Background. Mononuclear cell infiltration is a common feature of cell-mediated renal transplant rejection. Chemokines and their corresponding receptors likely play a central role in directing specific classes of leukocytes to graft sites during rejection. Localization of chemokine receptors may help us understand how specificity in leukocyte trafficking is achieved in renal inflammatory processes. The localization of the chemokine receptor CXCR4 in human kidney and in renal transplant rejection is unknown.

Methods. We generated a riboprobe specific for the detection of CXCR4 mRNA by in situ hybridization to evaluate cellular sites of synthesis of this receptor in native human kidneys (n=11) and in human allograft nephrectomies with features of severe rejection (n=14).

Results. By in situ hybridization, CXCR4 mRNA expression is undetectable in intrinsic glomerular, tubular, and renovascular cells in native kidneys. When renal interstitial inflammation is present, CXCR4 mRNA expression is localized to a large fraction of infiltrating leukocytes. Large numbers of CXCR4-expressing cells are detected in cell-mediated renal allograft rejection. Double immunolabeling for CD3 antigen identified a large fraction of infiltrating CXCR4 mRNA-expressing cells as T lymphocytes. CXCR4 mRNA-expressing cells were frequently seen in neointimal lesions of vascular rejection in allograft nephrectomies. CXCR4 mRNA expression was identified in infiltrating neointimal T lymphocytes, but not smooth muscle cells by immunolabeling.

Conclusions. We demonstrate the involvement of CXCR4 mRNA-expressing infiltrating cells in human renal interstitial and vascular allograft rejection. Signaling via the CXCR4 receptor may be one mechanism by which chemokines mediate leukocyte trafficking in renal allograft rejection.

Renal transplant rejection is characterized by widespread interstitial and focal tubular infiltration of T lymphocytes, monocytes/macrophages, and occasionally eosinophils (1). Although many factors are involved in facilitating leukocytic infiltration of allograft tissue, several studies during the past years have shown that chemokines and the corresponding receptors play a central role in regulation of inflammatory processes (2,3). Recent observations in animal models and in human biopsy tissue have demonstrated an increased expression of several chemokines in solid organ transplants undergoing allograft rejection (4-8). It is likely that local expression and signaling by chemokines controls infiltration of renal allografts by specific classes of leukocytes through interactions with receptors borne by each leukocyte type.

Chemokines represent a large family of small cytokines and are largely classified into two groups, according to the position of the first two cysteines, which are separated by one amino acid (CXC) or are adjacent (CC) (3,9). In addition to their well-defined role as chemotactic mediators of target leukocytes, many chemokines have other distinct proinflammatory properties, including regulation of leukocyte extravasation, activation of effector functions of leukocytes, and mediation of angiogenesis (3,10). Chemokines mediate their specific effect on target cells through a family of seven-transmembrane G-protein-coupled receptors. The chemokine receptor CXCR4 is one of four CXC chemokine receptors and eight CC chemokine receptors that have currently been cloned and characterized for their ligand specificity (9,11). CXCR4 exclusively binds to SDF-1 (stromal cell-derived factor-1) (12,13), which is a potent chemoattractant for T lymphocytes and hematopoeitic progenitor cells (14,15). Chemokine receptor CXCR4 expression has been found in leukocytes, including peripheral blood lymphocytes, neutrophils and monocytes (16), but, additionally, CXCR4 is by far the most widely expressed of the functional chemokine receptors in nonhematopoeitic cells (9). High transcript levels were demonstrable in several tissues, including heart, brain, liver and colon (17).

In this study, we report on the cellular sites of synthesis of the human chemokine receptor CXCR4 in mature adult human kidney and in rejecting human allograft kidneys. Despite the growing numbers of reports on the role of chemokines in the rejection process, nothing is known about the expression of the chemokine receptor CXCR4 in this setting. We begin to address this deficiency by an in situ hybridization study in which we have identified CXCR4 mRNA-expressing cells in human allograft nephrectomies with features of severe cellular rejection and variable degrees of vascular rejection. Combined immunohistochemical double labeling was established to determine the phenotypes of chemokine receptor CXCR4-expressing cells.

