Proteasomal Processing of Albumin by Renal Dendritic Cells Generates Antigenic Peptides
Daniela Macconi*,
Chiara Chiabrando,
Silvia Schiarea,
Sistiana Aiello,
Linda Cassis,
Elena Gagliardini*,
Marina Noris,
Simona Buelli*,
Carla Zoja*,
Daniela Corna*,
Caterina Mele,
Roberto Fanelli,
Giuseppe Remuzzi*,, and
Ariela Benigni*
* Mario Negri Institute for Pharmacological Research, Clinical Research Center for Rare Diseases "Aldo e Cele Daccò," and Unit of Nephrology and Dialysis, Azienda Ospedaliera Ospedali Riuniti di Bergamo, Bergamo, and Mario Negri Institute for Pharmacological Research, Milan, Italy
Correspondence: Dr. Giuseppe Remuzzi, Mario Negri Institute for Pharmacological Research, Via Gavazzeni 11, 24125 Bergamo, Italy. Phone: +39-035-319-888; Fax: +39-035-319-331; E-mail: gremuzzi{at}marionegri.it
Received for publication November 22, 2007.
Accepted for publication August 6, 2008.
The role of dendritic cells (DC) that accumulate in the renalparenchyma of non–immune-mediated proteinuric nephropathiesis not well understood. Under certain circumstances, DC captureimmunologically ignored antigens, including self-antigens, andpresent them within MHC class I, initiating an autoimmune response.We studied whether DC could generate antigenic peptides fromthe self-protein albumin. Exposure of rat proximal tubular cellsto autologous albumin resulted in its proteolytic cleavage toform an N-terminal 24–amino acid peptide (ALB1-24). Thispeptide was further processed by the DC proteasome into antigenicpeptides that had binding motifs for MHC class I and were capableof activating syngeneic CD8+ T cells. In vivo, the rat five-sixthsnephrectomy model allowed the localization and activation ofrenal DC. Accumulation of DC in the renal parenchyma peaked1 wk after surgery and decreased at 4 wk, concomitant with theirappearance in the renal draining lymph nodes. DC from renallymph nodes, loaded with ALB1-24, activated syngeneic CD8+ Tcells in primary culture. The response of CD8+ T cells of five-sixthsnephrectomized rats was amplified with secondary stimulation.In contrast, DC from renal lymph nodes of five-sixths nephrectomizedrats treated with the proteasomal inhibitor bortezomib losttheir capacity to stimulate CD8+ T cells in primary and secondarycultures. These data suggest that albumin can be a source ofpotentially antigenic peptides upon renal injury and that renalDC play a role in processing self-proteins through a proteasome-dependentpathway.
Dendritic cells (DC), which reside in most tissues in immaturestate, represent a double-edged sword in the immune system controllingimmunity and tolerance.1,2 Acting as a network of sentinel cells,they capture antigens and present them as peptides within MHCclasses I and II (MHC-I and MHC-II). Subsequent T cell activationand differentiation determine the outcome of infectious andautoimmune diseases. Studies have found that immunologicallyignored antigens, including self-antigens, can be taken up andtransported by DC under special circumstances, initiating anautoimmune response.3–5 Separate processing pathways forpresentation of exogenous and endogenous antigen by antigenpresenting cells (APC) have been described. Exogenous antigensare loaded on MHC-II in a post-Golgi compartment, whereas peptidesderived from intracellular synthesized proteins are presentedwithin MHC-I after proteasome processing. This strict dichotomywas recently challenged by studies showing that DC can presentexogenous proteins on MHC-I, through a process called "crosspresentation."6,7 Antigen presentation leads to either the initiationof immune response or tolerance, depending on the availabilityof co-stimulatory signals that are highly expressed upon DCactivation and maturation.8,9 Inflammatory stimuli also favorDC immunogenic capacity.8,9
Here we took as a pertinent paradigm the case of proteinuricnephropathy, whereby excess filtration of albumin upon breakdownof the glomerular barrier promotes tubular damage and releaseof chemokines into the interstitium.10 The ensuing inflammatoryenvironment might convert the tolerogenic capacity of residentDC into an immunogenic one, favoring recruitment of T cells.Actually, the significance of the presence of immune systemcells as DC and CD8+ T cells in the renal parenchyma of proteinuricnephropathies, even in the absence of an immune insult,11,12has never been explored before. The major APC population ofthe healthy kidney are DC, which, being in close contact withthe tubular epithelium, sense and respond to molecules thatcross the tubule layer and diffuse away.13,14 Upon injury, immunogenicDC might cross-present normally ignored self-antigens, namelyalbumin, triggering T cell effector response. Here we demonstratethat proteolysis of the self-protein albumin by proximal tubularcells provides the substrate to DC for the generation—viaa proteasome-dependent pathway—of antigenic peptides recognizedby CD8+ T cells.
