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J Am Soc Nephrol 14:1336-1348, 2003
© 2003 American Society of Nephrology

Normal Human Kidney HLA-DR–Expressing Renal Microvascular Endothelial Cells: Characterization, Isolation, and Regulation of MHC Class II Expression

Kimberly A. Muczynski*,{dagger}, David M. Ekle*,{dagger}, David M. Coder{ddagger} and Susan K. Anderson*,{dagger}

Departments of *Medicine, {dagger}Pediatrics, and {ddagger}Immunology, University of Washington, Seattle, Washington.

Correspondence to Kimberly A. Muczynski, University of Washington, Department of Medicine and Division of Nephrology, Box 356521, Seattle, WA 98195. Phone: 206-598-6190; Fax: 425-255-5318;


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
ABSTRACT. Human, but not murine, renal peritubular and glomerular capillaries constitutively express class II major histocompatibility (MHC) proteins at high levels in normal human kidney. Expression of class II proteins on renal microvascular endothelial cells (RMEC) makes it available to circulating lymphocytes and imparts a surveillance capacity to RMEC for controlling inflammatory responses. In this report, the co-expression of HLA-DR and the endothelial marker CD31 are used to identify RMEC as a distinct population of cells within a standard renal biopsy using flow cytometry. A three-laser, multicolor flow cytometry analysis using Alexa dyes, developed for characterizing the expression of cell surface antigens, identifies RMEC as a population separate from HLA-DR-expressing leukocytes. HLA-DR RMEC co-express HLA-DP and HLA-DQ. RMEC also express the T cell costimulatory factor CD58 but not CD80, CD86, or CD40. On the basis of high HLA-DR expression, RMEC are isolated for culture using fluorescence-activated cell sorting and magnetic beads. Cultured RMEC require normal basal physiologic concentrations of gamma interferon ({gamma}IFN) to maintain HLA protein expression. This expression is regulated by CIITA, the MHC class II-specific transcription factor. Four tissue-specific promoters have been described for CIITA. In freshly isolated RMEC, RT-PCR and hybridization using specific oligonucleotide probes to CIITA promoter sequences identify only the statin-sensitive {gamma}IFN-induced promoter IV of CIITA. Therefore, the constitutive expression of HLA-DR on RMEC in normal human kidney is located in a position for immune surveillance, depends on basal physiologic concentrations of {gamma}IFN, and may be amenable to regulation with statins. E-mail: kzynski@u.washington.edu


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
MHC proteins are of two classes distinguished on the basis of structure and function. Class I molecules, composed of a polymorphic subunit complexed with {beta}-2-microglobulin, are found on all nucleated cells and present antigenic peptides to CD8+ T lymphocytes; class II molecules, composed of polymorphic {alpha} and {beta} chains, are constitutively expressed on a limited number of cell types (dendritic cells, macrophages, B lymphocytes) and present antigenic peptides to CD4+ T lymphocytes. We recently described an unusual expression of MHC class II proteins in normal human kidneys that is not found in murine kidneys (1). The human MHC class II protein HLA-DR is abundantly expressed on peritubular and glomerular capillary endothelial cells but not on endothelial cells of larger blood vessels of normal kidney. Antibodies to HLA-DR and CD31, a protein highly expressed on endothelial cells, co-localize on peritubular and glomerular cells within sections of kidney tissue, indicating capillary endothelial cell location of HLA-DR. HLA-DR has also been identified on rare scattered circulating leukocytes found within the kidney, but over 98% of the DR identified by immunofluorescence microscopy in kidney cortex is located on capillary endothelial cells (1). We refer to these cells co-expressing HLA-DR and CD31 as renal microvascular endothelial cells (RMEC).

HLA class II molecules with their bound peptides play a pivotal role in directing the immune responses of CD4+ T lymphocytes. Presentation of peptides to unprimed CD4+ T cells in the absence of CD80 and CD86 on the antigen-presenting cell renders T cells unresponsive to bound peptide (2–4); in the presence of these T cell costimulatory factors, the T lymphocytes become activated. Further control of CD4+ T lymphocyte responses comes from the regulated expression of HLA class II molecules. Although constitutive class II expression is limited to a few cell types, most other cells can be induced to express class II with high concentrations of gamma interferon ({gamma}IFN; 100 to 500 units/ml).

Regulation of HLA class II expression occurs at the level of transcription. The three HLA class II isotypes, DR, DP, and DQ, are coordinately transcribed due to common promoters and transcription factors. Transcription factors required for expression of class II genes include CIITA (5–7), RFX-5 (8), RFX-B (9,10), and RFXAP (11). CIITA, the master switch for class II transcription, is a non-DNA binding protein that is specific for class II expression (12). The other transcription factors are DNA binding proteins ubiquitously present in most cells regardless of whether or not they express class II proteins. CIITA, which can be induced with {gamma}IFN, is the specific {gamma}IFN-inducible factor responsible for induced class II expression (6,7). Regulation of constitutive class II expression on RMEC likely depends on CIITA, although a CIITA-independent mechanism for class II expression on endothelial cells that involves NK cells has been described (13).

CIITA itself is highly regulated. Four different promoters and first exons have been sequenced for CIITA that are utilized in a tissue specific manner (14). Constitutive HLA class II expression in dendritic cells utilizes promoter I; B cells express class II genes using CIITA promoter III; and {gamma}IFN-induction of class II genes is associated with CIITA promoter IV expression (14,15). Recent reports also describe constitutive class II expression on melanoma and cortical thymic epithelial cells that is mediated by CIITA promoter IV (16–19).