Source of tissue. Normal-appearing human kidney tissue (n=11) was obtained fresh from uninvolved portions of kidneys surgically resected because of the presence of a localized neoplasm. Microscopic examination of this tissue demonstrated the focal presence of interstitial inflammation with chronic tubulointerstitial injury, generally attributable to the nephrosclerosis that is routinely encountered in aging kidneys. In most cases, these nephrosclerotic changes involved a minority of the cortical parenchymal tissue available for examination. We subsequently regarded the macroscopically normal-appearing kidney tissue as three different groups: three cases that demonstrated absent interstitial inflammation, four cases that demonstrated features of very mild and focal interstitial inflammation associated with fibrosis, and four cases in which the nephrosclerotic changes were more diffuse, involving up to approximately 50% of the cortical parenchyma.

Human allograft nephrectomies (n=14) excised for irreversible rejection were also utilized. Microscopic examination demonstrated the presence of severe cellular (interstitial) rejection in all cases (NIH Cooperative Clinical Trials in Transplantation pathology class I; 18). Four cases showed additional features of chronic vascular rejection (defined as neointimal proliferation in muscular arteries) with variable degrees of superimposed acute inflammatory infiltrates in some arteries (NIH Cooperative Clinical Trials in Transplantation pathology class II), as previously described and illustrated (19,20). The nephrectomy specimens in general were exposed to multiple courses of routine and intensified immunosuppression before excision, and represent a heterogeneous sample from a clinical standpoint.

All tissues were fixed in 10% phosphate-buffered formalin, processed, paraffin-embedded, and sectioned using conventional techniques.

Molecular probes. A 1.1-kilobase pair sequence of DNA was derived from cDNA encoding human CXCR4, and cloned into pcDNAI/amp (Invitrogen, Carlsbad, CA). This probe was kindly provided through the AIDS Research and Reference Reagent Program (Division of AIDS, NIAID, NIH) by Dr. Nathaniel Landau and has been characterized previously (21,22). The plasmid was linearized and transcribed into antisense and sense riboprobes, using reagents from Promega (Madison, WI), except [³⁵S]UTP, which was obtained from New England Nuclear (Boston, MA). The transcription reaction mixture contained 1 µg of CXCR4 cDNA (sense or antisense orientation), 250 µCi of [³⁵S]UTP, 500 µmol/L each of ATP, CTP, and GTP, 40 U of RNasin, 10 mmol/L dithiothreitol, 40 mmol/L Tris, and 10 U of either SP6 or T7 polymerase. After 75 min at 37°C, the template DNA was digested by adding 1 U of DNAse and incubation at 37°C for an additional 15 min. Free nucleotides were then separated with a Sephadex G-50 column. The collected fraction containing labeled probe was ethanol-precipitated, resuspended in nuclease-free water containing 10 mmol of dithiothreitol and used within 48 hr. The CXCR4 antisense riboprobe generated for Northern analysis was labeled with digoxigenin-labeled UTP (Boehringer-Mannheim Biochemicals, Indianapolis, IN).

The pTRI-RNA-28S antisense control template containing a 115-base pair cDNA fragment of the human 28S rRNA gene was purchased from Ambion (Austin, TX). Labeled transcripts were generated from the 28S antisense control template and employed as a probe for Northern blots. The in vitro transcription reaction was performed with digoxigenin-labeled UTP (Boehringer-Mannheim) and T7 polymerase.

Antibodies. A murine monoclonal antibody 1A4 (DAKO, Carpinteria, CA) directed against smooth muscle [alpha]-actin has previously been characterized by tissue immunohistochemistry and Western blotting (23). This antibody has been shown to recognize cells expressing smooth muscle [alpha]-actin in fixed tissues with no loss of sensitivity compared with frozen tissue specimen (24,25).

An affinity-isolated rabbit polyclonal antibody directed against human CD3 was purchased from DAKO. This antibody reacts with the T-lymphocyte-associated CD3 antigen and is suitable for use on formalin-fixed, paraffin-embedded tissue sections after heat-mediated antigen retrieval (26).

Cell lines. To confirm the specificity of the CXCR4 riboprobe, chemokine receptor-transfected cell lines were used as controls. Transfected HOS cells, individually expressing CCR1, CCR2B, CCR3, CCR4, CCR5, or CXCR4, were obtained through the AIDS Research and Reference Reagent Program (Division of AIDS, NIAID, NIH) from Dr. Nathaniel Landau (21,27). Transfected cell lines containing mRNA encoding CXCR1-3 are not currently available and could not be tested in a similar fashion. The cells were grown in Dulbecco's minimum essential medium (Sigma Chemical, St. Louis, MO), supplemented with 10% fetal bovine serum and 0.5 µg/ml puromycin (Fluka, Ronkonkoma, NY). After reaching confluence, the cells were trypsinized and centrifuged. The resulting cell pellets were either fixed in 10% phosphate-buffered formalin, paraffin-embedded, and analyzed by in situ hybridization or used for total RNA isolation.

