J Am Soc Nephrol 11: 1075–1084, 2000 Functional Relevance of Activated ␤1 in Mercury- Induced Nephritis

ESTHER ESCUDERO,* ANA MART´IN,* MARTA NIETO,† ELENA NIETO,* ELENA NAVARRO,* ALFONSO LUQUE,‡ CARLOS CABANAS,˜ ‡ FRANCISCO SANCHEZ-MADRID,´ † and FRANCISCO MAMPASO* *Department of Pathology, Hospital Ramo´n y Cajal, Universidad de Alcala´, †Immunology Section, Hospital de la Princesa, Universidad Auto´noma, and ‡Department of Biochemistry, Medical School, Universidad Complutense, Madrid, Spain.

Abstract. through different adhesion molecules and interstitial nephritis. Using the mercury model of nephritis, is a crucial event in the inflammatory response. Integrins can it was found that the expression of HUTS-21 epitope is induced only bind and mediate cellular adhesion after their activation in vivo in rat lymphocytes, and its appearance is correlated with by different specific stimuli. The state of ␤1 activation the other parameters at the onset of the disease. In addition, the can be assessed by a group of monoclonal antibodies (HUTS) administration of HUTS-21 monoclonal antibody to HgCl2- that selectively recognize ␤1 integrins in their active form. A treated rats offered evidence of its protective effects (1) against similar activated epitope in the rat was defined using the infiltration of renal interstitium by leukocytes, and (2)inthe anti-human monoclonal antibody HUTS-21, which recognizes reduction of anti-glomerular basement membrane synthesis an activation-dependent epitope on the ␤1 chain. It was found and glomerular deposition. Nevertheless, urinary val- that the divalent cations Mn2ϩ and Hg2ϩ were able to induce ues remained unaffected. These results demonstrate a key role in vitro the activation of ␤1 integrins on rat lymphocytes. The of ␤1-activated integrins in both leukocyte cell-cell interac- Hg2ϩ cation induces an autoimmune disease in the Brown tions and leukocyte infiltration pathway mechanism, and also Norway rat characterized by synthesis and glomerular deposits indicate that leukocyte migration may have less importance in of anti-glomerular basement membrane antibodies, proteinuria, the development of this disease than previously thought.

The leukocyte adhesion to endothelium and the subsequent Other investigators have reported the existence of a specific extravasation through the vascular wall into tissues are crucial and regulatory region in the common ␤1 subunit (CD29) of steps in the genesis of an inflammatory response, which are VLA integrins, whose expression is regulated upon cell acti- regulated by several proinflammatory factors (1–5). The inte- vation, and it correlates with the ligand binding activity of grins, an important family of the adhesion molecules, are these heterodimeric glycoproteins (11–16). The state of inte- central to many of these adhesion-dependent events. The ␤1or grin activation can be assessed by a group of monoclonal very late activation antigen (VLA) are a subgroup within the antibodies (HUTS) that selectively recognize integrins in their integrin family comprising at least 10 members, each with a active form. In addition, it has been shown that HUTS-21 mAb ␣ distinct subunit noncovalently associated with the common recognizes an epitope whose expression is induced upon ligand ␤ 1 subunit (6–8). The cellular adhesion through integrins can binding to the ␤1 integrins on T lymphoblasts (11,17). rapidly be regulated by reversible modulation of receptor func- In the present study, we have found that the human tion. One mechanism implicated in the upregulation of inte- HUTS-21 mAb also recognizes ␤1 integrins on rat lympho- grin-mediated adhesion consists in the induction of transitions ϩ ϩ cytes activated with divalent cations such as Mn2 and Hg2 . to a high affinity state in a small fraction of integrin receptors, It is well known that Hg2ϩ induces an autoimmune disease in probably as a consequence of conformational changes of these the Brown Norway (BN) rat mediated by T-dependent poly- molecules (9). These changes can be modulated by different factors such as the concentration of divalent cations, physio- clonal B cell activation and resulting in synthesis of autoanti- logic ligands, or monoclonal antibodies (mAb), among others bodies (mainly, anti-glomerular basement membrane [GBM] (7,9–11). antibodies) with glomerular linear deposits of IgG, proteinuria, and interstitial mononuclear cell infiltrates (18). Considering the recently described expression of activated Received March 2, 1999. Accepted October 7, 1999. ␤1 integrins in a small proportion of lymphoid cells at sites of Correspondence to Dr. Francisco Mampaso, Pathology Department, Hospital inflammation in human autoimmune chronic inflammatory dis- Ramo´n y Cajal, Carretera Colmenar km 9.100, 28034 Madrid, Spain. Phone: eases (19), we have examined the expression and possible role ϩ ϩ 91 336 8052; Fax: 91 336 9016; E-mail: [email protected] of activated ␤1 integrins in the mercury-induced model of 1046-6673/1106-1075 nephritis. Our results demonstrate the capacity of HUTS-21 Journal of the American Society of Nephrology ␤ Copyright © 2000 by the American Society of Nephrology mAb to recognize in vivo an activation-dependent 1 epitope 1076 Journal of the American Society of Nephrology J Am Soc Nephrol 11: 1075–1084, 2000