Characterization of the regulation of RMEC CIITA requires isolating the cells. Classic capillary endothelial cell isolation procedures involved separating the microvasculature from tissue and then allowing cells to grow from the vessels (20). Other investigators have isolated microvascular endothelial cells on the basis of induced expression of cell surface activation markers (21). In this report, we isolate RMEC based on normal basal level co-expression of DR and CD31, a marker of endothelial cells, using fluorescence-activated cell sorting (FACS) and magnetic beads. RMEC lose DR expression after 1 to 2 wk in vitro, but DR expression can be maintained with low concentrations of {gamma}IFN. Similar findings have been reported for microvascular endothelial cells isolated from human heart (22). To characterize RMEC as they exist in vivo without changes that occur in culture, a triple laser flow cytometry analysis is devised to evaluate expression of surface proteins in viable cells from normal kidney tissue. We find that RMEC in vivo express DR via CIITA promoter IV without the T cell costimulatory factors CD80, CD86, and CD40. These observations suggest that RMEC have the potential for mediating peripheral tolerance through DR expression and that statins may provide a pharmacologic means of regulating this immune activity by inhibiting the expression of CIITA promoter IV (23,24).


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Antibodies
Monoclonal antibodies that recognize monomorphic HLA class II determinants of the following specificities were purified and fluorophore-labeled in the laboratory: L243 recognizes DR (25), B7/21 recognizes DP (26), and SPVL3 recognizes DQ (27). FITC conjugation was performed in 0.29 M carbonate/bicarbonate buffer, pH 9.3, and labeled antibody was separated from free FITC using a Sephadex G-25 column. Conjugation to Alexa Fluor dyes (Alexa 568, Alexa 633, Alexa 680) was done with Alexa Fluor Protein Labeling Kits from Molecular Probes (Eugene, OR) according to manufacturer’s instructions. Two monoclonal antibodies to CD31, BD PharMingen (San Diego, CA) clone WM59 and Caltag (Burlingame, CA) clone MBC78.2, both directly conjugated to the same fluorophore (FITC or PE) were used in combination for flow cytometry. Anti-CD45, CD14, CD19, CD80, CD86, CD58, CD141, and HLA-DR (L243) phycoerythrin (PE) conjugates and CD40-FITC were obtained from BD PharMingen. CD105-PE was obtained from Caltag. L243, SPVL3, anti-CD14, and anti-CD58 are IgG2a antibodies; the other antibodies used are all IgG1. Anti-von Willebrand factor (vWF) rabbit serum and FITC or tetramethylrhodamine isothiocyanate (TRITC) conjugated secondary goat anti-rabbit antibodies were obtained from Sigma (St. Louis, MO).

Cells
HUVEC and lung microvascular endothelial cells (LMEC) were purchased from Clonetics and grown in EGM-2MV BulletKit medium (BioWhittaker, Walkersville, MD). Jurkat, a T cell line that does not express CD80, CD86, or CD40; EBV-transformed B-lymphoblastoid cell lines, which express high levels of HLA class II proteins, CD80, CD86, CD40, and CD58; T2, a T and B lymphoblastoid hybrid that is DR-negative on the basis of class II gene deletion (28); ThM, a melanoma line that does not express DR unless induced with {gamma}IFN (29); and HK-2, a human kidney proximal tubule cell line that does not express DR unless {gamma}IFN-induced (30), were grown in RPMI containing 10% bovine calf serum.

Isolated RMEC were grown in Clonetics EGM-2-MV BulletKit medium in a 5% CO2 humidified environment. RMEC grow equally well in GibcoBRL human endothelial-SFM medium (Life Technologies, Gaithersburg, MD) containing either 5% fetal calf serum or 5 to 10% pooled human AB sera.

RMEC Isolation
RMEC were isolated from normal areas of human kidney cortex recovered at the time of nephrectomy for renal cell carcinoma. Donors had normal renal function, no proteinuria, and grossly normal appearing parenchyma in areas unaffected by tumor. Tissue could be stored at 4°C overnight in Hank’s balanced salt solution (HBSS; GibcoBRL catalog number 24020–125) supplemented with 100 U/ml penicillin and 50 µg/ml streptomycin (HBSS) before cell isolation without affecting results.

Cell suspensions used for flow cytometric sorting and magnetic bead isolation were prepared in the same manner using sterile techniques. Excised grossly normal kidney cortex (1 to 10 g) was minced with a scalpel and digested for 1 h at 37°C in 30 ml of 0.2% collagenase P (Roche Molecular Biochemicals, Indianapolis, IN) in HBSS on an orbital shaker. This preparation was centrifuged at low speed (600 to 1000 x g), resuspended in 10 to 20 ml phosphate-buffered saline (PBS) containing 0.05% trypsin and 0.53 mM EDTA (GibcoBRL), and agitated for 10 to 15 min at 37°C. An equal volume of Clonetics EGM-2-MV BulletKit medium was added to neutralize trypsin, and the resulting cell suspension was passed through a 20-mesh sieve to remove remaining large particulates. Recovered cells were then labeled with antibody as described below for isolation of RMEC using flow cytometry or magnetic beads. Cells could also be stored at 4°C overnight before cytometry without loss of viability or change in phenotype.

RMEC were isolated by flow cytometry sorting as follows. Kidney cell suspensions were labeled on ice for 1 h with saturating concentrations of two anti-CD31 antibodies that recognize different CD31 epitopes, both conjugated with the same fluorophore, and with FITC-conjugated or PE-conjugated L243 (anti-HLA-DR) in RPMI 1640 containing 25 mM HEPES, 5% FBS, pH 8.0 (diluent). L243-FITC was used with anti-CD31-PE, and L243-PE with anti-CD31-FITC. Cells were washed with diluent and passed through a nylon strainer (40-µm pore size; Falcon 352340, Becton Dickinson, San Jose, CA; available from Fisher, Pittsburgh, PA). Propidium iodide (PI) was added at a concentration of 5 µg/ml just before cell sorting as a viability indicator. Cell sorting was performed with a FACSVantage SE (Becton Dickinson) equipped with a 70-µm-diameter nozzle tip and an argon laser-emitting 150 mW of 488 nm light. PI-negative cells with the highest level of CD31 and DR expression were collected under sterile conditions in a 15 ml conical polypropylene tube containing Clonetics EGM-2-MV BulletKit medium, centrifuged, and placed in culture with fresh medium.

