Identification and Functional Characterization of Dendritic Cells in the Healthy Murine Kidney and in Experimental Glomerulonephritis
Thilo Krüger*,
Dirk Benke,
Frank Eitner*,
Andreas Lang*,
Monika Wirtz*,
Emma E. Hamilton-Williams,
Daniel Engel,
Bernd Giese,
Gerhard Müller-Newen,
Jürgen Floege* and
Christian Kurts
Departments of *Nephrology and Clinical Immunology and Biochemistry, University Clinic of Aachen, Aachen, Germany; and Institute of Molecular Medicine and Experimental Immunology, University Clinic of Bonn, Bonn, Germany.
Correspondence to Dr. Christian Kurts, Institute of Molecular Medicine and Experimental Immunology, Friedrich-Wilhelms-Universität, 53105 Bonn, Germany. Phone: 49-228-287-1031; Fax: 49-228-287-1052; E-mail: ckurts{at}web.de
ABSTRACT. The kidney tubulointerstitium contains numerous bonemarrow-derived antigen-presenting cells, which are often referredto as resident tissue macrophages, although several previousstudies had demonstrated characteristics of dendritic cells(DC). In this study, we describe a subset of tubulointerstitialcells expressing the DC marker CD11c. A protocol was establishedto isolate these cells for in vitro analysis. Renal CD11c+ cellsresembled splenic DC, but not peritoneal macrophages, in morphology,lysosomal content, phagocytic activity, microbicidal effectorfunctions, expression of T cell costimulatory molecules, andability to activate T cells. Nevertheless, many CD11c+ renalcells expressed low or intermediate levels of F4/80 and CD11b,indicating that both markers are not absolutely specific formacrophages in the kidney. Subpopulations of renal DC couldbe distinguished based on their expression of MHC class II andcostimulatory molecules and may represent different maturationstages. In nephrotoxic glomerulonephritis, increased numbersof CD11c+ cells showing DC functionality were found in the tubulointerstitium.Focal accumulation was seen within tubulointerstitial mononuclearinfiltrates and adjacent to, but not within, inflamed glomeruli.These results are the first to identify and characterize renalCD11c+ cells as DC and to demonstrate marked changes in experimentalglomerulonephritis.
Interest in the tubulointerstitial compartment is growing asa result of its pivotal role in the progression of renal disease(1). The cells residing in this compartment are mostly fibroblastsand bone marrow-derived cells of the immune system (2,3). Thetubulointerstitial immunocytes have been shown to express MHCclass II molecules, indicating that they are professional antigen-presentingcells (APC), i.e., macrophages or dendritic cells (DC). Twentyyears ago, before the importance of the extensive system ofDC was fully appreciated, the tubulointerstitial immunocyteswere found to express the F4/80 molecule, then assumed to bea macrophage marker (4), resulting in their classification astissue macrophages. Although F4/80 has recently been molecularlycharacterized (5), its function remains unresolved. It has beenshown to be expressed also by some DC subsets (6), in particularthose representing earlier differentiation stages (7). Mostmurine DC populations specifically express the integrin chainCD11c (8). Previous studies have failed to detect CD11c+ cellsin the kidney (9,10). Macrophages lack CD11c but possess CD11b,also known as Mac-1. Although CD11b is often used to identifymurine macrophages by immunohistochemistry, it is known to beexpressed also by granulocytes and DC subsets (8). These andmany other DC subtypes have been defined by their expressionof various combinations of cell surface markers, but it is unclearwhether they represent individual cell types with distinct lineagesor differentiation stages of the same cell. Thus, the parametersthat unequivocally identify DC remain their morphology and theirfunctionality.
A careful morphologic analysis demonstrated by electron microscopythat tubulointerstitial cells possess features of DC, whereascells with macrophage morphology were confined to the capsule,the intravasal lumina, and the pelvic wall of healthy kidneys(2). The functionality of these DC-like tubulointerstitial cellswas not addressed. The principal DC function is the inductionof primary immune responses (11). Their precursors are producedin the bone marrow and then released into the blood stream (12,13),from which they enter tissues. Immature DC, the Langerhans cellin the skin being the best-studied example, capture antigenin peripheral tissues and sense "danger" signals (pathogens,tissue damage, local inflammation), which trigger their maturation.Maturation downregulates the phagocytic activity of DC but upregulatesthe molecular machinery for T cell activation (e.g., MHC andcostimulatory molecules) and induces their migration to draininglymph nodes (12). There, DC present antigens to specific T cells.These DC functions, however, can principally be performed byall classes of professional APC, albeit at lesser efficacy.For example, macrophages are most effective at phagocytosis,but far less effective at T cell activation, than DC. Typicalmacrophage effector functions, such as oxidative burst or nitricoxide (NO) production, are normally not performed by DC. Inthe present study, we demonstrate a hitherto undescribed kidneycell population expressing DC and macrophage markers and characterizethem in the healthy and inflamed kidney as DC.
Mice, Reagents, and the Nephrotoxic Nephritis Model
Female C57/BL6 and Balb/c mice 6 to 8 wk old in specific pathogen-freeconditions were purchased from Bomholtgard (Denmark). All experimentswere in accordance with local animal ethics procedures. Reagentswere from Sigma-Aldrich (Steinheim, Germany) if not specifiedotherwise. Nephrotoxic nephritis (NTN) was induced by injectionof 6 µl of nephrotoxic sheep serum per gram of mouse bodyweight as described (14,15).