as well as its positive/negative effects on this autoimmune Urinary Protein Excretion experimental disease. Animals were maintained in metabolic cages for 24 h to collect urine samples and had free access to food and water. Urine samples were taken at regular intervals starting on day 0. The amount of protein was mea- Materials and Methods sured in triplicate by using a Bio-Rad assay (Bio-Rad, Richmond, CA), Animals according to the manufacturer’s protocol. The optical density from each BN rats, weighing 150 to 180 g, were obtained from IFFA-Credo sample was measured in a Titertek Multiskan Plus spectrophotometer (Paris, France) and from our own breeding colony and maintained (Flow, Irvine, Scotland, United Kingdom) at 595 nm. under standard conditions, with free access to food and water.

Anti-GBM Antibody Assays Monoclonal Antibodies Rat GBM was isolated using essentially the same procedure de- The mouse anti-human HUTS-21 mAb, which reacts with an scribed by Bowman et al. (24). Briefly, glomeruli were obtained from activation-dependent epitope on the VLA-␤1 chain (CD29), has al- normal BN rats by differential sieving and centrifugation of minced ready been described (11,17). The mouse anti-human HP2/1 mAb is kidney cortices. The glomerular suspension was sonicated, washed, directed toward the ␣4 integrin (20) and cross-reacts with the rat ␣4 and lyophilized. The GBM was digested with type I collagenase integrin (21,22). The mouse anti-rat OX1 mAb, specific for the (Sigma Chemical Co., St. Louis, MO) at 0.7% wt/wt for1hat37°C. pan-leukocyte CD45 antigen (23), was purchased from Serotec (Ox- Anti-GBM antibodies were measured by enzyme-linked immunosor- ford, United Kingdom). bent assay (ELISA) as described previously (24). Briefly, 96-well microtiter plates (Nunc, Roskilde, Denmark) were coated with rat Flow Cytometry Assays GBM (100 ␮l/well of 10 ␮g/ml GBM in 0.1 sodium carbonate buffer, To assay the effects of MnCl and HgCl on the activated ␤1 pH 9.6) by overnight incubation at 4°C and then washed with PBS 2 2 ␮ subunit expression, whole-blood samples from normal control rats containing 0.05% Tween 20 (Sigma). Wells were filled with 100 lof were incubated with a saturating concentration of HUTS-21 mAb for sera (1:100 diluted) to be tested. Plates were incubated for1hat37°C. 15 min at 37°C in Hepes/NaCl buffer (20 mM Hepes, 150 mM NaCl, After incubation with peroxidase-labeled rabbit anti-rat IgG antibody 2ϩ 2 mg/ml D-glucose, pH 7.4) containing 0.9 mM Mn or 7.5 ␮M (Serotec), peroxidase activity was revealed and absorbance was mea- sured at 492 nm by using a Titertek Multiskan Plus (Flow). HgCl2. After washing, cells were incubated with FITC-conjugated goat-anti-mouse IgG (Becton Dickinson, Mountain View, CA) sec- Samples of a serum pool from untreated BN rats and from BN rats ondary antibody for 30 min at 4°C in the dark. The erythrocytes were that were treated with HgCl2 and bled on day 13 of the disease served lysed, leukocytes were fixed, and cellular membranes were stabilized as negative and positive controls, respectively. Results were expressed in fluorescence-activated cell sorter (FACS) lysing solution (Becton as the percentage of binding obtained with samples from positive Dickinson). OX1 mAb and only the direct application of the second- control serum. ary antibody were used as positive and negative controls, respectively. The samples were analyzed using a FACScan cytometer (Becton Dickinson). Kidney Tissue Processing On day 15 of the experiment (when the interstitial cell infiltration To investigate the effects of HgCl2 in the in vivo expression of activated ␤1 integrins, a group of rats (n ϭ 5) was injected subcuta- was still highly present) (22), rats from each group were sacrificed. ␮ Kidneys were harvested and further processed for histologic, inmu- neously with HgCl2 three times a week for 2 wk with 100 g of HgCl2 per 100 g body wt (18). To establish the kinetics of the expression of nohistochemical, and electron microscopy studies. For micros- ␮ HUTS-21 epitope throughout the course of the disease, animals were copy, 2- m paraffin-embedded kidney sections were stained with sequentially bled on different days of the experiment by tail artery hematoxylin and eosin and periodic acid-Schiff. For immunohisto- puncture. Then, whole blood samples were incubated with saturating chemistry studies, pieces of renal tissue were snap-frozen in isopen- Ϫ concentration of HUTS-21 mAb for 15 min at 37°C, washed, and tane precooled in liquid nitrogen, and stored at 70°C until used. incubated with FITC-conjugated goat-anti-mouse IgG (Becton Dick- Direct immunofluorescence studies were performed on ether/ethanol- inson) secondary antibody for 30 min at 4°C in the dark. Then, fixed serial cryostat sections by using FITC-conjugated rabbit anti-rat samples were processed using the same procedure as above and IgG (Serotec), as reported previously (25). To characterize OX1 and further analyzed by flow cytometry. HUTS-21-positive cells in the renal interstitium, frozen kidney sec- tions were stained with those mAb and revealed with an indirect immunoperoxidase method (26). Enumeration of interstitial infiltrat- Experimental Procedure ing cells bearing OX1ϩ cell surface markers was determined by Four groups of rats were used in this study. Groups I to III were counting, in two kidney tissue sections per each rat, the total number ␮ injected with 100 g of HgCl2 per 100 g body wt, following the same of positive-labeled cells examined in 10 randomly chosen areas of procedure as described above to induce the disease. Group I (n ϭ 12) interstitial infiltrates. The ratio of infiltrating cells expressing acti- did not receive any additional treatment. Rats from group II (n ϭ 12) vated ␤1 subunit (HUTS-21ϩ cells) to the total number of OX1ϩ cells and group III (n ϭ 12) also received an intraperitoneal injection (0.5 was obtained using a conventional light microscopy objective (ϫ60), mg) of HUTS-21 or OX1 mAb on days 0, 8, and 13, respectively. as described (27). For ultrastructural studies, the renal tissue was fixed Group III was used as control of HUTS-21 mAb administration. immediately in 2.5% glutaraldehyde in 1.1 M cacodylate buffer for Group IV (n ϭ 12) served as a normal control in which rats received 2 h, followed by post-fixation in osmium tetroxide. Then, tissue was

