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J Am Soc Nephrol 11:1426-1438, 2000
© 2000 American Society of Nephrology

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{dagger} and JAN J. WEENING*

* Department of Pathology, (Renal Transplant Unit), Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands.
{dagger} Department of Internal Medicine (Renal Transplant Unit), Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands.

Correspondence to Dr. Jan Aten, Department of Pathology, Academic Medical Center, Meibergdreef 9, L2-256, 1105 AZ Amsterdam, The Netherlands. Phone: +31 20 566 4935/5635; Fax: +31 20 696 0389; E-mail: j.aten{at}amc.uva.nl


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Abstract. CD134 (OX40) is a member of the tumor necrosis factor (TNF) receptor (TNFR) family that can be expressed on activated T lymphocytes. Interaction between CD134 and its ligand (CD134L) is involved in costimulation of T and B lymphocyte activation, and in T cell adhesion to endothelium. To examine the possible role of this interaction in the pathogenesis of systemic lupus erythematosus (SLE), expression of CD134 and CD134L on peripheral blood leukocytes was studied, and no significant differences between SLE patients and control individuals were found. Immunohistology on renal biopsies from patients with lupus nephritis or other renal disorders, using a recombinant human CD134-containing chimeric molecule to detect CD134L, demonstrated the abundant presence of CD134L in all cases of proliferative lupus nephritis in a granular distribution predominantly along the epithelial side of the glomerular capillary wall. Confocal laser scanning microscopy indicated colocalization with subepithelial immune deposits. In none of the other renal disorders examined, including nonproliferative forms of lupus nephritis, was glomerular staining for CD134L detected in a similar pattern. Endothelial CD134L expression was frequently observed in different types of vasculitis. CD134 was detected on perivascular infiltrating leukocytes and on part of the tubular epithelium, but not on glomerular resident cells. Immunohistology for several other TNF(R) family members revealed in proliferative lupus nephritis a similar distribution for TNFR1 as was observed for CD134L. In contrast, glomerular expression of TNFR2 was similar in all cases examined. The glomerular presence of CD134L and TNFR1 in proliferative lupus nephritis in association with subepithelial immune deposits may be of pathogenetic significance and have diagnostic value.


   Introduction
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 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The tumor necrosis factor receptor (TNFR) family consists of a number of type I transmembrane glycoproteins characterized by homologous cysteine-rich domains in their extracellular region (1). The intracellular parts of these proteins vary in size and structure, corresponding to the wide array of functions of TNFR proteins, ranging from regulation of cell activation and differentiation to induction of cell death (1,2). Twenty-two TNFR family members have been identified, including TNFR1, TNFR2, the low-affinity nerve growth factor receptor, CD27, CD30, CD40, CD95 (Fas), and CD134 (OX40). Except for the ligands of the low-affinity nerve growth factor receptor, the ligands of TNFR proteins form the family of TNF-related proteins. Most TNF family members can be expressed as type II transmembrane receptors, and some of these proteins mediate signal transduction via their intracellular domain (1). Several members of the TNFR family (e.g., CD95, TNFR1, and TNFR2) and the TNF family (e.g., TNF-{alpha}, CD40 ligand [CD40L], and CD95L) can be produced as functional soluble proteins, as a consequence of either alternative mRNA splicing or proteolytic cleavage of the transmembrane protein (3,4). The expression and function of members of the TNFR and TNF families have been mainly studied with respect to the immune system. More evidence now points to an important role of TNFR- and TNF-related proteins in regulating the function of nonlymphoid cells as well (5,6).

Qualitative and quantitative alterations in the interactions between several members of the TNF and TNFR families have been implicated in the pathogenesis of different forms of autoimmune disease, including systemic lupus erythematosus (SLE) (7,8,9,10,11,12,13,14,15,16). Nephritis is commonly part of the spectrum of disorders associated with SLE (17). The severity of lupus nephritis is an important prognostic factor for SLE (17,18,19). Staging of the histologic abnormalities observed in renal biopsies from patients with lupus nephritis according to the World Health Organization (WHO) classification (20) provides guidelines for therapy (17,18). WHO class III and class IV proliferative lesions represent the most severe forms of glomerular involvement in patients with lupus nephritis. The deposition of immune complexes at the endothelial side of the glomerular basement membrane (GBM) in these classes causes activation of the complement cascade, production of inflammatory mediators, and inflammatory by inflammatory cells. In turn, disruption of the capillary wall leads to extracapillary proliferation and extensive scarring (18).

