 in glomeruli in all renal biopsies examined without clear variation in expression level in various disease condi- References tions. Surprisingly, TNFR1 was found to be highly expressed 1. Gruss HJ, Dower SK: Tumor necrosis factor ligand superfamily: in proliferative lupus nephritis in contrast to all other renal Involvement in the pathology of malignant . Blood 85: 3378–3404, 1995 disorders examined. Provided that the TNFR are expressed as 2. Nagata S: Apoptosis by death factor. Cell 88: 355–365, 1997 transmembrane receptors in these conditions, it can be hypoth- 3. Moss ML, Jin SLC, Milla ME, Burkhart W, Carter HL, Chen esized that TNF-␣ is likely to signal via TNFR2 in membra- WJ, Clay WC, Didsbury JR, Hassler D, Hoffman CR, Kost TA, nous glomerulopathy. 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