distilled water adjusted to pH 3.8 (the same pH of the HgCl2 solution dehydrated in increasing concentrations of ethanol and embedded in used), following the same procedure described above for the mercury Epon 812. Thin sections for ultrastructural examination were stained administration. The animals were sequentially bled by tail artery with lead citrate and examined at 75 kV with a Jeol 100CX electron puncture and sacrificed on different days of the experiment. microscope. J Am Soc Nephrol 11: 1075–1084, 2000 ␤1-Activated Integrins in Autoimmune Nephritis 1077

Statistical Analyses analyzed by flow cytometry. As shown in Figure 1, the pres- ϩ ϩ The results are given as mean Ϯ SD values obtained from the levels ence of either Mn2 and Hg2 cations induced the expression of proteinuria and ELISA results. Data were analyzed using two-way of the epitope recognized by HUTS-21 mAb. ANOVA, and when the statistical difference was significant, data were also analyzed using the t test with the Bonferroni correction. Induced Expression of HUTS-21 Activated Epitope on Results Rat Lymphocytes through the Course of the HgCl2- Recognition of Activated ␤1 Integrins on Rat Induced Nephritis Lymphocytes by HUTS-21 mAb To establish the kinetics of HUTS-21 expression in vivo, ϭ To assess the cross-reactivity of the mouse anti-human ac- whole blood samples of HgCl2-treated rats (n 5), obtained at tivated ␤1 integrin (HUTS-21) mAb with the same subunit in different days of the disease, were incubated with a saturating the BN rat, rat lymphocytes obtained from whole blood were concentration of HUTS-21 mAb and analyzed by flow cytom- incubated with saturating concentrations of HUTS-21 mAb in etry. Figure 2 shows the time course of the expression of the the presence of Mn2ϩ and Hg2ϩ in the medium. Samples were activated ␤1 subunit epitope on rat lymphocytes. The maximal