Information on the expression and distribution of members of the TNFR and TNF families in lupus nephritis is limited. Yellin et al. recently demonstrated increased glomerular and tubular expression of CD40 in several types of renal inflammation, including WHO class III and IV lupus nephritis, but not in the membranous type of lupus nephritis, i.e., WHO class V (21). Interestingly, TNF-{alpha} was shown to be strongly expressed by glomerular visceral epithelial cells (GVEC) in WHO class V lupus nephritis and in membranous glomerulopathy, but not by GVEC in lupus nephritis of the proliferative types (22).

In an experimental model for drug-induced SLE-like autoimmunity, we have shown enhanced expression of CD134 on a subset of activated T lymphocytes (23,24). A functional role for the interaction of CD134 with its ligand (CD134L) has been demonstrated in maturation of dendritic cells (25), in costimulation of T lymphocyte proliferation and cytokine production (26,27,28,29,30), in B lymphocyte proliferation and Ig production (31,32), and in T lymphocyte adhesion to endothelial cells (33,34). Here, we hypothesize that the interaction between CD134 and CD134L is of relevance in the pathogenesis of SLE. Therefore, we studied expression of CD134 and CD134L on peripheral blood leukocytes from patients with SLE and in a series of renal biopsies representing the various classes of lupus nephritis. Several other proliferative and nonproliferative renal disorders were studied for comparison. The expression of other members of the TNFR and TNF families in renal tissue was investigated to assess the specificity of our observations. The main result of this study is the strong and specific localization of CD134L and TNFR1 in the glomerular capillary wall in patients with proliferative types of lupus nephritis.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Study Subjects and Samples
Peripheral blood samples were obtained from eight patients with SLE (seven women, 1 man; average age 40 yr; range, 30 to 56). Five of these patients were treated with prednisolone and azathioprine. Nine healthy volunteers served as control subjects (four women, five men; average age 32 yr; range, 25 to 45). In a second set of in vitro activation experiments, peripheral blood was studied from four patients with a recently diagnosed active phase of SLE (four women; 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). Proliferative lupus nephritis was diagnosed in these four patients (one with type III and three with type IV lupus nephritis) based on renal biopsies taken during the same period of hospitalization. One of these patients was treated with prednisone and one with azathioprine at the time when blood was sampled; for the other two patients examined, therapy was started after blood sampling. Blood samples were obtained with informed consent of the donors.

Renal tissue from 113 patients was selected for the present study from the files of the Department of Pathology, Academic Medical Center, Amsterdam (n = 110), and the Department of Pathology, University of Utrecht, Utrecht (n = 3), The Netherlands. These specimens comprised the diagnostic groups listed in Table 1. As control tissue, histologically normal parts of kidneys that had been resected because of renal cell carcinoma and a renal biopsy without histologic abnormalities were used. In addition, skin biopsies from the lesional area of patients with cutaneous discoid lupus erythematosus or with SLE-associated skin lesions were studied and compared with biopsies from healthy skin (Table 1).


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  		Table 1. Tissue specimens of kidney and skin examined by immunohistology for the presence and distribution of TNF and TNFR family membersa
 

Generation of Chimeric Molecules
To detect CD134L, CD134-containing molecules were used, which were a kind gift from Dr. J. Shields (Cantab Pharmaceuticals, Cambridge, United Kingdom). These constructs were generated as outlined below.

A cDNA construct encoding the extracellular region of human CD134 and the Fc part of human IgG1 was kindly provided by Dr. W. R. Godfrey (Department of Pathology, Stanford University School of Medicine, Stanford, CA) (27). The part encoding human IgG1 was substituted by cDNA encoding the hinge region and the CH2-CH3 domains of mouse IgG2a. The resulting construct was transfected into Chinese hamster ovary cells. Positive clones were selected by G418; fusion protein secretion was assessed by incubation of culture supernatants with CD134L-transfected Sp2/0 mouse myeloma cells, and detection of binding was done by flow cytometry. Fusion proteins were purified from supernatants of secreting cells using protein G-Sepharose; purity of the eluted material was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

The resulting protein, named hCD134-mFcIgG2a, was used to examine expression of CD134L on cell suspensions by flow cytometry and in tissue sections by immunohistology. The chimeric protein, which binds specifically to Sp2/0 cells that had been transfected with full-length human CD134L, could precipitate CD134L from these cells. No binding of hCD134-mFcIgG2a was observed either to hCD134-transfected SP2/0 cells or to untransfected, or to human CD40L-transfected mouse 3T3 fibroblasts (not shown).