N-Terminal Truncation of Albumin by a Proximal Epithelial Cell Protease
We first investigated the profile of the peptides present inthe supernatant of rat proximal tubular cells (NRK) after incubationwith 0.5 mg/ml fatty acid–free albumin, a concentrationmeasured in the glomerular ultrafiltrate of proteinuric rats.15,16Mass spectrometry profiles by matrix-assisted laser desorptionionization mass spectrometry (MALDI-TOF-MS; mass range 600 to3600 Da), obtained from NRK supernatants acidified to pH 2.5and extracted on C18 ZipTip, showed the presence of three prominentpeptides (Figure 1A). Figure 1B shows the amino acid sequenceof the three peptides, named here ALB1-24, ALB1-27, ALB1-30.These cleavage products, identified by liquid chromatography-electrosprayionization tandem mass spectrometry (LC-ESI-MS/MS) with Mascotdatabase search engine, derive from N-terminal truncation ofalbumin at position 24, 27, and 30. ALB1-24 was by far the mostabundant product (Figure 1A, Supplemental Figure 1). Identityof ALB1-24 was confirmed by comparison with the synthetic peptideALB1-24 prepared by F-moc chemistry (Supplemental Figures 2through 4). Further experiments with NRK cell supernatant usingstable isotope–labeled ALB1-24 as internal standard, showedthat the formation of ALB1-24 occurs in an acidic environment(pH 2.5 to 5.0; data not shown) and is completely suppressedby pepstatin A (Figure 1C).
Figure 1. Formation of N-terminal albumin fragments in NRK cell supernatants. (A) MALDI-TOF-MS analysis of supernatants from NRK cells exposed or not to rat serum albumin (0.5 mg/ml). Samples were acidified to pH 2.5 and extracted on C18 ZipTip. The most abundant newly formed compounds were identified as ALB1-24, ALB1-27, and ALB1-30. *Corresponds to a modified form of ALB1-24, with pyroglutamic acid at position 1 (see Supplemental Figure 1). (B) Sequence and monoisotopic molecular mass of the [M+H]+ ions of ALB1-24, ALB1-27, and ALB1-30. (C) MALDI-TOF-MS analysis of supernatants from NRK cells exposed to albumin in the presence or absence of pepstatin A (10 µg/ml). The two peak groups refer to the isotopic cluster of ALB1-24 and to the internal standard 15N, 13C-ALB1-24, respectively (labels refer to the monoisotopic peak). In the presence of pepstatin A, signals of ALB1-24 but not those of the internal standard disappeared, indicating complete suppression of ALB1-24 formation (n = 3).
DC Proteasome Processes ALB1-24 into CD8+ T Cell–Recognized Antigenic Peptides
Given that albumin degradation fragments are predominantly exocytosedto the basolateral side of the proximal tubular cell17 in amanner that in vivo allows further processing in the interstitialcompartment, we next asked whether DC—abundantly presentin the renal interstitium in proteinuric diseases—couldprocess albumin fragments and present them to T cells. Studieswere also undertaken to document the pathways of albumin peptideprocessing. Bone marrow DC from Brown Norway (BN) rats wereincubated overnight with synthetic ALB1-24 and then used toprime syngeneic CD8+ T cells. CD8+ T cell stimulation, measuredas IFN- release by enzyme-linked immunospot (ELISPOT) assay,was not evident at the first encounter (Figure 2A). When ALB1-24-primedCD8+ T cells were restimulated by syngeneic pulsed DC, theyshowed a significant activation (P < 0.05 versus unpulsedDC), disclosing the antigenic nature of the self-peptide (Figure 2B).To assess further the intracellular pathways involved in ALB1-24processing and presentation by DC, we performed experimentsin the presence of amiloride analog 5-(N-ethyl-N-isopropyl)amiloride (EIPA), which inhibits antigen uptake by macropinocytosis,18or brefeldin A (BfA), which interferes with the egress of newlysynthesized MHC molecules.19 Activation of CD8+ T cells wascompletely prevented by both EIPA and BfA (Figure 2B). We thendetermined the possible involvement of proteasome, known toproduce most antigenic peptides presented by MHC-I,20 in CD8+T cell activation by DC. The proteasome inhibitor lactacystin21completely prevented the stimulation of CD8+ T cells by DC preexposedto ALB1-24 (Figure 2B). These findings indicate that, throughthe proteasome, DC are effective in processing protein fragmentsinto smaller peptides, which are presented to CD8+ T cells,leading to their activation.
Figure 2. Proteasome-dependent processing by DC of ALB1-24 stimulates syngeneic CD8+ T cells. (A) Primary mixed leukocyte reaction (MLR). Bone marrow–derived DC from BN rats were incubated with () or without (
) the synthetic ALB1-24 (35 µg/ml) and used to stimulate syngeneic CD8+ T cells. CD8+ T cell activation was evaluated by IFN- ELISPOT assay. As positive control, unpulsed DC were cultured with allogeneic LW T cells (). (B) Secondary MLR. After 3-d primary MLR, primed CD8+ T cells were harvested and cultured again in a 2-d secondary stimulation with DC previously pulsed with or without ALB1-24. As positive control, unpulsed DC were cultured in secondary MLR with allogeneic LW T cells recovered at the end of primary MLR (). The inhibitors EIPA (50 µM), BfA (0.5 µg/ml), or lactacystin (Lac; 10 µM), were added to DC 30 min before pulsing with ALB1-24. Data are means ± SEM (n = 5 independent experiments). *P < 0.05 versus syngeneic CD8+ T cells re-incubated with unpulsed DC or pulsed DC in presence of inhibitors.