Immunohistology
For immunohistochemistry, tissues were fixed for 3 d in Immunohistofix(Intertiles, Bruxelles, Belgium) and then embedded in Immunohistowax(Intertiles) at 37°C according to the manufacturer’sinstructions (16). Dewaxed sections were incubated with 3% H2O2,BR blocking reagent (Roche, Mannheim, Germany), and Fc-Block®(Pharmingen, Heidelberg, Germany). Primary monoclonal antibodieswere biotinylated anti-CD11c (clone HL-3), anti-Iab (AF6-120.1),anti-CD11b (Mac-1) (all from Pharmingen), and anti-F4/80 (CI:A3-1;Caltag, CA). As isotype controls, biotinylated antibodies againstirrelevant antigens were used (Pharmingen). Bound antibody wasrevealed with the Vectastain ABC kit (Vector Laboratories, Burlingame,CA) and 3,3'-diaminobenzidine (DAB). Slides were counterstainedwith methyl green.
Preparation of Splenic DC, Renal CD11c+ Cells, and Peritoneal Macrophages
Previously described methods for APC isolation from spleens(17,18) were modified to isolate CD11c+ cells from murine kidneys.Kidneys were finely minced and digested for 45 min at 37°Cwith 2 mg/ml collagenase D (Roche) and 100 µg/ml DNAseI in RPMI 1640 medium (Life Technologies, Karlsruhe, Germany)in 10% heat-inactivated FCS (PAA Laboratories, Linz, Austria)containing 10 mmol/L Hepes. Cell suspensions were filtered through30-µm nylon mesh, washed with HBSS without Ca2+ and Mg2+(Life Technologies) containing 10 mmol/L EDTA, 0.1% BSA, and10 mM Hepes, and adjusted with NaCl to mouse tonicity. A densitycentrifugation was performed at 1700g for 20 min at 4°Cusing 1.080 g/ml Nycodenz (Axis Shield, Oslo, Norway). The interphasecells were harvested, Fc receptors were blocked with Fc-Block®(Pharmingen), and CD11c+ cells were isolated with microbead-labeledspecific monoclonal antibodies (clone N418) (Miltenyi, Bergisch-Gladbach,Germany). Magnetic bead separation was done according to themanufacturer’s instructions.
Splenic DC were isolated using previously described methods(17,18) and enriched with microbead-labeled anti-CD11c (Miltenyi).Peritoneal macrophages were obtained by flushing the peritonealcavity with HBSS without Ca2+ and Mg2+ containing 10 mM EDTAand 0.1% BSA. For most experiments, macrophages were furtherenriched with microbead-labeled anti-CD11b (Miltenyi).
Flow Cytometry
After treatment with Fc-Block® (Pharmingen), isolated cellswere stained for 15 min on ice with fluorochrome-labeled monoclonalantibodies specific for CD11c, I-Ab, CD11b, CD80 (16-10A1),CD86 (PO3) (all from Pharmingen), and F4/80 (Caltag). For stainingof lysosomes, cells were cultured for 15 min with Lysotracker®green (Molecular Probes, Leiden, Netherlands) in culture medium.Dead cells were always excluded with propidium iodide (PI).Multicolor flow cytometry was performed on a Becton DickinsonFACScalibur®.
Phagocytic Activity
Isolated cells (1 x 105) were cocultured with 1 mg/ml FITC-labeleddextran (40,000 kD; Molecular Probes) for 45 min. As a controlfor nonspecific dextran attachment, 0.02% azide was added orcells were cultured at 4°C to stop energy-dependent cellularfunctions. To determine phagocytic activity, the uptake of fluorescencebeads was detected by multicolor flow cytometry.
NO Release
NO production was measured as described (19). Briefly, 1 x 105cells were incubated in 200 µl of culture medium withoutphenol red containing 10 ng/ml IFN and varying LPS concentrations.After 48 h, 50 µl of 14 mmol/L 4'4-diaminodiphenylsulfoneand 50 µl of 4 mmol/L N-ethylenediamine were added tothe culture supernatant, and NO was detected by measuring theOD at 560 nm on a Dynatech MR5000 reader (Dynatech, Chantilly,VA).
Alloresponse
Single cell suspensions of lymph nodes and spleens of Balb/cmice were enriched for T cells by nylon wool adhesion or negativeselection using a T cell isolation kit (Miltenyi). T cell puritywas typically >95%. T cells (10 x 106) were incubated for8 min at 37°C with 5 µmol/L 5,6-carboxyfluoresceinsuccinimidyl ester (CFSE; Molecular Probes) in RPMI 1640 culturemedium (Life Technologies) containing 10% heat-inactivated FCS,50 µmol/L 2-mercaptoethanol, 100 µg/ml streptomycin,and 100 U/ml penicillin. CFSE-labeled T cells (4 x 105) werecocultured with 1 x 105 APC (DC or macrophages) from C57/BL6mice. After 72 h, cells were stained for CD4 (L3T4) or CD8 (Ly-2;Pharmingen), and CFSE dilution was analyzed by flow cytometry.
The Kidney Contains Multiple CD11c+ Cells That Are Distinct from CD11b+ and F4/80+ Cells
CD11c is widely used as a marker for murine DC in lymphatictissues. Previous studies have failed to detect CD11c+ cellsin the kidney (9,10,16). Using commercially available antibodies,we also did not detect such cells in paraffin sections of thekidney and spleen. In contrast, a recently developed immunohistologicaltechnique using a zinc-based formalin-free fixative in combinationwith an embedding wax that melts at lower temperature and thusavoids heating of the tissue to 56°C (16) revealed multipleCD11c+ cells in the tubulointerstitium of the renal cortex (Figure 1B).CD11c+ cells were located in the tubulointerstitial spacesand close to, but mostly outside of, peritubular capillaries.They possessed dendritic processes that protruded into the peritubularspaces. Only occasionally were CD11c+ cells seen in the glomeruli,and these were located within capillary loops. To ensure thatthis technique specifically detected CD11c+ cells, splenic sectionswere stained using the same protocol. Here, CD11c+ cells weredetected in their typical locations, the T cell areas and themarginal zone of the white pulp (Figure 1A).