Cell Isolation and Cell Culture
Peripheral blood mononuclear cells (PBMC) were isolated from heparinized blood using Ficoll-PaqueTM (Pharmacia Biotech, Uppsala, Sweden). Culture of PBMC was performed in 24-well plates (2.106/ml per well), using RPMI culture medium (RPMI supplemented with 10% heat-inactivated fetal calf serum, 100 IU/ml penicillin, 100 µg/ml streptomycin, and 2 mM glutamine), for 18 h at 37°C. Cell culture was performed in the presence or absence of a monoclonal antibody (mAb) anti-human CD3 (1XA, purified mouse IgA) (35), which was previously coated on the culture wells during 2 h at 37°C. Nonbound mAb were washed away before addition of the cells. In some cases, cells were stimulated by addition of phorbol 12-myristate 13-acetate (10 ng/ml; Sigma, St. Louis, MO) and ionomycin (100 ng/ml; Calbiochem, La Jolla, CA).

Flow Cytometry
All cell incubations for flow cytometry were performed in phosphate-buffered saline (PBS) containing 1% bovine serum albumin and 0.01% NaN3, for 30 min on ice. Flow cytometry on cultured cells was performed using biotin-conjugated mAb L106 (anti-CD134, mouse IgG1; kindly provided by Dr. V. C. Maino, Becton Dickinson Immunocytometry Systems, San Jose, CA) (27), the chimeric molecule hCD134-mFcIgG2a, or nonbinding mAb (biotinylated mouse IgG1, mouse IgG2a; Pharmingen, San Diego, CA) in the first step. Subsequently, either phycoerythrin (PE)-conjugated streptavidin (Dako, Glostrup, Denmark) or PE-conjugated goat anti-mouse IgG2a antibodies (Southern Biotechnology Associates, Birmingham, AL) in the presence of 1% normal human serum were applied. After blocking with 2% normal mouse serum, FITC-conjugated mAb anti-CD4 (SK3, mouse IgG1; Becton Dickinson), anti-CD8 (DK25, mouse IgG1; Dako), or anti-CD19 (HD37, mouse IgG1; Dako) was added. The cells were analyzed in the presence of propidium iodide (Molecular Probes, Leiden, The Netherlands) for identification of dead cells.

Flow cytometry on freshly isolated PBMC was performed as follows. In the first step, biotinylated ACT35 was applied (mAb anti-CD134, mouse IgG1; Ancell Corp., Bayport, MN) (36), followed by streptavidin-CyChrome (Pharmingen). Subsequently, FITC-conjugated mAb anti-CD4 (Becton Dickinson), anti-CD8 (Dako), or anti-CD3 (SK7, mouse IgG1; Becton Dickinson) was added, together with anti-CD45RO conjugated to PE (UCHL-1, mouse IgG2a; Becton Dickinson). Finally, cells were fixed using 1% paraformaldehyde in PBS. Appropriately conjugated nonbinding isotype-matched mAb served as negative controls.

Data acquistion was performed using FACScan or FACSCalibur flow cytometers (Becton Dickinson). During analysis, cell populations were gated based on scatter parameters and, when appropriate, negative staining for propidium iodide.

Immunohistology
Immunoperoxidase histology was performed on acetone-fixed 4-µm-thick cryostat sections. Nonspecific binding sites were blocked by preincubation with 10% normal goat serum in PBS. All incubations with first-step reagents were performed in PBS for 16 h at 4°C and were followed by inhibition of endogenous peroxidase activity using 0.1% NaN3 and 0.3% H2O2 in PBS for 15 min at room temperature. Additional antibody incubations were performed in PBS containing 10% normal human serum for 30 min at room temperature. In all cases, enzyme activity of horseradish peroxidase (HRP) was finally detected using 3-amino-9-ethyl-carbazole. Sections were counterstained with hematoxylin.

Expression of CD134, CD40L, and Fas was studied using the mAb ACT35 (anti-CD134, mouse IgG1; Pharmingen) (36), 24-31 (anti-CD40L, mouse IgG1; Ancell Corp.), and UB2 (anti-Fas, mouse IgG1; Immunotech, Marseille, France), respectively, in the first step. As a second step, HRP-conjugated goat anti-mouse IgG1 was used. For signal amplification, fluorescein-tyramide (DuPont, Boston, MA) was applied according to the instructions of the manufacturer, followed by HRP-conjugated rabbit anti-FITC (Dako).