RNA isolation. Total RNA was extracted from each of the chemokine receptor-transfected cell lines, using the Totally RNA™ total RNA isolation kit (Ambion). Briefly, resuspended cells were denatured using a tissue homogenizer (Tissumizer SDT-1810; Teckmar, Cincinnati, OH). After phenol/chloroform extraction, the RNA was recovered by isopropanol precipitation. Remaining salt was removed by ethanol wash and residual carbohydrate by lithium chloride precipitation. Finally, the pellets were resuspended in RNase-free water. The RNA concentration was measured by reading the absorbance in a spectrophotometer at the wavelength of 260 nm.

Northern analysis. Samples (10 µg of RNA) were electrophoresed through a 1% agarose-formaldehyde gel and transferred to a positively charged nylon membrane (BrightStar-Plus, Ambion). Blots were hybridized overnight with the digoxigenin-UTP-labeled CXCR4 antisense riboprobe. Hybridized probe was detected using an alkaline-phosphatase labeled anti-digoxigenin antibody (Boehringer-Mannheim) and the NorthernMax™ northern blotting kit (Ambion). To confirm the intactness and total amount of the blotted RNA, the membranes were subsequently stripped and hybridized with the digoxigenin-UTP-labeled 28S antisense riboprobe.

In situ hybridization. Four-micrometer sections of tissue and cell pellets that had been fixed in 10% neutral buffered formalin and embedded in paraffin were deparaffinized after standard protocols, which we have utilized previously (28). The sections were washed with 0.5 × standard saline citrate (SSC*) (1 × SSC=150 mM NaCl, 15 mM sodium citrate, pH 7.0) and digested with proteinase K (5 µg/ml; Sigma) in Tris buffer for 30 min at 37°C. Several 0.5 × SSC washes were followed by prehybridization for 2 hr in 100 µl of prehybridization buffer (0.3 M NaCl, 20 mM Tris, pH 8.0, 5 mM EDTA, 1 × Denhardt's solution, 10% dextran sulfate, 10 mM dithiothreitol). The hybridizations were started by adding 500,000 cpm of ³⁵S-labeled riboprobe in 50 µl of prehybridization buffer and allowed to proceed overnight at 50°C. After hybridization, sections were washed with 0.5 × SSC, treated with RNase A (20 µg/ml, 30 min at 37°C), washed in 2 × SSC (two 2-min washes), followed by three high-stringency washes in 0.1 × SSC/0.5% Tween 20 (Sigma) for 40 min each at 50°C, and several 2 × SSC washes. After the tissue was dehydrated and air-dried, it was dipped in NTB2 nuclear emulsion (Kodak, Rochester, NY) and exposed in the dark at 4°C for 4 weeks. After developing, the sections were counterstained with hematoxylin and eosin, dehydrated, and coverslipped.

Combined immunohistochemistry and in situ hybridization. To further identify the phenotypes of CXCR4 mRNA-expressing cells within the kidney, tissue sections of human allograft nephrectomies were immunostained for CD3 (T lymphocytes) or smooth muscle [alpha]-actin (vascular smooth muscle cells, mesangial cells) before in situ hybridization for CXCR4 mRNA. Formalin-fixed, paraffin-embedded tissues were prepared as described above. Briefly, sections were deparaffinized in xylene and rehydrated in graded ethanols. Endogenous peroxidase was blocked by incubation in 3% hydrogen peroxide. Sections that were subsequently incubated with the anti-CD3 antibody were pretreated by microwaving for 8 min in Antigen Retrieval Buffer (Vector, Burlingame, CA). The sections were then incubated for 1 hr with the primary antibody diluted in phosphate-buffered saline (PBS) containing 1% bovine serum albumin (Sigma). After washes in PBS, the sections were sequentially incubated with biotinylated goat anti-rabbit (Vector) or horse anti-mouse antibody (Vector) and the ABC-Elite reagent (Vector), and finally 3,3'-diaminobenzidine was used as the chromogen. After an overnight incubation in PBS, in situ hybridization for CXCR4 mRNA was performed as described. Negative controls for the immunohistochemical procedures consisted of (a) substitution of the primary antibody with irrelevant murine monoclonal antibodies or (b) nonimmune rabbit antibody, and (c) in all cases, a second substitution control in which the primary antibodies were replaced by PBS.

Cellular sites of synthesis of chemokine receptor CXCR4 were examined in native human kidneys and in human allograft nephrectomies by in situ hybridization.