␤ Figure 1. Recognition of activated 1 integrins on rat lymphocytes by HUTS-21 monoclonal antibody (mAb) in the presence of MnCl2 and ␤ HgCl2 in the medium. Cells were incubated with a saturating concentration of the anti-activated 1 integrins HUTS-21 mAb in medium with or without Mn2ϩ and Hg2ϩ. Lymphocytes were then washed and incubated with FITC-conjugated goat-anti mouse IgG. OX1 mAb and the direct application of only the secondary antibody were used as positive and negative controls, respectively. All samples were analyzed by flow cytometry. 1078 Journal of the American Society of Nephrology J Am Soc Nephrol 11: 1075–1084, 2000

␤ Figure 2. Expression of HUTS-21 activated 1 epitope on rat lymphocytes through the course of the HgCl2-induced nephritis. Whole blood samples were obtained on different days of the disease. Samples were incubated with a saturating concentration of HUTS-21 mAb. The erythrocytes were lysed, leukocytes fixed, and cellular membranes stabilized in FACs-Lysys solution. OX1 mAb and the direct application of only the secondary antibody were used as positive and negative controls, respectively. All samples were analyzed by flow cytometry. expression of HUTS-21 epitope was obtained between days 13 groups I and II showed similar striking abnormalities of glo- and 15, with the expression declining after day 23 to reach the merular epithelial cells with edema and extensive effacement background levels at day 30 of the disease. of the podocyte foot processes. No other ultrastructural glo- merular alterations were observed (Figure 4, A through C). Effect of HUTS-21 mAb Treatment on the Proteinuria of HgCl2-Injected BN Rats Effect of HUTS-21 mAb Treatment on Anti-GBM As shown in Figure 3, HgCl2-treated rats (group I) devel- Antibody Production and Glomeruli Deposition oped proteinuria in two different phases: a first short phase, As shown in Figure 5, the sera from rats injected with HgCl2 which started immediately after the first injection of HgCl2, (group I) presented circulating antibody anti-GBM as detected followed by a second phase starting on day 11 and declining by ELISA. The maximal concentration of anti-GBM antibody after day 16 of the disease. At the third week, all of the animals was detected on day 13 of the disease, decreasing thereafter as reached the background protein levels. The treatment with also occurred with the proteinuria. By direct immunofluores- anti-activated ␤1 epitope HUTS-21 mAb (group II) did not cence methods, rats treated only with mercury showed a linear modify the time course of the urinary protein excretion levels. IgG glomerular deposition along the GBM on examination at Electron microscopy examination of the kidney of rats from day 15 of the disease, as demonstrated in Figure 6A. Rats that J Am Soc Nephrol 11: 1075–1084, 2000 ␤1-Activated Integrins in Autoimmune Nephritis 1079

Ϯ Ⅺ u s Figure 3. Urine protein excretion ratio in all tested groups. Results are expressed as mean SD. , HgCl2; , HUTS-21; , control.