To examine expression of CD40, TNF-{alpha}, and TNFR1, the mAb CLB-14G7 (anti-CD40, mouse IgM; from the Central Laboratory of The Netherlands Red Cross blood transfusion service (CLB), Amsterdam, The Netherlands), 4C6-H6 (anti-TNF-{alpha}, mouse IgM; Instruchemie, Hilversum, The Netherlands), and H398 (anti-TNFR1, mouse IgG2a; Instruchemie), respectively, were used. Furthermore, hCD134-mFcIgG2a was used to examine expression of CD134L. Binding of these reagents was detected using HRP-conjugated isotype-specific goat anti-mouse antibodies (SBA).

For analysis of TNFR2 expression, the mAb M1 was used (anti-TNFR2, rat IgG2b; Instruchemie), followed by a mouse mAb anti-rat IgG2b (Zymed, San Francisco, CA) and HRP-conjugated goat anti-mouse Ig (SBA). FasL expression was analyzed using an affinity-purified rabbit antiserum directed against FasL (Santa Cruz Biotechnology, Santa Cruz, CA), followed by HRP-conjugated goat anti-rabbit Ig (Dako).

Negative controls were performed by replacement of the first-step antibody with incubation buffer only or with isotype- and species-matched mAb, which do not bind to human tissue. In addition, to control for specificity of binding of the hCD134-mFcIgG2a construct, immunohistology was performed with OKT3 (anti-human CD3, mouse IgG2a; purified from culture supernatant of the hybridoma obtained from American Type Culture Collection, Manassas, VA).

The staining distribution was analyzed, and the glomerular staining intensity was scored by two pairs of the authors (J.A. and N.C.; A.R. and N.C.). Negative staining was expressed as 0, and positive staining was semiquantitatively classified from 1 (weak or sparse, but unequivocal staining) to 5 (diffuse and global, intense staining).

For all cases of lupus nephritis examined by immunohistology, 4-µm-thick sections of renal biopsies that were fixed in buffered formalin and embedded in paraffin had been stained with hematoxylin and eosin (HE), periodic acid-Schiff reagent, and silver, according to Jones, for routine diagnostic assessment. These sections were analyzed to determine indices for activity and chronicity of the lesions, using the scoring system of Austin and coworkers (37).

Confocal Laser Scanning Microscopy
Two-color immunofluorescence histology was performed on acetone-fixed 4-µm-thick cryostat sections that were preincubated with 10% normal goat serum. To compare localization of CD134L with that of human Ig, sections were incubated with hCD134-mFcIgG2a for 16 h at 4°C, followed by FITC-conjugated rabbit F(ab')2 antibodies specific for human Ig {chi} light chain, human Ig {lambda} 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, Zoetermeer, 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, Burlingame, CA) to inhibit fluorescence fading. Confocal laser scanning microscopy was performed using a Leitz CLSM (Leica, Heidelberg, Germany), applying double excitation with the 488 and 563 nm lines of an Argon/Krypton laser and double detection with a 530 nm bandpass filter for FITC emission, and a 610 nm longpass filter for 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 Analysis Software (Leica) and a look-up table to convert FITC signals to green, Texas Red to red, and overlapping areas to white.

Statistical Analyses
Differences between patients with SLE and control individuals with respect to fractions of CD134+ lymphocytes were evaluated using the Mann—Whitney rank sum test. Scores for glomerular binding of hCD134-mFcIgG2a, anti-TNF-{alpha}, anti-TNFR1, and anti-TNFR2 were analyzed for possible differences between groups, the latter consisting of control renal tissue specimens as defined above (group 1); renal biopsies from patients with lupus nephritides of WHO class II (group 2), classes III or IV (group 3), and class V (group 4); and renal biopsies from patients with membranous glomerulopathy (group 5). To determine whether an overall difference exists between the groups with respect to the variable considered, the Kruskal-Wallis rank sum test was applied with correction for ties. For subsequent comparison of specific groups, differences in rank sum were analyzed using the Dunn procedure, applying correction for ties, for variable 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-{alpha}, anti-TNFR1, and anti-TNFR2 were analyzed for all cases of lupus nephritis by calculating the nonparametric Spearman rank correlation coefficient {rho}. Differences or correlations were considered statistically significant when P values were <0.05.


   Results
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 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
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 CD4+ T lymphocytes, CD134 was expressed only on the subset expressing CD45RO. In most donors tested, CD134 expression could not be detected on resting CD8+ T lymphocytes. Flow cytometry performed on unseparated blood samples revealed that granulocytes and monocytes, which were distinguished on basis of their scatter characteristics, did not express detectable levels of CD134 (not shown). Furthermore, all lymphocytes expressing CD134 coexpressed CD3.