Proteasome Generates Predicted MHC-I Ligands from ALB1-24
To assess whether the proteasome is able to cleave from ALB1-24peptide fragments possibly responsible for its antigenicity,we incubated ALB1-24 with highly purified 20S proteasome for0 to 8 h at 37°C and identified the most relevant cleavageproducts by mass spectrometry (MALDI-TOF-MS and LC-ESI-MS/MS).At 0 h, only the intact peptide was observed. The intact substratewas still present at 2 and 4 h but exhausted after 8 h. After4 h, the main products observed by MALDI-TOF-MS were from C-terminaltrimming of ALB1-24 (1 to 22, 1 to 19, and 1 to 18 fragments).These products then tended to disappear, whereas shorter peptides(8 to 10 amino acid size) accumulated in the 2- to 8-h interval(data not shown). Figure 3 lists the MS/MS-identified proteasome-cleavedfragments of ALB1-24 with a size compatible with MHC-I binding(8 to 10 amino acids). Supplemental Figures 5 and 6 show theinterpreted MS/MS spectra and peptide fragmentation for twopeptides, EAHKSEIAHR and GEQHFKGL.
Figure 3. Cleavage of ALB1-24 by proteasome. ALB1-24 was incubated for 0 to 8 h with purified proteasome, and the fragments were identified by MS. Only peptides with a size compatible with MHC-I binding (8 to 10 amino acids) are listed here. The sequence of ALB1-24 is reported, with the position of the amino acids.
To investigate the presence of a potential binder for MHC-Iwithin the 1 to 24 domain of albumin, we submitted the entiresequence of rat serum albumin precursor (SwissProt accessionno. P02770) to database search for potential MHC ligands, usingthree bioinformatics tools (SYFPEITHI, ProPred-I, and MAPPP).Among all of the 8- to 10-mers in the entire albumin sequence,a number of peptides belonging to the 1 to 24 domain indeedranked within the five best binders on various murine MHC-Ialleles (Table 1). For example, GEQHFKGLV (15 to 23) was consistentlyranked as the number 1 among 9-mer binders for H2-Kk and GEQHFKGL(15 to 22) as a top-ranked 8-mer ligand for H2-Kb. Interestingly,we identified GEQHFKGL as a product of in vitro proteasomaldegradation of ALB1-24 (Figure 3, Supplemental Figure 6).
Table 1.In silico predicted peptide binding to MHC-Ia
Proteasome Inhibition Reduces Renal Inflammation and T Cell Stimulatory Capacity of DC in Proteinuric Nephropathy
To assess the in vivo functional contribution of proteasomeactivity to the formation of self-antigens, we evaluated theeffect of the proteasome inhibitor bortezomib on the patternand activation of inflammatory cells infiltrating renal interstitiumin a rat model of proteinuric progressive renal disease as aresult of surgical ablation of renal mass. Despite no primaryimmune insult in this model, the accumulation of DC and CD8+T cells in the interstitium was found to be effectively controlledby an antiproteinuric therapy, indicating that the upstreammechanism of protein traffic is critical to initiate and maintaininflammation and immune injury.11 Rats underwent five-sixthsnephrectomy and were given bortezomib or vehicle from days 1to 28. As reported in Table 2, no significant changes in systolicBP and renal functional parameters were found after bortezomibtreatment. Morphologic evaluation of the kidneys by light microscopyshowed that the incidence of focal glomerulosclerosis, affectingon average 22 ± 5% of glomeruli in the vehicle-treatedrats with renal mass reduction (RMR), was reduced by bortezomibtreatment to 11 ± 2%, although the difference did notreach statistical significance. Hematocrit and white blood cellcount were also comparable between the two groups (hematocrit:RMR + vehicle, 45 ± 1.2%, RMR + bortezomib, 41 ±1.15%; white blood cell: RMR + vehicle, 11,067 ± 694cells/µl, RMR + bortezomib, 13,520 ± 528 cells/µl).All animals developed interstitial inflammation. The natureof interstitial infiltrates examined by immunohistochemistryrevealed the presence of macrophages (ED1+ cells), CD4+ T cells,CD8+ T cells, and DC (OX62+ cells; Table 3). Bortezomib significantlyprevented the extent of infiltration of both inflammatory andimmune cells (Table 3). To characterize renal DC better, weperformed immunophenotypic analysis by FACS by double labelingrenal cells with anti-OX62 and anti-CD11c antibodies. As shownin Supplemental Figure 7, the majority of OX62+ cells (approximately80%) also expressed CD11c. FACS analysis of CD11c+ DC in totalkidney infiltrating cells confirmed increased content of thesecells in remnant kidneys as compared with control kidneys, therebyvalidating immunohistochemistry findings (Figure 4A). The comparisonof CD11c+ DC isolated from kidneys and renal draining lymphnodes (LN) of RMR rats, both at 1 and 4 wk after surgical ablation,allowed us to document dynamic changes in renal DC at differentstages of the disease (Figure 4A). The percentage of CD11c+DC among renal cells peaked at 1 wk. At 4 wk, a decrease inthe percentage of DC in the kidney was paralleled by an enhancementof these cells in the renal LN, suggesting migration of DC fromthe renal interstitium. These changes were associated with asignificant increase in CD8+ T cells in total LN at 4 wk precededby a marked increase in CD4+ T cells at 1 wk (Figure 4B). Importantly,DC sorted at 4 wk from renal LN of RMR rats and exposed in vitroto ALB1-24 significantly activated CD8+ T cells isolated atthe same time points from RMR rats (syngeneic CD8+ T cells)already in a primary mixed leukocyte reaction, suggesting anin vivo cross-priming of CD8+ T cells by the albumin peptide(Figure 5A). Of note, CD8+ T cells isolated from RMR rats at1 wk were not significantly stimulated by ALB1-24–loadedDC in primary cultures, indicating that in vivo CD8+ T cellcross-priming was a chronic progressive event. Upon secondarystimulation in vitro by ALB1-24–loaded DC, CD8+ T cellsisolated at both 1 and 4 wk after RMR showed an amplified activationas compared with CD8+ T cells from control rats stimulated inthe same condition. Addition of either lactacystin or bortezomibalmost completely prevented RMR CD8+ T cell activation by ALB1-24–loadedDC (Figure 5B). Consistently, treatment of RMR rats with bortezomibfor 4 wk reduced the capacity of ALB1-24–loaded DC fromrenal LN to stimulate CD8+ T cells in both primary and secondarycultures (Figure 5B).