Ϯ in addition to mercury administration were treated with anti- interstitial cells found in HgCl2-treated rats was 36 7 cells/ activated ␤1 epitope HUTS-21 mAb (group II) exhibited a high-power field, and the proportion of HUTS-21ϩ cells that significant reduction in the anti-GBM antibody serum levels formed part of such infiltrates was Ͼ95% (Figure 7, B and C). (40%) (Figure 5). This drop in circulating anti-GBM antibody The effect of HUTS-21 mAb treatment on the intensity of was maintained throughout the course of the disease. Also, a interstitial cell infiltrate was also examined. Figure 7D showed reduction in the intensity of IgG linear deposits on the GBM that the number of OX1ϩ-infiltrated cells in the renal tissue was found in this group of rats (Figure 6B). from rats treated with HUTS-21 (OX1ϩ cells/high-power field) To saturate the target protein, the following alternative ther- was considerably reduced (4.5 Ϯ 0.9) compared with the ϩ Ϯ apeutic protocol was performed: Mercury-treated rats received OX1 cells from HgCl2-treated rats alone (36 7). daily an intraperitoneal injection (0.5 mg) of HUTS-21 mAb (n ϭ 6) or OX1 mAb (n ϭ 6), starting on day 0 and finishing Discussion on day 14 of the experiment. Despite having increased the total Cell communication through different adhesion molecules is dose of HUTS-21 mAb from 1.5 to 7.5 mg, the levels of an essential event in the inflammatory response (1–5). Cellular circulating anti-GBM antibody and the intensity of linear IgG adhesive properties are regulated through the selective expres- glomerular deposits were not further decreased, and proteinuria sion of the integrin repertoire, as well as by the modulation of remained unaltered (data not shown). These results suggest that the binding properties of these receptors (9). The ␤1 subfamily the doses of HUTS-21 mAb used in both protocols were of integrins function mainly as cellular receptors for extracel- saturating. lular matrix , and they are also implicated in cell-cell interactions (7,28). The expression of ␤1 integrin activation Expression of HUTS-21 ␤1 Activation Epitope in epitopes at sites of tissue injury in inflammatory processes has Kidney Tissue and Effects of Anti-HUTS-21 mAb also been described, suggesting a key role of ␤1 integrin Administration on Interstitial Cell Infiltrates activation in the development of inflammatory processes (19).

Renal tissue sections from rats treated with HgCl2 alone The state of integrin activation can be assessed by a group of ϩ ␤ (group I) and rats treated with HgCl2 HUTS-21 mAb (group monoclonal antibodies (HUTS) that selectively recognize 1 II) were examined by light microscopy on day 15 of the integrins in their active form. These epitopes are expressed on disease. Renal tissue sections from normal rats served as con- activated ␤1 integrins by different stimuli, and their expression trol (Figure 7A). correlates with the binding activity of ␤1 integrins to immo- Pronounced patchy areas of interstitial mononuclear cell bilized ligands (11,16,17). infiltrates were located preferentially in the perivascular re- In this study, we have analyzed the expression of the ␤1- gions of the renal interstitium in HgCl2-treated rats (Figure activated epitope recognized by HUTS-21 mAb in an animal 7B). To characterize the total number of lymphohemopoietic model of autoimmune nephritis, induced by HgCl2. Our results infiltrating cells and to assess the HUTS-21 expression in the show the expression of a similar activated ␤1 epitope in rat interstitial cell infiltrates, frozen kidney tissue sections were lymphocytes, as demonstrated by the reactivity of HUTS-21 stained with anti-CD45 (OX1) and anti-activated ␤1 subunit mAb with rat lymphocytes in the presence of the divalent epitope (HUTS-21) mAb. The number of OX1ϩ inflammatory cations Mn2ϩ and Hg2ϩ. It is well known that Hg2ϩ cation 1080 Journal of the American Society of Nephrology J Am Soc Nephrol 11: 1075–1084, 2000

acute tubulointerstitial nephritis (18,22,24,29,30). The devel- opment of this experimental autoimmune model is a conse- quence of the generation of an autoreactive T cell subset inducing a polyclonal B cell activation (31,32). It has been shown previously that interaction between circulating leuko- cytes expressing VLA molecules and endothelial cells is a crucial event in the development of this renal disease (22,29,30). Taking into account that activated ␤1 integrins are expressed in a small proportion of lymphoid cells in different human autoimmune chronic inflammatory diseases (19), it was of interest to know the possible expression of leukocyte-acti- vated ␤1 integrins in the mercury model of nephritis. Our data demonstrate the induced expression by lymphocytes in this in vivo model of the activation-dependent epitope of the ␤1 (VLA) integrins, which paralleled the other parameters in the onset of the disease. In this regard, we have found that the expression of the HUTS-21 epitope on rat peripheral blood lymphocytes was maximal on days 13 to 15 of the disease, as occurred with the levels of circulating autoantibodies, protein- uria, and intensity of interstitial nephritis (22,29,30). Our find- ings suggest a role of ␤1-activated receptors in the develop- ment of this experimental autoimmune disease, and thus we have investigated whether HUTS-21 mAb was able to exert any blocking effect when it was used as a therapeutic agent.