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  		Figure 1. CD134 is expressed on CD4+CD45RO+ T lymphocytes. Freshly isolated peripheral blood mononuclear cells (PBMC) from a healthy donor were used. The percentage of positive cells within gated CD4+ (left panel) and CD8+ lymphocyte populations (right panel) is indicated in the quadrants. Isotype-matched negative control antibodies were used to determine the position of the markers.

 

PBMC from SLE patients and from healthy donors were compared for their expression of CD134. Also in SLE patients, CD134 expression was predominantly restricted to lymphocytes expressing CD3, CD4, and CD45RO. The number of CD134+ cells within the CD4+ T lymphocyte population was more variable in the group of SLE patients than in the control group, and reached high levels in some patients with SLE (Figure 2). However, the difference between SLE patients and control subjects did not reach statistical significance, and a clear relationship between T cell CD134 expression and clinical disease parameters could not be detected. Similar results were obtained when the fractions of CD134+ T lymphocytes were calculated within the population of CD4+CD45RO+ cells, or within the CD3+ cell population (not shown).



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  		Figure 2. Expression of CD134 on CD4+ lymphocytes from control donors and from systemic lupus erythematosus (SLE) patients. Flow cytometry was performed on freshly isolated PBMC. CD4+ lymphocytes were gated for analysis.

 

Flow cytometry did not reveal CD134L expression on any population in peripheral blood at a significant level, either in SLE patients or in healthy control subjects (results not shown). However, using the same technique, clear expression of CD134L was detected on human umbilical vein endothelial cells (results not shown), as reported previously by others (33).

In vitro stimulation of PBMC from either patients with active SLE or control individuals by immobilized anti-CD3 or a combination of phorbol 12-myristate 13-acetate and ionomycin showed similar upregulation in the numbers of CD134+ cells, with a tendency of CD8+ cells from SLE patients to be higher in CD134 expression than those from control subjects (Figure 3). Similar results were obtained for the mean fluorescence intensity values (not shown). Anti-CD3 did not induce significant expression of CD134L on T or B lymphocytes, either in cells obtained from SLE patients or in cells from control donors.



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  		Figure 3. Anti-CD3 stimulation induces CD134 expression on CD4+ and CD8+ lymphocytes. PBMC from a control donor and from an SLE patient were stimulated in parallel with immobilized anti-CD3 at the indicated coating concentrations. CD4+ and CD8+ lymphocytes were gated. Cells permeable for propidium iodide were excluded from analysis. Results represent one of at least three experiments.

 

Expression of CD134 in Lupus Nephritis and other Renal Disorders
Few CD134-expressing leukocytes were detected in the glomeruli in 50% of patients with WHO class III or class IV lupus nephritis. In these cases, at most five CD134+ leukocytes were present per glomerular section in the minority of the glomeruli. In only one patient with WHO class II lupus nephritis and in one patient with WHO class V lupus nephritis were glomeruli observed to contain CD134+ leukocytes. Renal biopsies contained several CD134+ leukocytes per glomerular section in 20 to 30% of patients with postinfectious glomerulonephritis, type I membranoproliferative glomerulonephritis, or antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis. In all other biopsies studied, CD134+ leukocytes were only occasionally detected in glomeruli.

CD134+ leukocytes, amounting to 5% of the total number of leukocytes, were found in perivascular infiltrates in 50% of patients with proliferative types of lupus nephritis, as well as in 25% of patients with nonproliferative forms of lupus nephritis. Perivascular infiltrates also contained CD134+ leukocytes in cases of postinfectious glomerulonephritis (40%), membrano-proliferative glomerulonephritis (50%), IgA nephropathy (65%), membranous glomerulopathy (25%), ANCA-associated vasculitis (50%), and renal allograft rejection (75%). In the tubulointerstitial area, moderate to high numbers of scattered CD134+ leukocytes were observed in cases of renal allograft rejection (60%). In control renal tissue, sporadic CD134+ leukocytes were observed in three of eight cases.

CD134 was not found to be expressed by any glomerular resident cell type in any condition studied. However, strong CD134 expression was detected at the apical and lateral membrane of epithelial cells in a distinct segment of the tubules, presumably the duct of Henle, in all renal biopsies examined (Figure 4A). Because CD134 has not been described to be expressed by nonlymphoid cells earlier, we further examined the specificity of this finding. Binding of the ACT35 anti-CD134 mAb to this type of tubular epithelium was inhibited by preincubation of the renal tissue with a chimeric molecule consisting of human CD134L linked to the Fc portion of human IgG1, as well as by preincubation of the ACT35 mAb with the chimeric hCD134-mFcIgG2a molecule (results not shown).



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  		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: x75 in A and B; x240 in C through H.