Figure 4. Immunophenotypic analysis in RMR rats. (A) Cells obtained from kidneys and renal draining LN of RMR rats (at 1 wk [n = 4] and 4 wk [n = 5] after surgical ablation) and control rats (n = 5) were labeled with FITC-conjugated mouse anti-rat CD11c and FACS analyzed. (B) Cells obtained from total LN of RMR rats (at 1 wk [n = 4] and 4 wk [n = 5] after surgical ablation) and control rats (n = 5) were labeled with PE-conjugated mouse anti-rat CD4, FITC-conjugated mouse anti-rat CD8, and FITC-conjugated mouse anti-rat CD11c. Labeled cells were analyzed using FACSAria. Data are means ± SEM. *P < 0.05 versus control rats and RMR rats at 4 wk; P < 0.05 versus control rats and RMR rats at 1 wk.
Figure 5. Primary and secondary MLR in RMR rats. (A) Primary MLR. CD11c+ DC sorted from renal draining LN of vehicle or bortezomib-treated RMR rats and splenic CD11c+ DC from control rats were incubated with () or without (
) the synthetic ALB1-24 peptide (35 µg/ml) and used to stimulate syngeneic CD8+ T cells. CD8+ T cell activation was evaluated by IFN- ELISPOT assay. As positive control, unpulsed DC were cultured with allogeneic LW T cells (). (B) Secondary MLR. After 3-d primary MLR, primed CD8+ T cells were harvested and cultured in a 2-d secondary stimulation with DC previously pulsed with or without ALB1-24 peptide. As positive control, unpulsed DC were cultured in secondary MLR with allogeneic LW T cells recovered at the end of primary MLR (). The inhibitors lactacystin (10 µM) or bortezomib (20 ng/ml) were added 30 min before peptide pulsing. Data are means ± SEM (n = 4 independent experiments). *P < 0.05 versus no ALB1-24; P < 0.05 versus no ALB1-24 and + ALB1-24 in the presence of inhibitors.
The presence of DC and T cells in the interstitium, a commonfinding of non–immunologically induced nephropathies,indicates activation of the immune system. The present studydocuments the generation of antigenic peptides from a self-protein,namely albumin, through the concerted action of renal proximaltubules and DC. Exposure of proximal tubular cells to excessautologous albumin, as in the case of proteinuric nephropathies,results in the formation of the N-terminal 24–residuefragment of albumin. This peptide is acquired by DC, where itis further processed by proteasome into antigenic peptides thatactivate CD8+ T cells.
An acid protease with pepsin-like activity is responsible forthe N-terminal truncation of albumin as documented by the inhibitoryeffect of pepstatin. These results are in line with previouslyreported studies showing selective truncation of albumin atposition 24.22,23 The N-terminal 24–amino acid peptidehas been described in various species as a major product ofalbumin digestion by pepsin at acidic pH,24 a condition thatmakes albumin more susceptible to proteolytic cleavage.25
Peptides of 8 to 10 residues in length, generated by proteasomeof APC and presented on MHC-I, are normally recognized by CD8+T cells. Proteasome digestion of ALB1-24 in a cell-free systemindeed produced a number of peptides 8 to 10 amino acids long.Database search for potential peptide binders to MHC-I indicatesthat, within the products of in vitro proteosomal degradationof ALB1-24, a possible candidate for CD8+ T cell cross-primingis GEQHFKGL.
The crucial role of proteasome in the activation of immune cellsis further supported here by in vivo findings showing that theproteasome inhibitor bortezomib prevents infiltration of inflammatoryand immune cells in the interstitium in the rat model of progressivenephropathy caused by renal mass ablation. Accumulation of DCin the peritubular interstitium is a constant although unexplainedfinding of proteinuric nephropathies. Recent observations indicatethat interstitial DC, positioned to serve as a major interfacebetween innate and adaptive immunity, express on their surfacethe fractalkine receptor CX3CR1.26 Expression of fractalkineis increased in the renal interstitium of mice with proteinoverload proteinuria, which would explain DC recruitment intothe renal interstitium.27 The limited capacity of lysosomalproteolysis of DC allows internalized antigens to persist andenhances the chance for them to be degraded by proteasome andcross-presented on MHC-I, thereby enhancing immunogenic T cellresponse.28
Our findings that DC were increased in kidney draining LN fromRMR rats at the late stage of the disease suggests transportof the antigen taken up by DC from the renal interstitium tothe T cell area of the renal LN. Renal DC are important participantsof renal injury by virtue of their capacity to mediate antigentransfer from the renal parenchyma to renal LN, where Ag-specificT cell activation takes place.13 Furthermore, DC from RMR ratscan cross-present albumin peptides to CD8+ T cells. Processingof ALB1-24 by DC likely occurs through the proteasome-dependentpathway as suggested by the in vitro inhibitory effect of bothbortezomib and lactacystin on T cell activation. Of note, thatDC from renal LN of bortezomib-treated RMR rats lose their capacityto stimulate T cells further supports a role for the proteasomein immunologic and inflammatory processes that concur to parenchymaldamage. It has been proposed that rat CD4+/SIRP+ DC exhibitstrong CD8+ T cell stimulatory capacity.29 We have identifiedthe presence of this subset in the rat remnant kidney (see SupplementalFigure 8). Whether such a population acts as cross-presentingcell in this model of proteinuric nephropathy merits furtherinvestigations.