Specificity of the CXCR4 antisense riboprobe was established by Northern analysis and in situ hybridization. Northern analysis demonstrated positive hybridization with the CXCR4 riboprobe only with RNA isolated from CXCR4-transfected HOS cells (Fig. 1). No nonspecific cross-hybridization with RNA isolated from the several control cell lines transfected with genomic material coding for CCR1, CCR2B, CCR3, CCR4, or CCR5 could be detected. Cross-reactivity of the CXCR4 antisense riboprobe with other chemokine receptors could not be tested in a similar fashion because of the lack of appropriate transfected cell lines for these studies. By in situ hybridization, positive hybridization signal with the CXCR4 antisense probe was demonstrable on sections of paraffin-embedded cell pellets of the CXCR4-transfected HOS cell line (Fig. 2A), but not on any other cell lines transfected with different chemokine receptors (data not shown). Absent hybridization signal was seen when identical procedures were performed with substitution of a sense probe for hybridization (Fig. 2B).

In mature human kidneys CXCR4 mRNA-expressing cells were only rarely detectable in the absence of interstitial inflammation. A very small number of CXCR4 mRNA-expressing mononuclear cells, which appeared to be leukocytes, were localized within the interstitium and occasionally within the lumina of peritubular capillaries (Fig. 2C). Cells of the glomerular, tubular, or renal vascular compartments showed no detectable CXCR4 mRNA expression in adult normal human kidney tissue. However, an increased number of CXCR4 mRNA-expressing cells is demonstrable in the presence of renal interstitial inflammation (Fig. 2, D and E). CXCR4 mRNA expression was localized to infiltrating leukocytes, and the number of CXCR4-expressing cells correlated with the total number of infiltrating mononuclear cells. Within these foci of renal interstitial inflammation, no expression of CXCR4 mRNA was seen in intrinsic renal cells. Endothelial cells of the renal vasculature showed no detectable CXCR4 mRNA hybridization signal.

All 14 allograft nephrectomies included in this study demonstrated severe cell-mediated transplant rejection, which was characterized by diffuse, at times massive, interstitial mononuclear inflammatory cell infiltrates. In situ hybridization studies detected massive numbers of CXCR4 mRNA-expressing cells in 13 of the 14 analyzed cases (Fig. 2, F and G). CXCR4 was restricted to infiltrating mononuclear cells (Fig. 2, F and G). Double labeling for CXCR4 mRNA and the T lymphocyte-specific antigen CD3 identified a fraction of the CXCR4-positive cells as T lymphocytes (Fig. 3A). CXCR4 mRNA expression was not detectable in intrinsic renal cells of the glomerular or tubular compartment in human renal allograft nephrectomies (Fig. 2G). One of the 14 analyzed nephrectomy cases demonstrated no hybridization signal for CXCR4 mRNA, despite the presence of massive cellular infiltrates. A possible explanation for this result could be mRNA degradation by endogenous RNases either in vivo as a result of the severity of the transplant rejection, or ex vivo as a result of inadequate fixation of this tissue sample.

We further investigated four allograft nephrectomies with additional features of chronic vascular rejection and variable degrees of acute neointimal inflammatory infiltrates (Fig. 3, B-G). Within neointimal lesions of all four cases, CXCR4 mRNA-expressing cells were frequently detected by in situ hybridization. Simultaneous double labeling for CD3 protein and CXCR4 mRNA identified a large fraction of CXCR4-expressing neointimal cells as T lymphocytes (Fig. 3, B and C), but we additionally detected neointimal CD3-negative, CXCR4 mRNA-expressing cells (Fig. 3, B and C). Immunohistochemical staining for smooth muscle [alpha]-actin identified a large number of neointimal cells as smooth muscle cells (Fig. 3, D and F). However, combined immunohistochemistry for smooth muscle [alpha]-actin and in situ hybridization for CXCR4 mRNA failed to detect CXCR4 expression in vascular smooth muscle cells either within the neointima or the media of rejecting arteries (Fig. 3, D-H).

The present study is the first to demonstrate an increased chemokine receptor CXCR4 expression in human allograft kidneys undergoing severe transplant rejection and in adult human kidneys with interstitial nephritis. By in situ hybridization, we identified cellular sites of CXCR4 synthesis through localization of mRNA coding for this peptide. CXCR4 mRNA expression was undetectable in intrinsic renal parenchymal cells. However, an increased number of CXCR4 mRNA-expressing cells was demonstrable in the presence of renal interstitial inflammation either in interstitial nephritis and, very pronounced, in rejecting allograft kidneys. CXCR4 mRNA expression was localized to infiltrating mononuclear cells. Immunohistochemical double labeling identified a large fraction of the CXCR4-positive cells as T lymphocytes.