 

Distribution of CD134L in Lupus Nephritis and other Renal Disorders
CD134L was abundantly present in glomeruli in almost all cases of proliferative lupus nephritis, as detected by binding of the hCD134-mFcIgG2a construct (Figure 4, B and C). The localization of CD134L was predominantly along the glomerular capillary wall (Figure 4C and Figure 5A) and in most cases confined to the epithelial side of the basement membrane (Figure 6). Glomerular CD134L colocalized with (sub)epithelial human Ig deposits, as indicated by the double-positive white staining with anti-human {chi} light chain (Figure 6A, arrow) or anti-human {lambda} light chain (Figure 6B). In addition, single-positive red staining for CD134L was observed at the epithelial side of the GBM (Figure 6A, long arrow, and Figure 6B). Occasionally, focal colocalization with (sub)endothelial immune deposits was present, as demonstrated by double-positive white staining with anti-human {chi} light chain (Figure 6A, arrowhead) or anti-human IgA (Figure 6C, arrowhead), in the latter case on a section of a renal biopsy in which IgA was only deposited at the endothelial side of the GBM (Figure 6C). The prevalence of CD134L at the epithelial side of the GBM is emphasized by the granular single-positive red staining for CD134L adjacent to the urinary space (arrows in Figure 6, C and D) when double staining was performed with anti-human IgA (Figure 6C) or with anti-collagen type IV (Figure 6D). Double-staining experiments with mAb anti-factor VIII confirmed that part of the glomerular endothelium was positively stained by hCD134-mFcIgG2a and that most of the hCD134-mFcIgG2a present was bound to the glomerular visceral epithelium (not shown).



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  		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: x405.

 


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  		Figure 6. Confocal laser scanning microscopy of immunofluorescence staining for CD134L (A through E: red) and human Ig {chi} light chain (A and E: green), human Ig {lambda} 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: x485 in A; x430 in B; x1000 in C; x965 in D; and x685 in E.

 

In contrast to the strong binding of hCD134-mFcIgG2a along the glomerular capillary wall observed in almost all patients with proliferative lupus nephritis (Figure 7), glomeruli of patients with nonproliferative types of lupus nephritis showed no or only weak binding of hCD134-mFcIgG2a, mainly in the mesangial area (Figure 4D and Figure 7). Remarkably, in all other renal disorders characterized by the presence of subendothelial and/or subepithelial immune deposits, such as postinfectious glomerulonephritis, membranoproliferative glomerulonephritis, and membranous glomerulopathy (Figure 7), the at most weak staining for CD134L was not associated with immune deposits.



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  		Figure 7. Scatter diagrams of glomerular scores of staining for CD134L, TNF-{alpha}, TNFR1, and TNFR2 performed on control renal tissue (Ctrl) and on renal biopsies from patients with SLE-associated nephritides of WHO classes II, III/IV, and V, and with membranous glomerulopathy (MGP). Stars indicate the calculated P values for differences between groups, according to Dunn rank sum analysis: *P < 0.05; **P < 0.01; ***P < 0.001.

 

The extent of glomerular staining for CD134L was shown to be positively correlated with the histologic activity index of lupus nephritis, as determined according to the scoring system of Austin and coworkers (37). The nonparametric Spearman rank correlation coefficient {rho} is 0.753 (P < 0.0001) for all cases of lupus nephritis and 0.435 (P = 0.0371) for cases of proliferative lupus nephritis only (Figure 8A).



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  		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 ({blacktriangleup}, n = 4), III and IV ([UNK], n = 24), and V ({blacksquare}, n = 11) were analyzed.

 

Endothelial staining for CD134L in the extraglomerular vasculature was also observed (Figure 5B and Figure 6E) and was increased in frequency and intensity in several renal disorders, i.e., in proliferative lupus nephritis (15 of 24); in membranoproliferative glomerulonephritis (5 of 6); in allograft rejection (6 of 8); and in ANCA- and anti-cardiolipin-associated vasculitis (6 of 7). Confocal laser scanning microscopy analysis of double staining with anti-human {chi} light chain 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 proliferative lupus nephritis. In control renal tissue, in two of eight cases weak staining for CD134L was observed on some vessels.