In the kidney, the tubular acidic milieu provides the environmentfor proteolytic cleavage of albumin, which, upon sieving dysfunctionof the glomerular barrier, is filtered in abnormally high amountsresulting in excess protein loading of proximal tubular cells.The N-terminal 24-residue fragment of albumin can be acquiredby interstitial DC that have the ability to uptake moleculesfiltered into the urinary space.13 Extracellular acidosis, whichis usually associated with inflammatory responses, increasesthe uptake of exogenous antigens and MHC-I–restrictedpresentation of derived peptides by DC stimulating CD8+ T cellimmunity.30 Acidic regions were also detected in the renal corticalinterstitium by using a low pH–sensitive membrane peptideconstruct that targets acidic tissues in vivo.31 Thus, the highacidic milieu related to inflammation is likely to facilitatestrongly albumin peptide acquisition by interstitial DC andits recognition by CD8+ T cells.
DC were recently described to form an immune sentinel networkthroughout the entire kidney, where they probe the environmentin search of antigens.26 Cross-presentation by DC is a majormechanism for the immune surveillance of tissues against foreignantigens. Its outcome toward immunity depends on the availabilityof immunostimulatory signals that are acquired together withthe antigen.32 The current view that immunity is controlledby internal communication between the tissue and immune-competentcells suggests that tolerance is maintained by healthy organs,whereas immunity can be stimulated by a distressed tissue.33Conceivably, inflammatory stimuli released from damaged tubulesrepresent "danger" signals that, once in the presence of thealbumin peptide, alert DC to promote local immunity via CD8+T cells. Theoretically, in humans, any chronic condition characterizedby both the nephrotic syndrome and the presence of the interstitialinflammatory infiltrates may provide the environment for theDC processing of albumin peptide to occur in areas surroundingthe injured nephron. Our data indicate that a self-protein thatis normally ignored by immune competent cells becomes a reservoirof potentially antigenic peptides upon organ injury. It is plausiblethat the mechanism underlying the formation of self-antigensin conditions of kidney injury also operates for other proteinsand in other circumstances of organ damage. One notable situationto which this paradigm may apply is the processing of uniquepathogenic epitopes that are present in normal cardiac myosinin experimental myocarditis.34 Proteasomal degradation of self-proteinsinto antigenic peptides that drive the outcome of cross-presentationtoward immunity deserves further investigation as a novel triggerof disease states and break of immune tolerance in chronic inflammatorydiseases.
Proximal Tubular Epithelial Cell Culture and Incubation
NRK-52E cells (a rat proximal tubular epithelial cell line)obtained from Deutsche Sammlung von Mikroorganismen und Zellkulturen(Braunschweig, Germany) were cultured in DMEM supplemented with5% FBS, l-glutamine (2 mM), penicillin (100 U/ml), and streptomycin(100 µg/ml). Confluent cells were maintained 48 h in serum–freeDMEM before experiments and then exposed for 19 h to essentiallyfatty acid and globulin-free rat albumin (0.5 mg/ml; Sigma-Aldrich,St. Louis, MO) or medium alone. At the end of the incubation,supernatants (100 µl) were acidified to pH 2.5 beforeextraction and analysis by mass spectrometry, as described inthe next section. In additional experiments, NRK cells werepreincubated with or without the aspartic protease inhibitorpepstatin A (10 µg/ml; Sigma-Aldrich) 30 min before andduring the addition of rat albumin (0.5 mg/ml) for 19 h. Supernatants(50 µl) were enriched with internal standard 15N, 13C-labeledALB1-24 (2 pmol/µl), acidified to pH 2.5, and incubatedfor 24 h at 37°C before extraction and analysis by massspectrometry (see below).
Peptide Extraction
Peptides were extracted from NRK cells supernatant (acidifiedto pH 2.5 with 2.5% trifluoroacetic acid) on ZipTip C18 pipettetips (Millipore Corp., Bedford, MA). Peptides were identifiedby MALDI-TOF-MS and LC-ESI-MS/MS (see Supplemental Informationfor details).
Proteasome Digestion
Synthetic ALB1-24 peptide (2.4 nmol) was incubated with purifiedrabbit 20S proteasome (Sigma-Aldrich) at a molar ratio of 800:1(peptide:proteasome) at 37°C in 10 mM Tris-HCl (pH 7.5).35Aliquots were taken at different times of incubation (0, 2,4, and 8 h) and analyzed by MS in comparison with incubatedreaction blanks (peptide in buffer or buffer alone).