The administration of HUTS-21 mAb to HgCl2-treated rats abrogated the renal interstitial mononuclear cell infiltrates and reduced the levels of circulating anti-GBM antibodies and glomerular deposition. Nevertheless, urinary protein values were unaffected. It is feasible that the blocking effects observed after HUTS-21 mAb administration on leukocyte extravasation and migration into the renal interstitium were due to conforma- tional changes in the integrin molecules, inducing an increase of the ligand binding affinity. These in vivo results concur with previous in vitro studies showing that the engagement of ␤1 integrins by HUTS-21 causes functional inhibition of ␤1 inte- grin-dependent dynamic processes, such as cell migration on proteins or endothelial cell ligands (17). It has been reported previously (33–35) that the cell migration requires a gradient of adhesive strength from the front to the rear of the migrating cell that determines cell movement and that the cell migration could be inhibited by changes in this delicate balance of adhesion/release (35–38). It is therefore conceivable that HUTS-21 mAb treatment could affect this adhesion/release balance. On the other hand, engagement of ␤1

Figure 4. Ultrastructural micrographs from rats injected with HgCl2 epitope HUTS-21 inhibits migration of T lymphoblasts on alone (A) and rats also treated with HUTS-21 mAb (B), showing both vascular -1 and fibronectin substrates, edema of the epithelial cell with prominent foot processes but has no effect over intercellular adhesion molecule-1 mi- obliteration. Normal control rats show a well-preserved morphologic gration (17). Our results demonstrate that HUTS-21 mAb ϫ appearance of the foot-processes (C). Magnification: 5200 in A and administration was able to abrogate the renal interstitial cell ϫ B; 5600 in C. infiltrates, thus strongly supporting that leukocyte migration from blood to renal interstitium is mediated by the VLA/ vascular cell adhesion molecule-1 adhesion pathway in this induces an autoimmune disease in the BN rat characterized by particular disease model, in accordance with previous studies the synthesis of autoantibodies (mainly, anti-GBM antibody) (22,29,30). Moreover, our findings indicate that the expression with glomerular linear deposits of IgG, severe proteinuria, and of active conformations of ␤1 integrins is involved in the J Am Soc Nephrol 11: 1075–1084, 2000 ␤1-Activated Integrins in Autoimmune Nephritis 1081

o Figure 5. Levels of circulating anti-glomerular basement membrane autoantibodies in HgCl2-injected rats , in rats treated with HUTS-21 and OX1 mAb `, and in the normal control group Ⅺ. Values are expressed as mean Ϯ SD. *P Յ 0.01, difference with the normal control group. Ⅲ Յ P 0.5, difference with the HgCl2-treated group.

Figure 6. Immunofluorescence staining. Positive linear pattern deposition of rat IgG in rats treated with HgCl2 alone (A) and in rats that were also injected with HUTS-21 mAb (B). Panel B shows a reduction in the intensity of the deposits.

leukocyte recruitment into the renal interstitium and also in the uria. In this regard, it has been previously demonstrated that the development of this inflammatory disease. induction of injury with heterologous anti-GBM antibody can In addition to the protective effect of HUTS-21 mAb treat- be related to micrograms of antibody bound per gram of kidney ment in the development of renal cell infiltrates, we found a in terms of molecules per glomerulus. Quantitative studies reduction in the levels of circulating anti-GBM antibodies, as showed that 75 ␮g of antibody bound per gram of kidney, or well as in the intensity of glomerular IgG deposition. This last approximately one molecule for 26 ␮m2 of glomerular filtering observation suggests a role for activated ␤1 integrins in T surface, is necessary to induce proteinuria in the rat. Most cell–B cell interactions implicated in the synthesis of the au- probably and depending also on steric factors, at least half of toantibodies in this experimental model. Nevertheless, the in- the filtering surface would be covered by the antibody (39). By complete reduction of anti-GBM antibody production after electron microscopy, we have found similar ultrastructural

HUTS-21 mAb administration suggests that other mechanisms glomerular alterations in both HgCl2-treated rats with persis- are at work in the autoantibody synthesis. This issue is sup- tent proteinuria and in those rats that also received HUTS-21 ported by our previous observation, in which anti-␣4 chain mAb treatment, independently of the intensity of anti-GBM HP2/1 mAb treatment abolished anti-GBM antibody synthesis antibody deposition. Once proteinuria develops, it may exert a (22). Of interest, the persistence of high urinary protein excre- pathologic effect by itself as enhanced uptake of plasma pro- tion levels after in vivo administration of HUTS-21 mAb to teins by glomerular epithelial cells does occur in proteinuric