CD134 on activated T cells was reported to mediate adhesion to CD134L-expressing endothelial cells in vitro (33,34). Interestingly, perivascular infiltrates often contained CD134+ leukocytes in cases of vasculitis in which endothelial cells were observed to express CD134L. Perivascular infiltrates frequently contained CD134+ leukocytes, whereas only low numbers of large CD134L+ 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 CD134+ 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 CD134+ 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 nonproliferative lupus nephritis, membranous glomerulopathy, and other renal disorders examined. In contrast, TNF-{alpha} was found to be present in similar quantity and distribution in proliferative lupus nephritis and membranous lupus nephropathy. In idiopathic membranous glomerulopathy, TNF-{alpha} tended to be expressed at even higher levels. Also, in histologically normal tissue from kidneys that were resected because of urinary tract carcinoma, glomerular TNF-{alpha} 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 TNFR1 is correlated with the histologic activity index of lupus nephritis (Spearman rank correlation coefficient {rho} is 0.660; P < 0.0001) (Figure 8B). Both the intensity and the pattern of glomerular staining for TNFR1 showed a clear positive correlation with those for CD134L, as can be observed in two adjacent sections (Figure 4, C and E) and as confirmed by Spearman's {rho}, which is equal to 0.866 (P < 0.0001). Glomerular TNFR1 staining was not detected in control renal tissue, in WHO class II lupus nephritis, and in most cases of membranous glomerulopathy and WHO class V lupus nephritis (Figure 7). In contrast, glomerular staining for TNFR2 was readily observed in each renal biopsy examined, and no apparent differences between the various diagnostic groups were observed (Figure 4, G and H, and Figure 7). Neither glomerular staining for CD134L, nor for TNF-{alpha}, TNFR1, and TNFR2, were correlated with the histologic chronicity index of lupus nephritis.

Glomerular expression of CD40, Fas, and FasL was detected to a variable degree in all diagnostic groups examined. None of these TNFR and TNF family members, however, was observed in a glomerular expression pattern as described here for CD134L and TNFR1. The various diagnostic groups could not be discerned on the basis of glomerular expression of CD40L, CD40, FasL, or Fas (not shown).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study, we demonstrate the abundant presence of the TNF family member CD134L and of TNFR1 in association with the glomerular capillary wall in proliferative lupus nephritis. The high amount of CD134L and TNFR1, in combination with its glomerular immune complex-like distribution pattern, is a highly specific characteristic of proliferative lupus nephritis compared with many other inflammatory as well as noninflammatory renal disorders. We have obtained analogous results in experimental models for immune-mediated nephritis. Glomerulonephritis in mice undergoing a chronic semi-allogeneic graft-versus-host reaction, which shares many features with lupus nephritis (39,40), was associated with binding of a human CD134-human FcIgG1 chimeric molecule to the glomeruli. In contrast, glomeruli of proteinuric mice that had received anti-aminopeptidase A (41) or anti-dipeptidyl peptidase IV mAb did not show staining for CD134L (unpublished observations in collaboration with Dr. K. J. M. Assmann, Department of Pathology, St. Radboud Hospital, Nijmegen, The Netherlands).

The presence of CD134L was defined by detection of binding of a recombinant human CD134-containing chimeric molecule. This method of detection does not identify the exact nature of the local ligand to which hCD134-mFcIgG2a binds. In this respect, it is noteworthy that the TNFR family member CDw137, previously known as 4-1BB, was reported to bind to several extracellular matrix proteins, including laminin and collagen type IV (42,43). However, in view of the restricted staining patterns obtained in immunohistology, specificity of CD134 for matrix proteins is unlikely. Western blotting with the hCD134-mFcIgG2a molecule on lysates from human umbilical vein endothelial cells revealed a protein band at 31 kD after reduction (O. J. de Boer et al., submitted for publication), corresponding to the molecular weight of the CD134L monomer (26). The limited size of human renal biopsies has thus far hampered further characterization of the ligand in glomeruli from patients with proliferative lupus nephritis by immunoprecipitation.

The observation that in proliferative lupus nephritis CD134L and TNFR1 were localized in an immune complex-like pattern may suggest that deposition and accumulation of these proteins into the capillary wall takes place from the circulation. Staining for CD134L and also for TNFR1 was also observed in association with immune deposits along the dermal-epidermal junction in one patient with lupus-associated skin lesions. Soluble forms of CD134L have not yet been demonstrated in vivo, but are likely to occur in view of the existence of soluble forms of most members of the TNF family, such as TNF-{alpha} and CD95L. Circulating levels of soluble TNF receptors, including TNFR1, have been described to be elevated in SLE (10,11,44). Therefore, at least part of the TNFR1 detected in the glomeruli in proliferative lupus nephritis may have been derived from the circulation.