Immunophenotypic Analysis
Immunophenotypic analysis was performed using FACS (FACSAria;Becton Dickinson, Franklin Lakes, NJ). Cells obtained from kidneysand kidney draining LN of RMR (both at 1 and 4 wk after surgicalablation) and sham-operated rats were labeled with FITC-conjugatedmouse anti-rat CD11c (clone 8A2, AbD; Serotec, Oxford, UK).Cells from total LN of RMR and sham-operated rats were labeledwith anti-CD11c, FITC-conjugated mouse anti-rat CD8 (clone OX8,AbD), and PE-conjugated mouse anti-rat CD4 (clone W3/25, AbD).In a selected experiment, renal DC were analyzed for Sirp (CD172a,clone OX41, AbD) CD4 coexpression (see Supplemental Informationfor details). Negative controls were performed using controlisotype FITC- and PE-conjugated antibodies.
Primary and Secondary CD8+ T Cell Stimulation
For primary and secondary stimulation, DC were used as stimulatorsand syngeneic CD8+ T cells as responders. Preparation of DCand CD8+ T cells is described in supplemental materials. DCwere bone marrow–derived CD11c+ DC from BN rats, splenicFACS-sorted CD11c+ DC from Sprague-Dawley (SD) control sham-operatedrats, renal draining LN FACS-sorted CD11c+ DC from SD RMR rats,and bortezomib-treated RMR rats. Because SD is an outbred strain,to maintain syngeneic condition, we obtained co-cultured DCand CD8+ T cells from the same animals (both for RMR rats andfor control sham-operated rats).
DC were incubated (105 cells/100 µl RPMI/5% rat serum,37°C, 5% CO2) for 18 h with or without the synthetic ALB1-24(35 µg/ml) and used as stimulators in a 3-d primary stimulationwith syngeneic CD8+ T cells (DC-to-responders ratio: 1:100 forBN and 1:40 for SD). Allogeneic Lewis (LW, RT1l) T cells wereused as positive control to verify DC allostimulatory capacityin each experiment. After primary stimulation, CD8+ T cellswere harvested and cultured again in a 2-d secondary stimulationwith DC previously pulsed with or without ALB1-24 (at the sameDC-to-responder ratio of primary stimulation). CD8+ T cell activationwas evaluated by IFN- ELISPOT assay (Euroclone, Ltd., Paington,UK). For experiments with BN cells, inhibitors EIPA (50 µM;Sigma-Aldrich), BfA (0.5 µg /ml; Sigma-Aldrich), or lactacystin(10 µM; Sigma-Aldrich) were added to DC 30 min (37°C,5% CO2) before pulsing with ALB1-24. For experiments with SDcells, proteasome inhibitors such as lactacystin or bortezomib(20 ng/ml) were added to DC 30 min (37°C, 5% CO2) beforepulsing with ALB1-24. DC were washed with RPMI before addingCD8+ T cells to avoid possible inhibitors’ toxic effect.DC viability, evaluated by Trypan blue dye exclusion, accountedfor 70% on average in all settings.
Experimental Model of RMR in Rat
RMR was induced in male SD rats (320 to 340 g; Charles RiverItalia, Calco, Italy) as described previously.11 Animals receivedvehicle (saline; n = 11) or bortezomib36 (JANSSEN-CILAG SpA,Milan, Italy; n = 10, 0.3 mg/kg intravenously twice a week fromdays 1 to 28). An additional group of untreated RMR rats (n= 4) were killed 1 wk after surgical ablation. Nine sham-operatedrats served as controls. At the end of the study, kidneys, LN,and spleens were removed under anesthesia for immunophenotypiccharacterization of DC and T cells by FACS (see also SupplementalInformation) and cross-presentation studies. Kidney specimenswere also used for the evaluation of glomerular sclerosis andinterstitial infiltrates by immunohistochemistry (see SupplementalInformation). Systolic BP, proteinuria, serum creatinine, andglomerular damage were determined as described previously.11Animal care and treatment were conducted in conformity withthe institutional guidelines that are in compliance with nationaland international laws and policies (EEC Council Directive 86/609,OJL 358, Dec 12 1987; Guide for the Care and Use of LaboratoryAnimals, US National Research Council, 1996).
Statistical Analysis
Results are expressed as means ± SEM. Data on IFN- spotsand FACS analysis of DC and T cells were compared by ANOVA andBonferroni post hoc analysis. Systolic BP, renal functionalparameters, and histology in RMR rats treated with vehicle orbortezomib were analyzed by unpaired t test. Results of renalinterstitial infiltrates were analyzed by nonparametric Kruskal-Wallistest for multiple comparisons. Statistical significance wasdefined as P < 0.05.
L.C. and S.B. are recipients of a fellowship of Fondazione ART(Milan, Italy) and Fondazione Aiuti per la Ricerca sulle MalattieRare (ARMR; Bergamo, Italy), respectively.
We are profoundly indebted to Dr. Alfred L. Goldberg for thoughtfuldiscussion of the rationale of this investigation. We thankDrs. Mauro Abbate and Marina Morigi for helpful discussion;Dr. Mario Salmona for the synthesis of ALB1-24; Dr. FrancescoGregis for providing bortezomib; and Anna Pezzotta, FedericaRocchetta, Marilena Mister, and Marta Todeschini for technicalassistance.
Footnotes
Published online ahead of print. Publication date availableat www.jasn.org.