Figure 1. Localization of CD11c+, F4/80+, CD11b+, and MHC II+ in the kidney. Immunohistowax sections of the spleen (A, C, E, and G) and kidney (B, D, F, and H) were stained for CD11c (A and B), CD11b (C and D), F4/80 (E and F), and MHC class II (G and H). The thin arrows in C and D indicate CD11blo cells in the splenic T cell area and the renal tubulointerstitium, respectively. The thick arrows indicate large, round CD11bhi cells in the marginal zone of the splenic white pulp and in a renal tubulointerstitial vessel. Original magnification: x100 for the spleen; x400 for the kidney.
To compare CD11c+ cells with macrophages, we stained for themarkers F4/80 and CD11b using the same technique. In spleensections, large, round, mononuclear cells highly positive forCD11b (CD11bhi) were located in the marginal zones and the redpulp, the typical locations of macrophages (Figure 1C, thickarrow). In addition, cells weakly positive for CD11b (CD11blo)with dendritic morphology were found in the T cell areas (Figure 1C,thin arrow), where the CD11c+ cells were found (Figure 1A),compatible with splenic CD11b+ DC (8). F4/80+ cells were prominentin the splenic red pulp but not in T cell areas (Figure 1E).In kidney sections, the glomerular capillaries contained large,round, mononuclear CD11bhi cells (Figure 1D, thick arrow) ina frequency of approximately one cell per 5 to 10 glomeruli.Occasionally, such cells were seen in tubulointerstitial capillaries.CD11blo cells with dendritic morphology were found in the extravasculartubulointerstitium (Figure 1D, thin arrow) at a frequency belowthat of CD11c+ cells (Table 1). The entire tubulointerstitiumshowed prominent staining for F4/80 (Figure 1F), as describedpreviously (4). The number of renal F4/80+ cells was approximatelythree times higher than that of CD11c+ cells, but cell numbersin individual animals varied considerably, resulting in highSD (Table 1).
Professional APC generally express MHC class II (8,11). Immunohistowaxsections of the spleen revealed large mononuclear MHC II+ macrophagesin the red pulp and numerous smaller MHC II+ cells in the whitepulp (Figure 1G), representing DC in the T cell areas or B cellsin lymphatic follicles. In the kidney, MHC II+ cells were detectedin numbers (Table 1) and locations similar to those of F4/80+cells (Figure 1H), compatible with previous studies (4,20,21).
Isolation and Phenotypical Characterization of Renal CD11c+ Cells
To quantitatively assess expression levels and the coexpressionof cell surface markers on CD11c+ cells, we developed a techniqueto isolate these cells from the kidney and to analyze them bymulticolor flow cytometry. This isolation technique was basedon existing protocols for APC isolation from the spleen involvingcollagenase digestion and density centrifugation. Splenic CD11c+DC usually possess a density of less than 1.080 g/ml, whereasthe isolation of macrophages requires a higher density (18).Renal CD11c+ cells were present exclusively in the low-densityfraction (data not shown), but purity was usually less than10% (data not shown). Overnight adherence on plastic to increasethe purity of APC was avoided here, because it resulted in thedeath of many renal CD11c+ cells and caused phenotypical changesin the surviving cells (data not shown). Instead, we added anadditional isolation step using CD11c-specific microbeads. Thistechnique yielded 0.05 to 0.1 x 106 viable CD11c+ cells perkidney, compared with 1 to 3 x 106 CD11c+ cells per spleen.Peritoneal macrophages isolated from the peritoneal cavity werealso used as a control cell population. Per peritoneal cavity,2 to 5 x 106 CD11b+ cells were obtained. Importantly, no inflammatorystimuli were instilled.
These three cell populations were examined by flow cytometry.The optical scatter properties for renal CD11c+ cells were comparableto those of splenic DC, whereas macrophages displayed largerside scatter (Figure 2A), indicating higher laser light diffractionat intracellular organelles such as lysosomes. To assess thepurity of our isolation method, the proportion of CD11c+ cellsamong the viable cells was determined by immunofluorescenceusing a monoclonal antibody for a different CD11c epitope andcompared with an appropriate isotype control. Typically, approximately95% of renal cells and more than 98% of splenic cells were CD11c+,with splenic cells expressing higher levels of this molecule(Figure 2B). Few peritoneal macrophages expressed CD11c, andexpression levels were low (Figure 2B).
Figure 2. Flow cytometry comparison of renal CD11c+ cells with splenic dendritic cells (DCs) and peritoneal macrophages. (A) Optical forward (FSC) and side (SSC) scatter dot plots. (B) Purity of isolated cells was assessed by staining with biotinylated anti-CD11c (clone HL-3) (thick line) or isotype control (thin line with gray shading) after blocking of Fc receptors. The numbers indicate the proportion of cells expressing more CD11c than the isotype control. (C) Expression of CD11b, F4/80, MHC class II, and CD80 was determined on renal CD11c+ PI- cells, splenic DCs, and peritoneal macrophages after blocking of Fc receptors. The numbers in the histograms give the proportion of cells expressing each marker. For CD11b, the proportions of cells displaying low and high levels are given separately. The results are representative of four individual experiments.