Apart from deposition from the circulation, glomerular CD134L and TNFR1 in proliferative lupus nephritis may also have been locally produced by glomerular resident cells. Double staining with mAb anti-factor VIII demonstrated that CD134L can be present on, and possibly be synthesized by, glomerular endothelial cells in proliferative lupus nephritis. CD134L expression by endothelial cells in vitro has been described previously (33). Recently, we detected upregulation of CD134L membrane expression on endothelial cells by incubation with interleukin-4 or TNF-{alpha} (O. J. de Boer et al., submitted for publication). Preliminary experiments indicated CD134L and TNFR1 mRNA expression in a human glomerular visceral epithelial cell line, transformed by SV-40; thus far, we did not detect CD134L or TNFR1 protein expression in this cell line. Additional experiments are under way to establish whether CD134L and TNFR1 are synthesized in situ by glomerular resident cells in proliferative lupus nephritis.

In view of the absence of CD134 on resident glomerular cells, it is unlikely that CD134L will affect glomerular cell function through direct binding to a specific receptor in the glomerulus. Whether CD134L from the glomerular epithelium is secreted into the urinary space and whether it may subsequently affect epithelial cell function in the CD134+ segment of the tubules is at present unknown. This is the first report of CD134 expression on a nonlymphoid cell type. The high constitutive expression of CD134 on a distinctive part of the tubule also suggests a role in physiologic epithelial cell function for this TNFR family member, as has been hypothesized for CD40 (5,6).

TNF-{alpha} is expressed by glomerular visceral epithelial cells in membranous glomerulopathy, as was first described by Neale et al. (22) and is supported by the present study. Interestingly, similar to CD134L in proliferative lupus nephritis, TNF-{alpha} in membranous glomerulopathy was localized mainly along the capillary wall in association with the immune deposits. TNF-{alpha} is likely to be secreted as a soluble factor by the GVEC, since its presence in the urine of patients with membranous glomerulopathy was demonstrated (22). In addition, TNF-{alpha} may be present in its transmembrane form on podocytes.

Reverse signaling through transmembrane TNF-{alpha} is not known to occur, and to have a biologic effect, TNF-{alpha} has to bind one of its specific receptors, i.e., TNFR1 or TNFR2. Signaling via the high-affinity receptor TNFR1 (45) by soluble TNF-{alpha} has in several cases been shown to require the coordinate expression of TNFR2 (46,47,48,49) and may induce apoptosis (47,48,49). In contrast, TNFR2 can be triggered by transmembrane TNF-{alpha} in the absence of TNFR1 (50,51) and may cause, among other effects, cell proliferation and cytokine production (50,52). When TNFR1 and TNFR2 are coexpressed, transmembrane TNF-{alpha} can efficiently induce apoptosis as well (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 conditions. Surprisingly, TNFR1 was found to be highly expressed in proliferative lupus nephritis in contrast to all other renal disorders examined. Provided that the TNFR are expressed as transmembrane receptors in these conditions, it can be hypothesized that TNF-{alpha} is likely to signal via TNFR2 in membranous glomerulopathy. In proliferative lupus nephritis, TNFR1 may be triggered in addition, possibly causing apoptosis, which is one of the classic characteristics of active, proliferative lupus nephritis.

In contrast to TNF-{alpha}, transmembrane CD134L can transduce signals and activate the cell on which it is expressed, as was shown for murine B lymphocytes (31), human dendritic cells (25), and human endothelial cells (54). Activated CD134+ leukocytes have been demonstrated to adhere specifically to CD134L expressed on endothelial cells in vitro (33,34). Whether this interaction may play a role in in vivo adhesion and infiltration is not known at present, but is suggested by the increased number of CD134+ leukocytes found in glomeruli and in perivascular infiltrates in proliferative lupus nephritis and in skin lesions of SLE patients, as well as in vasculitis. Indeed, these CD134+ leukocytes were frequently found around vessels where the endothelial cells stained positive for CD134L. Although some SLE patients showed high expression of CD134 on CD4+CD45RO+ T lymphocytes in peripheral blood, this was not significantly different from the control individuals.

Finally, ligation of transmembrane CD134L on the glomerular visceral epithelial cell may affect its function. However, as discussed above, we did not detect CD134 in the glomerulus. Another important possibility would be the presence of auto-antibodies against CD134L in SLE. Autoantibodies against TNFR2 have been reported to occur in patients with SLE previously (10). Such autoantibodies to CD134L and possibly also to TNFR1 may not only trigger the cells expressing these signaling proteins, but may in addition cause in situ immune complex formation, possibly explaining the striking distribution patterns of CD134L and TNFR1 in proliferative lupus nephritis.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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Received for publication May 6, 1999. Accepted for publication December 11, 1999.




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