D.M. and C.C. contributed equally to this work.
Supplemental information for this article is available onlineat http://www.jasn.org/.
See related editorial, "Cross Dendritic Cells Anger T Cellsafter Kidney Injury," on pages 3–5.
Ludewig B, Odermatt B, Ochsenbein AF, Zinkernagel RM, Hengartner H: Role of dendritic cells in the induction and maintenance of autoimmune diseases.
Immunol Rev 169
: 45
–54, 1999[CrossRef][Medline]
Greter M, Heppner FL, Lemos MP, Odermatt BM, Goebels N, Laufer T, Noelle RJ, Becher B: Dendritic cells permit immune invasion of the CNS in an animal model of multiple sclerosis.
Nat Med 11
: 328
–334, 2005[CrossRef][Medline]
Heath WR, Belz GT, Behrens GM, Smith CM, Forehan SP, Parish IA, Davey GM, Wilson NS, Carbone FR, Villadangos JA: Cross-presentation, dendritic cell subsets, and the generation of immunity to cellular antigens.
Immunol Rev 199
: 9
–26, 2004[CrossRef][Medline]
Wilson NS, Villadangos JA: Regulation of antigen presentation and cross-presentation in the dendritic cell network: Facts, hypothesis, and immunological implications.
Adv Immunol 86
: 241
–305, 2005[CrossRef][Medline]
Huang FP, MacPherson GG: Continuing education of the immune system: Dendritic cells, immune regulation and tolerance.
Curr Mol Med 1
: 457
–468, 2001[CrossRef][Medline]
Finkelman FD, Lees A, Birnbaum R, Gause WC, Morris SC: Dendritic cells can present antigen in vivo in a tolerogenic or immunogenic fashion.
J Immunol 157
: 1406
–1414, 1996[Abstract]
Zoja C, Benigni A, Remuzzi G: Cellular responses to protein overload: Key event in renal disease progression.
Curr Opin Nephrol Hypertens 13
: 31
–37, 2004[Medline]
Remuzzi G, Zoja C, Gagliardini E, Corna D, Abbate M, Benigni A: Combing an antiproteinuric approach with mycophenolate mofetil fully suppresses progressive nephropathy of experimental animals.
J Am Soc Nephrol 10
: 1542
–1549, 1999[Abstract/Free Full Text]
Remuzzi A, Gagliardini E, Donadoni C, Fassi A, Sangalli F, Lepre MS, Remuzzi G, Benigni A: Effect of angiotensin II antagonism on the regression of kidney disease in the rat.
Kidney Int 62
: 885
–894, 2002[CrossRef][Medline]
Dong X, Swaminathan S, Bachman LA, Croatt AJ, Nath KA, Griffin MD: Antigen presentation by dendritic cells in renal lymph nodes is linked to systemic and local injury to the kidney.
Kidney Int 68
: 1096
–1108, 2005[CrossRef][Medline]
John R, Nelson PJ: Dendritic cells in the kidney.
J Am Soc Nephrol 18
: 2628
–2635, 2007[Abstract/Free Full Text]
Landwehr DM, Carvalho JS, Oken DE: Micropuncture studies of the filtration and absorption of albumin by nephrotic rats.
Kidney Int 11
: 9
–17, 1977[Medline]
Lewy JE, Pesce A: Micropuncture study of albumin transfer in aminonucleoside.
Pediatr Res 7
: 553
–559, 1973[Medline]
Gudehithlu KP, Pegoraro A, Dunea G, Arruda JA, Singh AK: Degradation of albumin by the renal proximal tubule cells and the subsequent fate of its fragments.
Kidney Int 65
: 2113
–2122, 2004[CrossRef][Medline]
Nakase I, Niwa M, Takeuchi T, Sonomura K, Kawabata N, Koike Y, Takehashi M, Tanaka S, Ueda K, Simpson JC, Jones AT, Sugiura Y, Futaki S: Cellular uptake of arginine-rich peptides: Roles for macropinocytosis and actin rearrangement.
Mol Ther 10
: 1011
–1022, 2004[CrossRef][Medline]
Yewdell JW, Bennink JR: Brefeldin A specifically inhibits presentation of protein antigens to cytotoxic T lymphocytes.
Science 244
: 1072
–1075, 1989[Abstract/Free Full Text]
Rock KL, Gramm C, Rothstein L, Clark K, Stein R, Dick L, Hwang D, Goldberg AL: Inhibitors of the proteasome block the degradation of most cell proteins and the generation of peptides presented on MHC class I molecules.
Cell 78
: 761
–771, 1994[CrossRef][Medline]
Dick LR, Cruikshank AA, Grenier L, Melandri FD, Nunes SL, Stein RL: Mechanistic studies on the inactivation of the proteasome by lactacystin: A central role for clasto-lactacystin beta-lactone.
J Biol Chem 271
: 7273
–7276, 1996[Abstract/Free Full Text]
Gorevic PD, Levo Y, Chatpar PC, Frangione B, Franklin EC: Some effects of the administration of endotoxin in mice: Specific cleavage of serum albumin by an acid protease and the generation of amyloid serum component.
J Clin Invest 63
: 254
–261, 1979[CrossRef][Medline]
Kaiser VL, Sifri ZC, Senthil M, Dikdan GS, Lu Q, Xu DZ, Deitch EA: Albumin peptide: A molecular marker for trauma/hemorrhagic-shock in rat mesenteric lymph.