Next, we compared the expression of the other cell surface markersexamined in Figure 1 on CD11c+ cells (Figure 2C). In the spleen,both CD11b- and CD11blo DC were visible, as described (8). RenalCD11c+ cells were mostly CD11blo, compatible with the findingof CD11blo cells in kidney sections (Figure 1D). The CD11bhicells detected by histology (Figure 1, C and D) were found onlyamong peritoneal macrophages and neither in splenic nor renalcell preparations, indicating that such cells were excludedby our isolation protocol. Likewise, high levels of F4/80 wereexpressed only on peritoneal macrophages, whereas only somesplenic DC expressed this marker at low levels (Figure 2C),consistent with the absence of F4/80+ cells in splenic DC areas(Figure 1E). Interestingly, most of the renal CD11c+ cells expressedhigh F4/80 levels, albeit not as high as those of macrophages(Figure 2C). The levels of MHC class II and of the costimulatorymolecules CD80 (Figure 2C) and CD86 (data not shown) on renalCD11c+ cells and splenic DC varied over more than 2 orders ofmagnitude. In contrast, macrophages generally expressed lowlevels of these molecules. Expression of costimulatory moleculesby some renal CD11c+ cells was higher than that of splenic DC.This consistent finding was not artificially induced by ourDC isolation method, because similar levels were found whenazide was added during isolation (data not shown), which stopsall energy-dependent cell functions, such as surface moleculeupregulation or downregulation.
Dot-plot analysis of renal CD11c+ cells revealed cellular subpopulationsdiffering in MHC II expression. A distinct population of 4 to6% of CD11c+ cells was devoid of MHC II expression (Figure 3A).These cells colocalized with a similar CD11c+ MHC II- populationin the blood (Figure 3B), which recently was identified as acommon DC precursor population (13). To determine whether therenal CD11c+ MHC II- cells were blood-borne, mice were perfusedthoroughly before the kidneys were taken. This did not affectthe number of CD11c+ cells isolated per kidney (data not shown)but reduced the CD11c+ MHC II- population slightly to 3 to 5%(Figure 3C), indicating that this renal cell population onlypartially represented blood-borne DC precursors. Renal CD11c+cells expressed MHC II+, in good correlation with the costimulatorymolecules CD80 (Figure 3D) and CD86 (data not shown), suggestingthat the populations differing in MHC expression may representDC maturation stages.
Figure 3. Subpopulations of renal CD11c+ cells. CD11c+ isolated from the kidney (A and C to F) or from blood (B) were analyzed by multicolor flow cytometry. In C, donor mice were perfused with 50 ml of PBS before the kidney was taken. Shown are typical dot-plot analyses of MHC class II versus CD11c (A to C), MHC class II versus CD80 (D), and F4/80 versus CD11c (E) expression on PI- cells. F shows F4/80 versus CD11b dot plot analysis of the cells in E after gating for CD11c+ PI- events. The numbers indicate the cellular proportions in each region. Shown are results representative of four experiments.
The histogram analysis shown in Figure 2C suggested the existenceof subpopulations of renal CD11c+ cells differing in their expressionof CD11b and F4/80. Dot-plot analysis verified this hypothesisby revealing distinct populations of F4/80- and F4/80+ renalCD11c+ cells (Figure 3E), which could be further subdividedinto cells expressing CD11b or not (Figure 3F). These findingsdemonstrate that renal CD11c+ cells were not a homogeneous cellpopulation.
Functional Comparison of Renal CD11c+ Cells with DC and Macrophages
The data described above showed that renal CD11c+ cells expressedboth macrophage and DC markers. Because of this conflictingmarker status, we decided to base the classification of renalCD11c+ cells on their functionality. We examined typical macrophagefunctions, such as phagocytosis, lysosomal content, or NO productionin response to microbial stimuli, and as a typical DC function,T cell activation. To ensure comparability, all cell populationswere prepared using microbeads. For macrophages, CD11b-specificbeads were used, because these cells did not express CD11c (Figure 2C)(8)).
Phagocytosis was determined by flow cytometry evaluation ofFITC-labeled dextran uptake. Here, only macrophages showed strongactivity. Splenic DC and renal CD11c+ cells showed significantFITC-labeled dextran uptake, but this was 1 order of magnitudelower than that of macrophages (Figure 4A). Phagocytosed materialis intracellularly degraded in lysosomes. The lysosomal contentcan be measured with fluorescent dyes that accumulate afteruptake into acidic compartments. In this assay, macrophagesshowed higher lysosomal contents than did splenic DC and renalCD11c+ cells (Figure 4B). T cells, which do not possess lysosomes,served as a negative control. NO production was tested by culturingcells with LPS and IFN and by measuring NO in the supernatant.Macrophages produced large amounts of NO, in contrast to splenicDC and renal CD11c+ cells (Figure 4C). These data demonstratethat renal CD11c+ cells did not show macrophage functionality,excluding the possibility that the subpopulations identifiedabove represented macrophages.
Figure 4. Macrophage functionality by renal CD11c+ cells, DCs, and macrophages. (A) Renal CD11c+ cells, splenic DCs, and peritoneal macrophages were cocultured with FITC-labeled dextran in the absence (black bars) or presence (white bars) of azide. After 45 min, the uptake of fluorescence beads was determined by flow cytometry. (B) T cells (negative control), splenic DCs, renal CD11c+ cells, and peritoneal macrophages were cultured with Lysotracker® green in culture medium at 37°C (Molecular Probes). After 15 min, fluorescence accumulation in lysosomes was detected by flow cytometry. In A and B, the geometric means of the fluorescence intensities (MFI) of 104 CD11c+ (for renal and splenic cells) and 104 CD11b+ (for macrophages) cells are shown. (C) Renal CD11c+ cells, splenic DCs, and peritoneal macrophages were cultured with IFN and 0 (white bars), 20 (gray bars), or 100 (black bars) ng/ml LPS. After 2 d, nitric oxide (NO) was measured in the culture supernatant. The results are representative of three individual experiments.