RMEC were isolated using magnetic beads as follows. Kidney cortical cells prepared above were incubated on ice with saturating concentrations of magnetic anti-CD14- and anti-CD19-coated Dynabeads (Dynal, Oslo, Norway) in diluent for 30 to 60 min and then dropped onto a tilted cell culture dish placed over a magnet. Cells that did not adhere to the magnet were collected, washed, and labeled with L243 in diluent on ice for 30 min. A trace amount of L243-FITC or L243-Alexa 633 was added so the isolation procedure could be assessed by immunofluorescence microscopy. Excess unbound L243 was removed by washing the cells. L243-bound cells were isolated using goat anti-mouse IgG-coated Dynabeads as described above or by using goat anti-mouse Miltenyi microbeads (Miltenyi Biotec, Auburn, CA) according to the manufacturer’s specifications. Cells adherent to the magnetic beads were placed in culture in Clonetics EGM-2-MV BulletKit medium.

Cell Suspensions from Renal Biopsies
Cell suspensions for multicolor flow cytometric analysis were prepared from 16-gauge needle biopsy tissue cores obtained under direct visualization of surgically removed longitudinally bisected kidneys from grossly normal cortex. Four to eight biopsies were taken from each kidney in different areas of normal cortex. Each biopsy contained enough cells for labeling with 6 to 8 different sets of antibodies, with each set consisting of 15,000 to 20,000 cells. Single biopsy cores were placed in 1.5-ml microfuge tubes, digested for 1 h at 37°C with 0.2% collagenase P in HBSS on an orbital shaker, followed by 10 min at 37°C with 0.05% trypsin-0.53 mM EDTA in PBS. Trypsin was neutralized with an equal volume of Clonetics EGM-2-MV BulletKit medium. Cells could be stored overnight at 4°C before labeling with antibody without affecting results. Flow cytometry results of different biopsy tissue cores from the same donor were identical.

Antibody Labeling of Cells for Flow Cytometric Analysis
Cells from renal biopsies or cells prepared for RMEC isolation were labeled with the desired combination of antibodies, each conjugated to a different fluorophore (FITC, PE, Alexa Fluor 633, or Alexa Fluor 680) in RPMI 1640 or diluent containing 0.02% sodium azide on ice. Excess antibody was removed by washing cells. 4',6-diamidino-2-phenylindole (DAPI) was added at a concentration of 1 µg/ml in RPMI as a viability indicator just before analysis with a LSR bench top flow cytometer (Becton Dickinson Biosciences). PI was added at a concentration of 5 µg/ml as a viability indicator with single 488-nm laser instruments.

Flow Cytometry
Single laser flow cytometry was performed as described previously (29).

A three-laser multicolor flow cytometry analysis was developed to characterize cell surface antigens on freshly isolated RMEC to gain information about the in vivo phenotype of the cells. A Becton Dickinson LSR bench top analyzer equipped with HeCd (325 nm), Ar (488 nm), and HeNe (633 nm) lasers was configured to allow discrimination of DAPI, FITC, PE, Alexa 633, and Alexa 680 fluorophores. Specific description of LSR configuration and filters are available upon request. Compensation was adjusted using B lymphoblastoid cells labeled with single L243 conjugates: L243-FITC, -PE, -Alexa 633, or -Alexa 680. A B lymphoblastoid cell line in which HLA class II genes are deleted, T2, was used as a negative control for L243 binding. After compensation, each fluorophore detected the same level of DR expression. Conjugation of purified antibody to FITC, Alexa 633, and Alexa 680 dyes is a straightforward procedure and increases the flexibility of antibody-fluorophore combinations available for use with this 5-color flow cytometry analysis. Only PE conjugates need to be purchased from commercial suppliers.

Cell Quest and WinMDI software were used for data analysis.

Immunofluorescence Microscopy
Immunofluorescence microscopy on human kidney tissue was performed as described previously (1). Cultured RMEC viewed by immunofluorescence microscopy were treated with trypsin to remove them from culture dishes, labeled according to the procedure used for flow cytometry, and then fixed in 1% paraformaldehyde in phosphate-buffered saline. Trypsin does not affect the level of detectable HLA-DR expression. Cells were mounted in Vectashield Mounting Medium with DAPI (Vector Laboratories).

RT-PCR and Southern Blots
Single-stranded cDNA was made from RMEC using cesium chloride gradient isolated total RNA and a Superscript first-strand synthesis system (Invitrogen Life Technologies, Carlsbad, CA). Oligo dT or CIITA oligonucleotide 5'-TCTTGCTGCTGCTCCTCT-3' were used to prime the reverse transcriptase. PCR using the oligo dT-primed cDNA and internal CIITA common primers 5'-CCTGATGCACATGTACTGGGC-3' and 5'-ACGTCCATCACCCGGAGGGAC-3' generated a 711-bp product common to all CIITA. Promoter-specific RT-PCR was performed with cDNA template, a common reverse primer (RP) 5'-GCACCTCACCATGGTAGATGA-3' and a specific forward primer (FP). CIITA III FP sequence used was 5'-GAGGCTAGTGATGAGGCTGTG-3' and CIITA IV FP, 5'-ACTTGCCGCGGCCCCAGAGCT-3'. PCR amplification was performed with 55 cycles of 94°C x 30 s (denaturation), 50°C x 1 min (annealing), and 72°C x 2 min (extension). Glyceraldehyde-3-phosphate dehydrogenase (GADPH) was amplified as an internal control using Stratagene (La Jolla, CA) primers that generated a 600-bp product.