HgCl2-treated rats suggests that the reduction in the levels of states and may eventually lead to the production of mediators circulating anti-GBM antibody and glomerular IgG linear de- with an injurious effect on the cellular components of the posits are not enough to prevent the development of protein- filtration barrier (40). On the other hand, the complete abro- 1082 Journal of the American Society of Nephrology J Am Soc Nephrol 11: 1075–1084, 2000

Figure 7. Immunoperoxidase staining with OX1 and HUTS-21 mAb. (A) OX1ϩ infiltrating cells in normal control rats. (B) OX1ϩ infiltrating ϩ ϩ cells in rats treated with HgCl2 alone. (C) HUTS-21 infiltrating cells in rats treated with HgCl2 alone. (D) OX1 infiltrating cells in rats treated with HgCl2 and also injected with HUTS-21 mAb. gation of interstitial cell infiltrates in the presence of protein- renal inflammatory disease, in both leukocyte cell-cell interac- uria, found after in vivo administration of HUTS-21 mAb to tions and leukocyte infiltration pathway mechanisms. Our re-

HgCl2-treated rats, indicates that the leukocyte migration may sults confirm the important role played by VLA integrins in have less importance in the development of renal tissue dam- this model and provide further evidence that cellular activation age than previously thought. These findings are consistent with and synthesis of anti-GBM antibody can be implicated in the ␣ those reported by our group, in which anti- 4 mAb HP2/4 renal dysfunction observed in rats with HgCl2-induced disease. treatment was able to abrogate the interstitial leukocyte infil- tration without affecting the proteinuria levels (30). They are Acknowledgments also in agreement with those described in allergic inflamma- This work was supported by Fondo de Investigaciones Sanitarias ␣ tory reactions, in which the administration of anti- 4 mAb Grant 98/1211 (to Dr. Mampaso); Comunidad Auto´noma de Madrid HP1/2 suppressed the leukocyte accumulation but had no effect 08.4/0015/98 (to Dr. Mampaso), 08.1/0015/97(to Dr. Caban˜as) and on stimulated edema formation (41). Nevertheless, there are 08.1/0011/97 (to Dr. Sa´nchez-Madrid); CICYT SAF 98/0088 (to Dr. conflicting data in the literature regarding the protective effect Caban˜as); and CICYT SAF 99/0034-C01 and 2FD97-0680-C02 (to of integrin blockade on renal function but not on inflammation. Dr. Sa´nchez-Madrid). A similar apparent discrepancy on the functional effects of the leukocyte adhesion molecules has been reported in allergic References airway inflammatory disorders (42–44). Although it is well 1. Springer TA: Traffic signals on endothelium for lymphocyte established that leukocyte adhesion molecule blockade is tis- recirculation and leukocyte emigration [Review]. Annu Rev sue-protective in ischemic reperfusion injury in muscle or Physiol 57: 827–872, 1995 heart, the interpretation of responses of the kidney is still a 2. Jutila MA: Leukocyte traffic to sites of inflammation. Acta matter of controversy. CD11/CD18 and intercellular adhesion Pathol Microbiol Immunol Scand 100: 191–201, 1992 molecule-1 blockade are usually protective in experimental 3. Cronstein BN, Weissmann G: The adhesion molecules in inflam- renal ischemia-reperfusion injury. In contrast, induction of mation. Arthritis Rheum 36: 147–157, 1993 systemic neutropenia and function blockade do not 4. Carlos TM, Harlan JM: Leukocyte-endothelial adhesion mole- have a protective effect, suggesting a neutrophil-independent cules [Review]. Blood 84: 2068–2101, 1994 5. Rabb HA: Cell adhesion molecules and the kidney. Am J Kidney mechanism for renal protection (45). Leukocyte adhesion mol- Dis 23: 155–166, 1994 ecules might mediate renal ischemia-reperfusion injury by 6. Giancotti FG, Mainiero F: Integrin-mediated adhesion and sig- mechanisms other than simply leukocyte migration, such as naling in tumorigenesis. Biochem Biophys Acta 1198: 47–64, signal transduction and cell transport (46). 1994 In conclusion, we have reported a key role of the ␤1- 7. Hynes RO: Integrins: Versatility, modulation, and signaling in activated integrins in the development and progression of this cell adhesion. Cell 69: 11–25, 1992 J Am Soc Nephrol 11: 1075–1084, 2000 ␤1-Activated Integrins in Autoimmune Nephritis 1083

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