The ability to activate T cells was determined in a mixed lymphocyteculture using responder T cells from Balb/c mice. Their proliferationwas assessed using the CFSE dilution technique. Division ofCFSE-labeled cells results in the dilution of this fluorescentdye. The number of divided cells indicates T cell proliferation(22). Both splenic DC and renal CD11c+ cells activated T cells,with splenic DC consistently being more effective (Figure 5, A and B).In contrast, peritoneal macrophages induced less Tcell proliferation (Figure 5F), consistent with their low expressionof MHC class II (Figure 2C). No proliferation resulted whencongenic C57/BL6 T cells were added to renal DC (Figure 5E)or to the other cell populations (data not shown), indicatingthat T cell activation was MHC-restricted. These results demonstratethat renal CD11c+ cells possessed DC activity. However, theprincipal DC function, namely T cell activation, cannot be determinedfor individual APC. Thus, it is possible that some renal CD11c+cells did not contribute to the observed T cell activation andmay represent CD11c+ non-DC devoid of macrophage functionality.
Figure 5. Ability to activate T cells. Renal CD11c+ cells (A, C, and E), splenic DCs (B and D), and peritoneal macrophages (F) from C57/BL6 donor mice were cocultured with 5,6-carboxyfluorescein succinimidyl ester (CFSE)-labeled T cells from Balb/c mice (A to D and F) or C57/BL6 mice (E). After 3 d, T cell proliferation was determined by flow cytometry. Shown are the CFSE levels of the CD3+ cells in culture. The proportion of proliferated T cells showing reduced CFSE intensity is indicated by the numbers. The results are representative of four individual experiments. NTN, nephrotoxic nephritis.
Localization and Functionality of Renal CD11c+ Cells in Nephrotoxic Glomerulonephritis
To investigate CD11c+ cells in inflammatory kidney disease,we induced NTN by injection of nephrotoxic sheep serum as described(14,15). Seven days after disease induction, the mice had developedsevere proteinuria and showed severe glomerular damage withfocal glomerular necrosis and tubulointerstitial damage, includingtubular atrophy and mononuclear infiltrates (Figure 6, B and C).The number of renal CD11c+ cells was increased in Immunohistowaxsections (Figure 6, A and B) and after isolation from the kidneyusing our protocol (healthy, 61,000 ± 34,000 per kidney,versus NTN, 391,000 ± 149,000 per kidney; n = 4). Inaddition, marked changes in the localization were seen: severelydamaged glomeruli showed periglomerular accumulation of CD11c+cells (Figure 6C). Interestingly, also in NTN despite significantglomerular injury, intraglomerular CD11c+ cells were almostcompletely absent (Figure 6C and data not shown). Focal accumulationof CD11c+ cells was seen within mononuclear tubulointerstitialinfiltrates (Figure 6B) and within the adventitia of small intrarenalarteries (Figure 6D). These morphologic changes were dose-dependent,because in mice that received a 33% lower dose of nephrotoxicsheep serum, changes were similar but less severe. Animals thatreceived a 50% higher dose showed anuric kidney failure at day7 and signs of wasting, and their kidneys contained very fewCD11c+ cells (data not shown).
Figure 6. Localization of renal CD11c+ cells in NTN. Seven days after induction of NTN, Immunohistowax sections were stained for CD11c. Shown are low magnifications (x200) of the tubulointerstitium healthy, untreated (A) versus diseased animals (B) and higher magnifications (x400) of periglomerular (C) infiltration and of a small intrarenal artery (D). Note the increased numbers of CD11c+ cells at sites of tubulointerstitial (B) and glomerular (C) damage and the perivascular adventitial infiltrate of CD11c+ cells (D). These images illustrate representative changes seen in three experiments in groups of four to five mice.
CD11c+ cells isolated from mice with NTN induced T cell proliferation(Figure 5C), demonstrating that these cells still possessedDC functionality. This ability was increased compared with thatof renal CD11c+ cells from healthy mice (Figure 5A), suggestingthat renal CD11c+ cells were activated. NTN did not increasethe T cell proliferation induced by splenic DC (Figure 5, B and D),demonstrating organ specificity. Macrophage functionsof renal CD11c+ cells such as phagocytic ability and NO productionwere not significantly changed in NTN (data not shown).
Bone marrow-derived APC were identified in the kidney tubulointerstitiummore than 20 yr ago (4). Their intense staining for the macrophagemarker F4/80 (4) and failed attempts to detect the DC markerCD11c (9,10) resulted in their classification as resident tissuemacrophages. Using an alternative, recently published immunohistochemicalmethod (23), the present study clearly demonstrates expressionof the DC marker CD11c on numerous tubulointerstitial cells.These cells were less numerous than F4/80+ cells, and therewas considerable overlap, because most of the CD11c+ cells expressedF4/80. Many studies have attempted to classify APC by cell surfacemarkers. In particular, numerous DC subpopulations have beendefined by their expression of surface markers, such as myeloidDC expressing CD11b or immature DC subsets positive for F4/80[6–9,17]. Most of these studies, however, have studiedDC in lymphatic tissues, which are easier to isolate. In nonlymphoidtissues, in particular in the kidney, it remains to be shownthat CD11c+ cells are DC. There is also functional overlap betweenmacrophages and DC, so that the definition of clear barriersis difficult, and maybe even impossible. For example, principalDC functions such as the activation of T cells can also be performedby macrophages, albeit less efficiently, and depending on theactivation and differentiation state. Likewise, some DC populationscan perform bactericidal functions normally ascribed to macrophages(24). At least in vitro, these cell types could be convertedto each other by cytokines (25). Consequently, it has been suggestedthat the macrophage-DC dichotomy be replaced with a differentiationcontinuum of mononuclear phagocyte cells, with DC and macrophagesas functional extremes (26). Our findings are consistent withthis idea. We determined both macrophage and DC functions forrenal CD11c+ cells compared with defined populations of DC andmacrophages. Splenic DC were effective T cell activators withlittle phagocytic and bactericidal activity, as expected. Incontrast, peritoneal macrophages were very efficient at theselatter functions, whereas their ability to activate T cellswas small. Compared with these functional extremes, renal CD11c+cells showed functionality very close to that of DC, which inour opinion allows the classification of renal CD11c+ as DC.Slightly higher phagocytic activity and less efficient T cellactivation were noted and may indicate some macrophage functionalityor immature DC functionality. These results do not exclude thatrenal CD11c+ cells may perform macrophage effector functionsunder certain situations, such as renal infections (24). Inthe healthy kidney and in NTN, however, the functionality ofrenal CD11c+ cells was confined to that of DC, despite the expressionof CD11b and F4/80. These findings have implications for theuse of these markers in immunohistochemistry. They are not absolutelyspecific for macrophages in the kidney, because most CD11c+cells also expressed F4/80. Consequently, a fraction of thetubulointerstitial F4/80 cells must be DC. This is a remarkabledifference from lymphoid tissues such as the spleen, in whichfew DC expressed F4/80 and only at low levels.