Since CIITA is a 4.5-kb mRNA of low abundance, it is difficult to detect CIITA promoters when fewer than 1 x 106 RMEC are available and cDNA is made from oligo dT primed reverse transcriptase reactions. Therefore, a 1500-bp cDNA PCR template corresponding to the 5' end of CIITA was generated. CIITA promoter RT-PCR product yields are greater using this shortened cDNA template, although identical results are obtained with cDNA generated from oligo dT priming.

Southern blots of RT-PCR products were prepared by standard techniques and hybridized to promoter-specific CIITA oligonucleotides labeled at the 5' end with T4 polynucleotide kinase (New England Biolabs, Beverly, MA) and {gamma}32P-ATP. Oligonucleotide probe for CIITA promoter III sequence was 5'-AGACTCCGGGAGCTGCTGCCTGGCTGGGATT-3'; oligonucleotide probe for CIITA promoter IV was 5'-GGCCACCAGCAGCGCGCGCGGGA-GCCCGGGGAACAG-3'.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Multicolor Flow Cytometry Analysis of Freshly Isolated RMEC
A three-laser multicolor flow cytometry analysis was developed to characterize cell surface antigens on freshly isolated RMEC to gain information about the in vivo phenotype of the cells. The flow cytometric analysis allows the use of CD31 and HLA-DR antibodies to precisely define RMEC within the population of kidney cortex cells while simultaneously using antibodies conjugated to other fluorophores to evaluate the expression of additional RMEC cell surface proteins. We devised a filter system that allows the discrimination of Alexa 633 and Alexa 680 antibody conjugates excited by the far red laser. These fluorophores are used in combination with standard FITC and PE conjugates. DAPI, excited with a UV laser and excluded by cells that maintain membrane integrity, is used as a viability indicator.

Multicolor flow cytometric analysis of viable cells contained within a renal biopsy of normal human kidney cortex reveals two populations of cells expressing CD31 and HLA-DR (Figure 1A). RMEC, which express high levels of CD31 and HLA-DR, do not express CD45, the leukocyte common antigen; the population of cells expressing lower levels of CD31 and HLA-DR also express CD45, which identifies them as leukocytes (Figure 1B). Leukocytes that constitutively express HLA-DR include monocytes/macrophages and B lymphocytes, which can be identified on the basis of CD14 and CD19 expression, respectively. RMEC do not express CD14 and CD19, but a subset of the population of cells expressing low levels of CD31 and HLA-DR expresses CD14 and CD19, consistent with these cells being CD45+ leukocytes of monocytic and B-lymphocytic origin (Figure 1, C and D). The two populations of CD31+/HLA-DR+ cells are not evident by immunofluorescence microscopy of kidney tissue sections (Figure 2A). This may be due to the loss of circulating CD45 expressing leukocytes from tissue during preparation for microscopy or it may be that the levels of CD31 and HLA-DR expression on leukocytes are below the level of detection by immunofluorescence microscopy. For example, flow cytometry detects cells with low level HLA-DR expression that are undetectable by immunofluorescence microscopy (Figure 3A).



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  		Figure 1. Multicolor flow cytometry of normal human kidney cortical cells from a representative renal biopsy identifies two populations of cells co-expressing HLA-DR and CD31. Renal microvascular endothelial cells (RMEC), the cells with the highest expression of HLA-DR and CD31 (region R2; A and F), do not express CD45 (B), CD14 (C), or CD19 (D) (solid lines), but they co-express the other HLA class II proteins, HLA-DQ and HLA-DP (E through G). Cells within region R3 (A), which express lower levels of CD31 and HLA-DR, also express CD45 (B), CD14 (C), and CD19 (D) (dotted lines). (E) FSC versus 4',6-diamidino-2-phenylindole (DAPI) fluorescence dot plot of total cells within a renal biopsy. Viable cells (DAPI-negative) are indicated in region R1. (A and F) CD31 versus HLA-DR dot plots of viable cells within a renal biopsy. (G) HLA-DP versus HLA-DQ dot plots of viable RMEC in regions R1 and R2. Grid is set based on labeling of T2, a cell line in which HLA class II genes are deleted. Fluorophores: anti-CD31-FITC or -PE as indicated; L243 (anti-HLA-DR)-Alexa 680; anti-CD45-, CD14-, or CD-19-PE; SPVL3 (anti-HLA-DQ)-Alexa 633; B7/21 (anti-HLA-DP)-FITC. Histograms (B through D) are normalized for cell number.

 


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  		Figure 2. (A) HLA-DR expression on normal human kidney microvascular endothelial cells. Immunofluorescence microscopy of normal human kidney cortex labeled with monoclonal antibodies to CD31, a marker of endothelial cells (FITC-conjugated, green) and HLA-DR (L243-Alexa 568–conjugated, red). Co-localization of CD31 and HLA-DR on peritubular and glomerular capillary endothelial cells appears in yellow. T, proximal tubule lumen; G, glomerulus; Scale bar, 50 µm. (B) Flow cytometry bivariate dot plot of viable cells within human kidney cortex labeled with anti-CD31-PE and L243-FITC. RMEC (shown in the yellow region) express high levels of CD31 and HLA-DR, corresponding to the capillary endothelial cells that show co-localization of CD31 and HLA-DR in panel A. RMEC recovered by flow cytometry sorting are placed in culture. (C) Light microscopy of cultured RMEC recovered from the yellow region in panel B. Scale bar, 50 µm.