Chemokines are low-molecular-weight proteins with chemotactic properties for leukocytes (3,29). They mediate their specific effect on target cells largely through two related subfamilies of seven-transmembrane G-protein-coupled receptors (9). The chemokine receptor CXCR4 was originally cloned and characterized by several groups as an orphan receptor (16,17,30-33). High levels of transcripts were found in leukocytes and in a wide variety of tissues. Additional major interest in the chemokine receptor CXCR4 came from reports on the role of CXCR4 in the pathogenesis of human immunodeficiency virus (HIV) infection. CXCR4 was identified as an infection cofactor for T lymphocyte tropic strains of HIV-1, together with CD4 on mammalian cells. Recent work identified the chemokine SDF-1 as the only known ligand for CXCR4. The function and cellular localization of human SDF-1 has not yet been clearly established. By Northern blot analysis, SDF-1 mRNA expression has been found in almost all organs tested, and is expressed most abundantly in pancreas, spleen, ovary, and small intestine (34). A feature of CXCR4 that is unusual for chemokine receptors is its wide distribution in nonhematopoietic tissues, although currently available data about renal expression of CXCR4 are conflicting. Although one study reported on high CXCR4 transcript levels in total RNA isolated from bovine and rat kidneys (30), another study failed to detect CXCR4 mRNA in total RNA isolated from rhesus macaque kidneys (17). To our knowledge, nothing is known about the potential cellular sites of synthesis of CXCR4 in kidney tissue.

In this study, CXCR4-expressing cells were undetectable by in situ hybridization in renal parenchymal cells of the glomerular, tubular, or vascular compartments. However, human kidneys with features of interstitial inflammation showed large amounts of CXCR4 mRNA-expressing cells. In this setting, CXCR4 mRNA expression was restricted to infiltrating leukocytes.

Recent studies have demonstrated chemokine receptor CXCR4 expression in endothelial cells (35,36). CXCR4 protein and mRNA expression were detected in cultured endothelial cells isolated from human umbilical veins, human coronary arteries, human brain microvasculature, bovine aorta, bovine pulmonary arteries, and additionally in the endothelial cell layer in rabbit thoracic aortic tissue. CXCR4 expression was not demonstrable in vascular smooth muscle cells in these studies. Both studies showed identical CXCR4 expression patterns on the transcript and on the protein level, indicating that CXCR4 mRNA is subsequently translated into protein. In our study, CXCR4 mRNA expression was undetectable in endothelial cells of the renal vasculature. This might in part explain differences in response to injury of the renal vasculature compared to the vasculature of other localizations.

Perhaps the most important new finding of this study is the massive increase of CXCR4 mRNA-expressing leukocytes in rejecting renal allografts within the interstitial and the neointimal compartment. Despite a growing number of reports of elevated expression of different chemokines in the process of allograft rejection, nothing is known about the expression of the corresponding chemokine receptors. Here, we demonstrate that CXCR4-expressing T lymphocytes are involved in human renal allograft rejection. CXCR4 mRNA expression was detectable in many but not all infiltrating T lymphocytes; furthermore, CXCR4 mRNA was expressed in a large group of non-T cells. In the setting of vascular rejection, we demonstrated that CXCR4 mRNA expression by cells other than T cells did not correspond to vascular smooth muscle cells. We believe that CD3-negative, CXCR4 mRNA-positive cells within rejecting allograft tissues are largely of monocytic/macrophage origin, although, because of technical limitations, we were unable to establish combined immunohistochemical labeling for monocyte/macrophage-specific antigens and in situ hybridization for CXCR4 mRNA. However, as we have shown previously that the principal cell populations in the neointimal lesions of rejecting renal arteries in humans are T lymphocytes, monocyte/macrophages, and smooth muscle cells (19), our results in toto would then indicate that monocytes/macrophages will comprise the majority of non-T lymphocyte CXCR4-positive cells in this site of injury. With better definition of chemokine mediated leukocyte trafficking events, we believe new therapeutic approaches in renal allograft rejection using anti-chemokine agents may be envisioned.

Acknowledgments. The authors thank Dr. Nathaniel Landau and the AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH for kindly providing the cDNA encoding human CXCR4 and the different chemokine receptor-transfected cell lines.

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