Renal CD11c+ cells did not represent a homogeneous cell population.A CD11c+ MHC II- subset resembled DC precursors. The expressionpatterns of MHC class II and costimulatory molecules suggestedthat different DC maturation or activation stages were presentin the kidney (8). These results are consistent with a previousstudy showing that MHC II+ cells from the kidney performed someDC functions (9). CD11c, however, was not detected in this previousstudy; thus, their equivalence to the renal CD11c+ cells investigatedhere is unclear, in particular because the kidney was shownto contain non-bone marrow-derived cells expressing MHC II,such as endothelial cells (20,21,27). Additional subpopulationswere identifiable when expression of CD11b and/or F4/80 wasexamined on renal CD11c+ cells. The CD11b- CD11c+ populationis unlikely to be related to CD11b- CD11c+ "lymphoid" DC inlymphatic tissues (8), because renal CD11c+ cells did not expressthe CD8 molecule (D. Benke, unpublished observations), whichis characteristic for these DC (8).
Also in experimental glomerulonephritis, CD11c+ cells showedDC functionality, which was even higher than that of cells fromhealthy kidneys. Inflammation is known to induce the recruitmentof DC to affected nonlymphoid organs and also their emigrationfrom these organs to draining lymph nodes (8,11). For MHC II+cells in the kidney, this has been reported after systemic LPSinjection (12,28). Similar dynamic changes may occur to renalCD11c+ DC in NTN. The accumulation of these cells in kidneyarterial walls observed in the present study may reflect blood-borneDC precursors entering the inflamed kidney. The result was amarked increase in renal DC numbers in NTN. Interestingly, thesecells were preferentially attracted to inflammatory sites, suggestingthat soluble mediators such as inflammatory chemokines are crucialin DC recruitment (29). The functional role of renal DC in NTNremains to be elucidated. They may be involved in the resolutionof inflammation, but they may also aggravate disease, similarto pulmonary DC, which have recently been shown to contributeto the perpetuation of asthma bronchiale (30). Experiments addressingthese questions will be possible based on the present study,as soon as techniques to specifically deplete DC in vivo becomeavailable.
In conclusion, we have described renal CD11c+ cells, characterizedtheir phenotype and functionality as that of DC, and demonstratedstriking changes in experimental glomerulonephritis.
Acknowledgments
The authors thank Steffi Schweistal and Alexandra Korzen forexcellent animal husbandry. Dr. K. Assmann, University of Nijmegen,The Netherlands, kindly provided nephrotoxic sheep serum. Thiswork was funded by a project grant of the German state of Nordrhein-Westfalenand by the Interdisziplinäres Zentrum für KlinischeForschung (IZKF) Biomat at the University of Aachen. C. Kurtswas supported by a Heisenberg fellowship from the Deutsche Forschungsgemeinschaft(Grant Ku1063/2-1).
Footnotes
Thilo Krüger and Dirk Benke contributed equally to thisstudy.
Bohle A, Muller GA, Wehrmann M, Mackensen-Haen S, Xiao JC: Pathogenesis of chronic renal failure in the primary glomerulopathies, renal vasculopathies, and chronic interstitial nephritides. Kidney Int 54 [Suppl]: S2–S9, 1996[CrossRef]
Kaissling B, Hegyi I, Loffing J, Le Hir M: Morphology of interstitial cells in the healthy kidney. Anat Embryol 193: 303–318, 1996[Medline]
Kaissling B, Le Hir M: Characterization and distribution of interstitial cell types in the renal cortex of rats. Kidney Int 45: 709–720, 1994[Medline]
Hume DA, Gordon S: Mononuclear phagocyte system of the mouse defined by immunohistochemical localization of antigen F4/80: Identification of resident macrophages in renal medullary and cortical interstitium and the juxtaglomerular complex. J Exp Med 157: 1704–1709, 1983[Abstract/Free Full Text]
McKnight AJ, Macfarlane AJ, Dri P, Turley L, Willis AC, Gordon S: Molecular cloning of F4/80, a murine macrophage-restricted cell surface glycoprotein with homology to the G-protein-linked transmembrane 7 hormone receptor family. J Biol Chem 271: 486–489, 1996[Abstract/Free Full Text]
Vremec D, Shortman K: Dendritic cell subtypes in mouse lymphoid organs: Cross-correlation of surface markers, changes with incubation, and differences among thymus, spleen, and lymph nodes. J Immunol 159: 565–573, 1997[Abstract]
Martinez del Hoyo G, Martin P, Arias CF, Marin AR, Ardavin C: CD8+ dendritic cells originate from the CD8- dendritic cell subset by a maturation process involving CD8, DEC-205, and CD24 up-regulation. Blood 99: 999–1004, 2002[Abstract/Free Full Text]
Shortman K, Liu YJ: Mouse and human dendritic cell subtypes. Nat Rev Immunol 2: 151–161, 2002[CrossRef][Medline]