 


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  		Figure 3. Expression of cell surface endothelial markers and T cell costimulatory factors on freshly isolated and cultured RMEC. (A) Immunofluorescence microscopy and flow cytometry histograms of gamma interferon ({gamma}IFN)–induced (5 units/ml) HLA-DR on cultured RMEC isolated from a single representative donor. Immunofluorescence microscopy detects HLA-DR expression after 24 h of {gamma}IFN induction. Scale bar, 25 µm. HLA-DR is detected 12 h after {gamma}IFN induction by flow cytometry. Uninduced RMEC (dotted gray line); RMEC after 12 h (dotted black line), 24 h (bold gray line), and 48 h (bold black line) of {gamma}IFN induction. In vivo refers to HLA-DR expression on RMEC as they are isolated from kidney cortex by fluorescence-activated cell sorting (FACS) and is included as a reference (shaded gray histogram). RMEC are labeled with L243-FITC. Endothelial cell markers, CD31 (B), CD105 (C), and CD141 (D), and T cell costimulatory factors, CD80 (E), CD86 (F), CD40 (G), and CD58 (H) expression by flow cytometry. Shaded gray histograms represent freshly isolated RMEC prior to culture. (B through D) Bold gray and black lines are RMEC from two representative donors. In panel B, controls include B lymphoblastoid cells (dotted gray line), a negative control; cultured HUVEC (fine solid black line); and cultured lung microvascular endothelial cells (LMEC; dotted black line). (E through H) Cultured RMEC from a representative donor (bold gray lines). Controls include B-lymphoblastoid cells (black lines), which are capable of presenting antigen and activating T cells, express CD80, CD86, CD40, and CD58; Jurkat (dotted black lines) expresses CD58 but not CD80, CD86, or CD40. Only viable cells are included in the histograms.

 
Our initial immunofluorescence microscopy characterization of HLA class II proteins on RMEC described HLA-DR expression without the other HLA class II isotypes, DP and DQ (1). This was unusual given that HLA class II proteins are coordinately regulated through common promoters and transcription factors, including one transcription factor specific for class II genes, CIITA. Among cells that express HLA class II proteins on their surface, HLA-DP and HLA-DQ are generally of lower abundance than HLA-DR. Therefore, one possible explanation for the apparent lack of HLA-DP and HLA-DQ on RMEC by immunofluorescence microscopy is that the level of expression is below the detectable limit of this technique. Using multicolor flow cytometric analysis, which is more sensitive for detecting low-level fluorescence, RMEC from normal kidney cortex are found to express HLA-DR, HLA-DP, and HLA-DQ (Figure 1, E through G), although the level of DQ expression has been variable for the donors studied. This is consistent with HLA class II protein expression in normal human RMEC being regulated by the mechanisms reported in simpler cell culture systems and indicates that RMEC have the potential for presenting antigen peptides via all three HLA class II isotypes.

Isolation of RMEC Co-Expressing CD31 and HLA-DR
The abundant and relatively restricted expression of HLA-DR on RMEC in normal kidney allows isolation of RMEC using antibodies to HLA-DR (Figure 2). Single cell suspensions prepared from normal human kidney cortex using collagenase and trypsin are labeled with antibodies to HLA-DR and CD31, each conjugated with a different fluorophore. L243, a monoclonal antibody that recognizes an epitope common to all DR alleles, is used so that HLA-DR polymorphism does not affect RMEC isolation. Viable cells with the highest expression of both HLA-DR and CD31 are selected by flow cytometry for culture (Figure 2B). Cells are sorted at a rate of 5000 to 8000 events per second. Two to three percent of the total sorted cells are collected as RMEC, and purity is greater than 98%. Isolated RMEC grow well in culture with commercially available media (Figure 2C).

RMEC can also be isolated on the basis of HLA-DR expression using magnetic beads coated with an anti-mouse IgG antibody. However, cell suspensions derived from whole kidney cortex also contain circulating HLA-DR expressing monocytes, macrophages, and B lymphocytes; therefore, these cells must be removed before labeling with L243. Anti-CD14 and anti-CD19 beads remove monocytes/macrophages and B lymphocytes, respectively. Kidney cortical cell suspensions cleared of CD14- and CD19-expressing cells are labeled with L243 to isolate the remaining HLA-DR–expressing cells, which are the RMEC. Ig-coated magnetic beads from Dynal and Miltenyi work equally well for isolating RMEC. The purity of RMEC isolated by magnetic bead immunoselection is greater than 95%. The morphology and phenotype of the cultured cells are identical to RMEC isolated by flow cytometry. Greater numbers of viable cells are recovered using magnetic bead isolation techniques.

Phenotype of Cultured versus Freshly Isolated RMEC
Cultured RMEC isolated by either flow cytometry or magnetic beads maintain a uniform phenotype that varies little between donors, although the expression of some surface antigens differ from that observed on tissue and freshly isolated RMEC. Cultured RMEC lose expression of HLA class II proteins after 7 to 10 d unless {gamma}IFN is maintained in the media. RMEC that lose HLA-DR expression in culture can be induced to re-express DR with {gamma}IFN (Figure 3A). The superior sensitivity of flow cytometry over immunofluorescence microscopy for detecting low levels of surface protein expression is also apparent in Figure 3A; low levels of HLA-DR are readily detected by flow cytometry 12 h after addition of {gamma}IFN, whereas immunofluorescence microscopy does not identify HLA-DR expression until levels are higher at 24 h.

CD31 cell surface expression is also diminished on cultured RMEC compared with levels present at the time of cell isolation (Figure 3B), and it is not restored by {gamma}IFN. The low level of CD31 expression in cultured RMEC distinguishes these cells from other primary human endothelial cell lines such as human umbilical vein endothelial cells (HUVEC) and lung microvascular endothelial cells (LMEC), which retain high levels of cell surface CD31 protein in culture (Figure 3B). Despite the low CD31 expression in cultured RMEC, persistence of CD31 mRNA encoding both extracellular and cytoplasmic domains of the protein at a level similar to that in other cultured endothelial cells can be demonstrated by RT-PCR (data not shown).

RMEC express other surface proteins characteristic of endothelial cells. Endoglin (CD105), a receptor for transforming growth factor–{beta} (TGF-{beta}) (Figure 3C), and thrombomodulin (CD141) (Figure 3D) are expressed on the cell surface of cultured RMEC at levels similar to that in vivo. RMEC from some donors have two populations of cells with respect to CD141 expression: one that lacks CD141 and represents about one third of the RMEC within the population and another that expresses CD141 at the level shown in Figure 3D. The significance of the two CD141-expressing RMEC populations is a subject of current investigation.

HLA-DR+/CD31+ RMEC are heterogeneous with respect to vWF expression in kidney tissue, freshly isolated and cultured RMEC (Figure 4). Some RMEC express vWF (Figure 4, A through F, yellow arrows; K through N), while others do not (Figure 4, G through J), and this heterogeneity in vWF is maintained in cultured cells (Figure 4, O and P). RMEC vWF heterogeneity has been a consistent observation in all donors examined.



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  		Figure 4. Expression of von Willebrand factor (vWF) is heterogeneous in tissue (A through F), freshly isolated RMEC (G through N), and cultured RMEC (O and P). Yellow arrows indicate RMEC expressing HLA-DR and vWF (A through C) or CD31 and vWF (D through F) in tissue. RMEC freshly isolated by magnetic beads co-express HLA-DR and CD31 (G, I, and J; K, M, and N), and some also express vWF (L and N). Cultured RMEC that express vWF (O and P) can have a diffuse intracellular pattern or discrete intracellular structures resembling Weibel-Palade bodies (P). Pseudocolors used: A, D, G, and K are in green channel; B, E, H, and L are in red channel; I and M are in blue channel; DAPI nuclear label in O and P are in red channel. Scale bars: 50 µm in A through F; 20 µm in G through N; 20 µm in O; 30 µm in P.

 
Antigen-presenting cells require costimulatory molecules to initiate T cell responses. We evaluated cultured RMEC from seven donors for the presence of costimulatory proteins involved in lymphocyte activation and found consistent results for cells from all donors. Cultured RMEC do not express CD80 (B7–1) or CD86 (B7–2), but CD40 and CD58 (LFA-3) are expressed at levels comparable to other antigen-presenting cells (Figure 3, E through H). Similar results are found for the expression of CD80, CD86, and CD58 on freshly isolated RMEC (Figure 3, E, F, and H). CD40 is not detectable on freshly isolated RMEC from most donors but is induced when the cells are cultured (Figure 3G).

Low Physiologic Concentrations of {gamma}IFN Are Necessary and Sufficient to Maintain RMEC HLA-DR Expression In Vitro
In most cells, HLA-DR expression can be induced with prolonged exposure to high concentrations of {gamma}IFN. Cultured RMEC are unusual in that their HLA-DR expression can be induced with very low concentrations of {gamma}IFN. RMEC express HLA-DR after 36 h of culture in 0.5 units/ml {gamma}IFN (Figure 5A), a concentration equivalent to a normal measured serum level of 15 pg/ml of {gamma}IFN. In contrast, HK-2 cells, derived from human kidney proximal tubule cells (30), require prolonged exposure to 500 units/ml {gamma}IFN to induce similar levels of DR expression (Figure 5B). The induction of HLA-DR expression on cultured RMEC with basal physiologic concentrations of {gamma}IFN suggests that the constitutive in vivo expression of HLA-DR observed on RMEC in human kidney tissue may depend upon circulating or locally produced {gamma}IFN.



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  		Figure 5. {gamma}IFN induction of HLA-DR on cultured RMEC and HK-2. FACS histograms of cultured RMEC (A) and HK-2 (B) grown in indicated concentrations of {gamma}IFN (units/ml) and labeled with L243-FITC. Cells were grown with {gamma}IFN for 36 h, except where indicated (HK-2 grown 1 wk in 500 units/ml {gamma}IFN). Only viable, PI-negative cells are included in the histograms. Solid thin gray line, no {gamma}IFN; bold black line, 0.5 units/ml {gamma}IFN; bold gray line, 2 units/ml {gamma}IFN; dotted black line, 5 units/ml {gamma}IFN; dashed black line, 500 units/ml {gamma}IFN; solid thin black line, 500 units/ml {gamma}IFN for 1 wk (HK-2 only).

 
Statin-Sensitive CIITA Promoter IV Transcription in Freshly Isolated RMEC
The loss of HLA-DR expression in cultured RMEC and its restoration by basal physiologic concentrations of {gamma}IFN suggested that the {gamma}IFN-inducible CIITA promoter IV might mediate in vivo RMEC HLA-DR expression. RMEC, freshly isolated from single donors using magnetic microbeads to select for HLA-DR–expressing cells, were processed for RT-PCR. Primers to a common internal sequence of CIITA amplified the appropriately sized RT-PCR product in all donors evaluated (Figure 6B, lane 4). Promoter-specific CIITA primers (Figure 6A) amplify only CIITA promoter IV from freshly isolated RMEC (Figure 6B, lane 12). Primers for CIITA promoter I (data not shown), II (data not shown), and III (Figure 6B, lane 8) do not generate RT-PCR products from RMEC cDNA.