Austyn JM, Hankins DF, Larsen CP, Morris PJ, Rao AS, Roake JA: Isolation and characterization of dendritic cells from mouse heart and kidney. J Immunol 152: 2401–2410, 1994[Abstract]
Steptoe RJ, Patel RK, Subbotin VM, Thomson AW: Comparative analysis of dendritic cell density and total number in commonly transplanted organs: Morphometric estimation in normal mice. Transpl Immunol 8: 49–56, 2000[CrossRef][Medline]
Mellman I, Steinman RM: Dendritic cells: Specialized and regulated antigen processing machines. Cell 106: 255–258, 2001[CrossRef][Medline]
Roake JA, Rao AS, Morris PJ, Larsen CP, Hankins DF, Austyn JM: Dendritic cell loss from nonlymphoid tissues after systemic administration of lipopolysaccharide, tumor necrosis factor, and interleukin 1. J Exp Med 181: 2237–2247, 1995[Abstract/Free Full Text]
del Hoyo GM, Martin P, Vargas HH, Ruiz S, Arias CF, Ardavin C: Characterization of a common precursor population for dendritic cells. Nature 415: 1043–1047, 2002[CrossRef][Medline]
Heeringa P, van Goor H, Itoh-Lindstrom Y, Maeda N, Falk RJ, Assmann KJ, Kallenberg CG, Jennette JC: Lack of endothelial nitric oxide synthase aggravates murine accelerated anti-glomerular basement membrane glomerulonephritis. Am J Pathol 156: 879–888, 2000[Abstract/Free Full Text]
Janssen U, Ostendorf T, Gaertner S, Eitner F, Hedrich HJ, Assmann KJ, Floege J: Improved survival and amelioration of nephrotoxic nephritis in intercellular adhesion molecule-1 knockout mice. J Am Soc Nephrol 9: 1805–1814, 1998[Abstract]
Pajak B, De Smedt T, Moulin V, De Trez C, Maldonado-Lopez R, Vansanten G, Briend E, Urbain J, Leo O, Moser M: Immunohistowax processing, a new fixation and embedding method for light microscopy, which preserves antigen immunoreactivity and morphological structures: Visualisation of dendritic cells in peripheral organs. J Clin Pathol 53: 518–524, 2000[Abstract/Free Full Text]
den Haan JM, Lehar SM, Bevan MJ: CD8(+) but not CD8(-) dendritic cells cross-prime cytotoxic T cells in vivo. J Exp Med 192: 1685–1696, 2000[Abstract/Free Full Text]
Vremec D, Zorbas M, Scollay R, Saunders DJ, Ardavin CF, Wu L, Shortman K: The surface phenotype of dendritic cells purified from mouse thymus and spleen: Investigation of the CD8 expression by a subpopulation of dendritic cells. J Exp Med 176: 47–58, 1992[Abstract/Free Full Text]
Ding AH, Nathan CF, Stuehr DJ: Release of reactive nitrogen intermediates and reactive oxygen intermediates from mouse peritoneal macrophages: Comparison of activating cytokines and evidence for independent production. J Immunol 141: 2407–2412, 1988[Abstract]
Sims TN, Halloran PF: MHC class II regulation in vivo in the mouse kidney. Microbes Infect 1: 903–912, 1999[CrossRef][Medline]
Muczynski KA, Cotner T, Anderson SK: Unusual expression of human lymphocyte antigen class II in normal renal microvascular endothelium. Kidney Int 59: 488–497, 2001[CrossRef][Medline]
Lyons AB, Parish CR: Determination of lymphocyte division by flow cytometry. J Immunol Methods 171: 131–137, 1994[CrossRef][Medline]
Maldonado-Lopez R, De Smedt T, Michel P, Godfroid J, Pajak B, Heirman C, Thielemans K, Leo O, Urbain J, Moser M: CD8+ and CD8- subclasses of dendritic cells direct the development of distinct T helper cells in vivo. J Exp Med 189: 587–592, 1999[Abstract/Free Full Text]
Serbina NV, Salazar-Mather TP, Biron CA, Kuziel WA, Pamer EG: TNF/iNOS-producing dendritic cells mediate innate immune defense against bacterial infection. Immunity 19: 59–70, 2003[CrossRef][Medline]
Nelson EL, Strobl S, Subleski J, Prieto D, Kopp WC, Nelson PJ: Cycling of human dendritic cell effector phenotypes in response to TNF-: Modification of the current ’maturation’ paradigm and implications for in vivo immunoregulation. FASEB J 13: 2021–2030, 1999[Abstract/Free Full Text]
Hume DA, Ross IL, Himes SR, Sasmono RT, Wells CA, Ravasi T: The mononuclear phagocyte system revisited. J Leukocyte Biol 72: 621–627, 2002[Abstract/Free Full Text]
Li S, Kurts C, Kontgen F, Holdsworth SR, Tipping PG: Major histocompatibility complex class II expression by intrinsic renal cells is required for crescentic glomerulonephritis. J Exp Med 188: 597–602, 1998[Abstract/Free Full Text]
Zernecke A, Weber KS, Erwig LP, Kluth DC, Schroppel B, Rees AJ, Weber C: Combinatorial model of chemokine involvement in glomerular monocyte recruitment: Role of CXC chemokine receptor 2 in infiltration during nephrotoxic nephritis. J Immunol 166: 5755–5762, 2001[Abstract/Free Full Text]
Julia V, Hessel EM, Malherbe L, Glaichenhaus N, O’Garra A, Coffman RL: A restricted subset of dendritic cells captures airborne antigens and remains able to activate specific T cells long after antigen exposure. Immunity 16: 271–283, 2002[CrossRef][Medline]
Received for publication May 2, 2003.
Accepted for publication November 20, 2003.