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  		Figure 6. CIITA RT-PCR of freshly isolated RMEC. (A) The four different promoters and first exons for CIITA are diagrammed indicating location of the specific forward primers (FP) and a common reverse primer (RP) used to detect the promoter-specific CIITA. The predicted nucleotide lengths of the promoter-specific CIITA RT-PCR products are indicated. Promoter-specific RT-PCR products were amplified from cDNA generated with oligo dT or with an oligonucleotide common to all CIITA, approximately 1500 bp from the 5'-end of the CIITA mRNA (RT nt). Locations of promoter III-specific and promoter IV-specific oligonucleotides used for the southern blots (C and D) are indicated (III nt*; IV nt**). Sequence of the diagrammed oligonucleotides appears in Materials and Methods. RT, reverse transcriptase. (B) CIITA internal primers (lanes 1 through 4) amplify a product common to all CIITA in cells that express HLA class II genes (lanes 2 through 4). A forward primer specific for promoter III CIITA and a common reverse primer (lanes 5 through 8) amplify a 904-bp product specific for CIITA promoter III in B lymphoblastoid cells (lane 7). A forward primer specific for promoter IV CIITA and a common reverse primer (lanes 9 through 12) amplify a 801-bp product specific for CIITA promoter IV in {gamma}IFN-induced HK-2 (lane 10) and RMEC freshly isolated from kidney (lane 12). GAPDH amplification (lanes 13 through 16) serves as a positive control for intact cDNA and active polymerase. Oligo dT–generated cDNAs were used for the RT-PCR. HK-2 (lanes 1, 5, 9, 13); HK-2 + {gamma}IFN (lanes 2, 6, 10, 14); B lymphoblastoid cells (lanes 3, 7, 11, 15); freshly isolated RMEC (lanes 4, 8, 12, 16). (C and D) CIITA promoter southern blots confirm the presence of CIITA promoter IV in freshly isolated RMEC. CIITA RT-PCR products generated with primers to promoter III (C) or promoter IV (D) were transferred to a nylon membrane and hybridized with radiolabeled promoter-specific oligonucleotide probes (A). *III and **IV indicate specificity of promoter probe used for hybridization. ThM, negative control for CIITA expression (lanes 1, 5); ThM + {gamma}IFN, positive control for CIITA promoter IV expression (lanes 2, 6); B lymphoblastoid cells, positive control for CIITA promoter III expression (lanes 3, 7); freshly isolated RMEC (lanes 4, 8). CIITA cDNAs used for the RT-PCRs were generated from a common CIITA-specific oligonucleotide primer.

 
Southern blots confirm that the RMEC RT-PCR products are actually CIITA promoter IV. Freshly isolated RMEC and control {gamma}IFN-induced ThM, a melanoma cell line that requires {gamma}IFN for class II expression (29), generate RT-PCR products with promoter IV primers that hybridize to an oligonucleotide specific for promoter IV (Figure 6D, lanes 6 and 8) but not to an oligonucleotide specific for promoter III (Figure 6D, lanes 2 and 4). These cells do not generate RT-PCR products with primers to CIITA promoter III (Figure 6C, lanes 2, 4, 6, and 8). Results were consistent in all eight kidney donors evaluated, i.e., only CIITA promoter IV is detected in freshly isolated RMEC. Thus, "constitutive" RMEC HLA-DR expression in vivo is associated with CIITA promoter IV expression.

cDNA from a B lymphoblastoid cell line generates a major RT-PCR product with CIITA promoter III primers that hybridizes to a promoter III, but not promoter IV, oligonucleotide (Figure 6C, lanes 3 and 7) and a minor RT-PCR product with CIITA promoter IV primers that hybridizes to a promoter IV oligonucleotide (Figure 6D, lane 7). The significance of the two hybridizing products for each RT-PCR reaction is unknown but has been a consistent observation.

Statins have recently been reported to inhibit {gamma}IFN-inducible HLA class II expression in endothelial cells by their action on CIITA promoter IV (23,24). We evaluated atorvastatin for its effect on {gamma}IFN-induced HLA-DR expression in cultured RMEC; it inhibits RMEC HLA-DR expression at a concentration as low as 1 µM (Figure 7).



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  		Figure 7. Atorvastatin inhibition of {gamma}IFN-induced HLA-DR on cultured RMEC. Flow cytometry histograms of cultured RMEC grown without {gamma}IFN (solid gray line), with {gamma}IFN (dotted gray line), with {gamma}IFN + 1 µM atorvastatin (dotted black line), and with {gamma}IFN + 10 µM atorvastatin (bold black line). {gamma}IFN concentration was 10 units/ml. Cells were evaluated 48 h after addition of reagents and only viable, PI-negative cells are included in histograms.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Flow cytometry is a valuable technique for studies of circulating cells. In this report, we extend the application of the technique to solid tissue. Noteworthy is the development of a protocol for reliably assessing the phenotype of viable cells present in a standard renal biopsy with five different fluorophores, three of which are easily conjugated to any purified antibody. The protocol we use for sample preparation allows all cells within a biopsy to be evaluated, whereas in immunofluorescence microscopy cells and antigens can be lost in tissue preparation. Furthermore, the ability to select specific populations of cells reveals information that might not otherwise be obvious. For example, RMEC constitute about 5% of the total cells within a renal biopsy. Multicolor flow cytometry allows RMEC to be precisely defined by their CD31 and HLA-DR co-expression while simultaneously assessing their expression of other surface proteins that may contribute to renal inflammation, such as the TGF-{beta} receptor, thrombomodulin, and T cell costimulatory factors. The techniques reported here should allow a more precise assessment of inflammatory cell types and altered expression of surface proteins on parenchymal cells obtained from routine renal biopsies. The number of cells obtained, the flexibility of fluorochrome choice, and the ease and reproducibility of sample preparation mean that these techniques may be useful for assessing renal inflammation in native and transplanted kidneys. This in turn should provide a more rational basis for evaluating the effect of immunosuppressants therapy.

Flow cytometry produces a pure CD31+/HLA-DR+ population of cells; we therefore regard the loss of CD31 and HLA-DR expression in cultured RMEC as a phenotypic change rather than an overgrowth of contaminating non-endothelial cells. In addition, the inability to identify HLA-DR expressing CD14 and CD19 cells within the RMEC population with multicolor flow cytometry indicates that DR-expressing RMEC are not of myelomonocytic or B-lymphocytic origin. The potential for isolating cells other than RMEC is increased when isolation is based on Ig-coated magnetic bead selection of CD14-depleted and CD19-depleted HLA-DR–expressing cells, yet RMEC isolated by this technique have the same morphology and phenotypic characteristics as RMEC isolated by flow cytometry. This is a useful result because RMEC isolation by magnetic bead techniques generates a greater quantity of viable cells which can be used for study without in vitro alterations.