This article has been cited by other articles:
D. R. Engel, J. Maurer, A. P. Tittel, C. Weisheit, T. Cavlar, B. Schumak, A. Limmer, N. van Rooijen, C. Trautwein, F. Tacke, et al. CCR2 Mediates Homeostatic and Inflammatory Release of Gr1high Monocytes from the Bone Marrow, but Is Dispensable for Bladder Infiltration in Bacterial Urinary Tract Infection
J. Immunol.,
October 15, 2008;
181(8):
5579 - 5586.
[Abstract][Full Text][PDF]
N. Eter, D. R. Engel, L. Meyer, H.-M. Helb, F. Roth, J. Maurer, F. G. Holz, and C. Kurts In Vivo Visualization of Dendritic Cells, Macrophages, and Microglial Cells Responding to Laser-Induced Damage in the Fundus of the Eye
Invest. Ophthalmol. Vis. Sci.,
August 1, 2008;
49(8):
3649 - 3658.
[Abstract][Full Text][PDF]
V. W. S. Lee, Y. M. Wang, Y. P. Wang, D. Zheng, T. Polhill, Q. Cao, H. Wu, I. E. Alexander, S. I. Alexander, and D. C. H. Harris Regulatory immune cells in kidney disease
Am J Physiol Renal Physiol,
August 1, 2008;
295(2):
F335 - F342.
[Abstract][Full Text][PDF]
R. Veelken, E.-M. Vogel, K. Hilgers, K. Amann, A. Hartner, G. Sass, W. Neuhuber, and G. Tiegs Autonomic Renal Denervation Ameliorates Experimental Glomerulonephritis
J. Am. Soc. Nephrol.,
July 1, 2008;
19(7):
1371 - 1378.
[Abstract][Full Text][PDF]
K. L. Edgtton, J. Y. Kausman, M. Li, K. O'Sullivan, C. Lo, P. Hutchinson, H. Yagita, S. R. Holdsworth, and A. R. Kitching Intrarenal Antigens Activate CD4+ Cells via Co-stimulatory Signals from Dendritic Cells
J. Am. Soc. Nephrol.,
March 1, 2008;
19(3):
515 - 526.
[Abstract][Full Text][PDF]
J. Scholz, V. Lukacs-Kornek, D. R. Engel, S. Specht, E. Kiss, F. Eitner, J. Floege, H.-J. Groene, and C. Kurts Renal Dendritic Cells Stimulate IL-10 Production and Attenuate Nephrotoxic Nephritis
J. Am. Soc. Nephrol.,
March 1, 2008;
19(3):
527 - 537.
[Abstract][Full Text][PDF]
V. Lukacs-Kornek, S. Burgdorf, L. Diehl, S. Specht, M. Kornek, and C. Kurts The Kidney-Renal Lymph Node-System Contributes to Cross-Tolerance against Innocuous Circulating Antigen
J. Immunol.,
January 15, 2008;
180(2):
706 - 715.
[Abstract][Full Text][PDF]
J. Menke, J. A. Lucas, G. C. Zeller, M. E. Keir, X. R. Huang, N. Tsuboi, T. N. Mayadas, H. Y. Lan, A. H. Sharpe, and V. R. Kelley Programmed Death 1 Ligand (PD-L) 1 and PD-L2 Limit Autoimmune Kidney Disease: Distinct Roles
J. Immunol.,
December 1, 2007;
179(11):
7466 - 7477.
[Abstract][Full Text][PDF]
R. John and P. J. Nelson Dendritic Cells in the Kidney
J. Am. Soc. Nephrol.,
October 1, 2007;
18(10):
2628 - 2635.
[Abstract][Full Text][PDF]
U. Panzer, O. M. Steinmetz, H.-J. Paust, C. Meyer-Schwesinger, A. Peters, J.-E. Turner, G. Zahner, F. Heymann, C. Kurts, H. Hopfer, et al. Chemokine Receptor CXCR3 Mediates T Cell Recruitment and Tissue Injury in Nephrotoxic Nephritis in Mice
J. Am. Soc. Nephrol.,
July 1, 2007;
18(7):
2071 - 2084.
[Abstract][Full Text][PDF]
D. Engel, U. Dobrindt, A. Tittel, P. Peters, J. Maurer, I. Gutgemann, B. Kaissling, W. Kuziel, S. Jung, and C. Kurts Tumor Necrosis Factor Alpha- and Inducible Nitric Oxide Synthase-Producing Dendritic Cells Are Rapidly Recruited to the Bladder in Urinary Tract Infection but Are Dispensable for Bacterial Clearance
Infect. Immun.,
November 1, 2006;
74(11):
6100 - 6107.
[Abstract][Full Text][PDF]
R. D. Pawar, P. S. Patole, D. Zecher, S. Segerer, M. Kretzler, D. Schlondorff, and H.-J. Anders Toll-Like Receptor-7 Modulates Immune Complex Glomerulonephritis
J. Am. Soc. Nephrol.,
January 1, 2006;
17(1):
141 - 149.
[Abstract][Full Text][PDF]
A. Lang, D. Benke, F. Eitner, D. Engel, S. Ehrlich, M. Breloer, E. Hamilton-Williams, S. Specht, A. Hoerauf, J. Floege, et al. Heat Shock Protein 60 Is Released in Immune-Mediated Glomerulonephritis and Aggravates Disease: In Vivo Evidence for an Immunologic Danger Signal
J. Am. Soc. Nephrol.,
February 1, 2005;
16(2):
383 - 391.
[Abstract][Full Text][PDF]
Top
Abstract
Introduction
and varying LPS concentrations. After 48 h, 50 µl of 14 mmol/L 4'4-diaminodiphenylsulfone and 50 µl of 4 mmol/L N-ethylenediamine were added to the culture supernatant, and NO was detected by measuring the OD at 560 nm on a Dynatech MR5000 reader (Dynatech, Chantilly, VA). 
molecule (D. Benke, unpublished observations), which is characteristic for these DC (8). 