Endothelial cells are known to be phenotypically heterogeneous in a manner that varies not only with vessel size (31,32) but also within microvascular populations (33–37). Our results support this: RMEC constitutively express HLA-DR in vivo, whereas endothelial cells from larger vessels do not; lower levels of CD31 on cultured RMEC distinguish them from HUVEC and cultured human lung microvascular endothelial cells; vWF expression is variable within a population of RMEC that otherwise have a uniform phenotype. Cardiac microvascular endothelial cells have a phenotype similar to RMEC with respect to HLA-DR expression in that endothelial cells of small but not large cardiac vessels express HLA-DR; and HLA-DR expression on isolated cardiac microvascular endothelial cells is lost after 2 wk of culture (22,38).

The capacity of RMEC to effectively present antigenic peptides resulting in T cell activation cannot be predicted from the costimulatory factors evaluated in this study. Whereas CD80 and CD86, which provide the major costimulation for unprimed T cell responses, are lacking on RMEC, CD58, which has been associated with HLA-DR–initiated T cell activation in allogeneic assays with cardiac microvascular endothelial cells (39), is abundant on RMEC. CD40 is not normally expressed on RMEC in vivo, but a low level of CD40 is consistently induced by unknown factors in our cultured RMEC. Studies are underway to evaluate the role of RMEC in presentation of antigenic peptides. Such information will be useful for determining whether HLA-DR, expressed so abundantly on RMEC, contributes to inflammation or tolerance in human kidneys. Knowledge of the role of RMEC HLA-DR will influence how DR levels should be manipulated to curtail renal inflammation.

Freshly isolated RMEC express all three HLA class II isotypes, DR, DP, and DQ, although the level of HLA-DQ expression varies with the donor. For other cultured parenchymal cells we have evaluated that require {gamma}IFN induction to express class II (renal proximal tubular cells, mesangial cells, and melanoma cells), HLA-DQ is detected only after prolonged high concentrations of {gamma}IFN; HLA-DR and HLA-DP are generally expressed within 48 h. It may be that the variable levels of HLA-DQ expressed on RMEC in vivo reflect variable concentrations of circulating {gamma}IFN in the donors.

CIITA mRNA is present in freshly isolated human renal peritubular and glomerular capillary endothelial cells, where its encoded protein likely mediates HLA-DR expression. Therefore, a CIITA-independent mechanism for HLA class II expression, as had been described for endothelial cells (13), does not need to be invoked to explain the in vivo expression of RMEC HLA-DR.

Although HLA-DR expression on RMEC appears constitutive in normal human kidneys, only the promoter IV form of CIITA, associated with {gamma}IFN induction of class II genes, is amplified from freshly isolated RMEC-derived cDNA. This suggests that RMEC HLA-DR expression may require circulating or locally produced {gamma}IFN. We do not believe the tumors present in the kidneys used in this study provided this {gamma}IFN, because RMEC from kidneys with benign masses also expressed HLA-DR. In addition, our studies with cultured RMEC indicate that in vitro induction of CIITA and HLA-DR can be achieved with basal physiologic concentrations of {gamma}IFN that are normally present in circulating blood.

Our studies examine the regulation of HLA-DR expression only in normal human kidneys. Under conditions of renal inflammation, including glomerulonephritis and acute rejection of transplanted kidneys, HLA-DR is also expressed on renal proximal tubule cells (1,40–42). HLA-DR regulation has not been evaluated in these states of renal inflammation. Cell culture studies with the HK-2 proximal tubule cell line, and similar studies with primary cultures of renal proximal tubule cells, show that HLA class II expression can be induced with prolonged high concentrations of {gamma}IFN. It may be the level of {gamma}IFN that determines which cells in the kidney express class II proteins. This would imply the existence of a {gamma}IFN sensing "meter" to regulate class II transcription. The duration of activation of Stat1, which transduces the {gamma}IFN signal leading to CIITA transcription, might be such a meter (43). Consistent with the hypothesis that {gamma}IFN levels determine in vivo HLA-DR expression is the finding of increased {gamma}IFN mRNA levels in rejecting transplanted kidneys where proximal tubule cells express class II (44).

Alternatively, HLA class II induction on renal parenchymal cells other than RMEC may require cytokines in addition to {gamma}IFN, e.g., tumor necrosis factor alpha (TNF-{alpha}). TNF-{alpha} synergizes with {gamma}IFN to increase class II expression (45,46) but has no effect on CIITA promoter activity (47). Elevated serum levels of TNF-{alpha} appear 2 to 3 d before acute rejection of transplanted kidneys (48). While the regulatory mechanism of induced HLA-DR expression under conditions of renal inflammation remains speculative, the absence of HLA-DR expression on tubule cells in normal kidney tissue indicates that the local concentration of {gamma}IFN sensed in normal kidneys is low, yet adequate, for RMEC HLA-DR expression.

The in vivo use of CIITA promoter IV for RMEC HLA-DR expression raises the possibility that statins could provide a pharmacologic means for regulating RMEC class II. We demonstrate that the HMG-CoA reductase inhibitor atorvastatin reduces {gamma}IFN-induced RMEC DR expression in vitro; however, our donor population did not allow us to answer the question of whether or not statins affect RMEC HLA-DR expression in vivo, because none of the donors were taking statins at the time of nephrectomy. Statins have been associated with a reduced rate of rejection in kidney transplants (49,50), although the mechanism for this effect has not been delineated. Although it is possible that statins may affect HLA class II expression on RMEC, the benefit of this under normal physiologic conditions depends on whether or not RMEC are capable of promoting an inflammatory response. It is possible that the value of statins in reducing kidney rejection may be in controlling the induced expression of HLA class II proteins on non-RMEC that occurs with renal inflammation.


   Acknowledgments
 
Funding for this work was provided by grants to K. A. Muczynski from the Northwest Kidney Centers (Seattle, WA) and the Atorvastatin Research Awards, and by NIH RO1 AI30527 awarded to Tom Cotner. We are indebted to the urologists of the University of Washington Medical Centers for their assistance with procurement of renal tissue.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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Received for publication May 25, 2002. Accepted for publication January 30, 2003.




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