Peptides 26
: 2491
–2499, 2005[CrossRef][Medline]
Peters T Jr, Hawn C: Isolation of two large peptide fragments from the amino- and carboxyl-terminal positions of bovine serum albumin.
J Biol Chem 242
: 1566
–1573, 1967[Abstract/Free Full Text]
Weber G, Young LB: Fragmentation of bovine serum albumin by pepsin: I. The origin of the acid expansion of the albumin molecule.
J Biol Chem 239
: 1415
–1423, 1964[Free Full Text]
Soos TJ, Sims TN, Barisoni L, Lin K, Littman DR, Dustin ML, Nelson PJ: CX3CR1+ interstitial dendritic cells form a contiguous network throughout the entire kidney.
Kidney Int 70
: 591
–596, 2006[Medline]
Donadelli R, Zanchi C, Morigi M, Buelli S, Batani C, Tomasoni S, Corna D, Rottoli D, Benigni A, Abbate M, Remuzzi G, Zoja C: Protein overload induces fractalkine upregulation in proximal tubular cells through nuclear factor kappaB- and p38 mitogen-activated protein kinase-dependent pathways.
J Am Soc Nephrol 14
: 2436
–2446, 2003[Abstract/Free Full Text]
Delamarre L, Pack M, Chang H, Mellman I, Trombetta ES: Differential lysosomal proteolysis in antigen-presenting cells determines antigen fate.
Science 307
: 1630
–1634, 2005[Abstract/Free Full Text]
Voisine C, Hubert FX, Trinite B, Heslan M, Josien R: Two phenotypically distinct subsets of spleen dendritic cells in rats exhibit different cytokine production and T cell stimulatory activity.
J Immunol 169
: 2284
–2291, 2002[Abstract/Free Full Text]
Vermeulen M, Giordano M, Trevani AS, Sedlik C, Gamberale R, Fernandez-Calotti P, Salamone G, Raiden S, Sanjurjo J, Geffner JR: Acidosis improves uptake of antigens and MHC class I-restricted presentation by dendritic cells.
J Immunol 172
: 3196
–3204, 2004[Abstract/Free Full Text]
Andreev OA, Dupuy AD, Segala M, Sandugu S, Serra DA, Chichester CO, Engelman DM, Reshetnyak YK: Mechanism and uses of a membrane peptide that targets tumors and other acidic tissues in vivo.
Proc Natl Acad Sci U S A 104
: 7893
–7898, 2007[Abstract/Free Full Text]
Rock KL, Shen L: Cross-presentation: Underlying mechanisms and role in immune surveillance.
Immunol Rev 207
: 166
–183, 2005[CrossRef][Medline]
Matzinger P: The danger model: A renewed sense of self.
Science 296
: 301
–305, 2002[Abstract/Free Full Text]
Cunningham MW: Cardiac myosin and the TH1/TH2 paradigm in autoimmune myocarditis.
Am J Pathol 159
: 5
–12, 2001[Free Full Text]
Vigneron N, Stroobant V, Chapiro J, Ooms A, Degiovanni G, Morel S, van der Bruggen P, Boon T, Van den Eynde BJ: An antigenic peptide produced by peptide splicing in the proteasome.
Science 304
: 587
–590, 2004[Abstract/Free Full Text]
Sinn DI, Lee ST, Chu K, Jung KH, Kim EH, Kim JM, Park DK, Song EC, Kim BS, Yoon SS, Kim M, Roh JK: Proteasomal inhibition in intracerebral hemorrhage: Neuroprotective and anti-inflammatory effects of bortezomib.
Neurosci Res 58
: 12
–18, 2007[CrossRef][Medline]
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Cross Dendritic Cells Anger T Cells after Kidney Injury
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Abstract
Introduction
release by enzyme-linked immunospot (ELISPOT) assay, was not evident at the first encounter (Figure 2A). When ALB1-24-primed CD8+ T cells were restimulated by syngeneic pulsed DC, they showed a significant activation (P < 0.05 versus unpulsed DC), disclosing the antigenic nature of the self-peptide (Figure 2B). To assess further the intracellular pathways involved in ALB1-24 processing and presentation by DC, we performed experiments in the presence of amiloride analog 5-(N-ethyl-N-isopropyl) amiloride (EIPA), which inhibits antigen uptake by macropinocytosis,18 or brefeldin A (BfA), which interferes with the egress of newly synthesized MHC molecules.19 Activation of CD8+ T cells was completely prevented by both EIPA and BfA (Figure 2B). We then determined the possible involvement of proteasome, known to produce most antigenic peptides presented by MHC-I,20 in CD8+ T cell activation by DC. The proteasome inhibitor lactacystin21 completely prevented the stimulation of CD8+ T cells by DC preexposed to ALB1-24 (Figure 2B). These findings indicate that, through the proteasome, DC are effective in processing protein fragments into smaller peptides, which are presented to CD8+ T cells, leading to their activation. 
+ DC exhibit strong CD8+ T cell stimulatory capacity.29 We have identified the presence of this subset in the rat remnant kidney (see Supplemental Figure 8). Whether such a population acts as cross-presenting cell in this model of proteinuric nephropathy merits further investigations. 
) or without (
) the synthetic ALB1-24 (35 µg/ml) and used to stimulate syngeneic CD8+ T cells. CD8+ T cell activation was evaluated by IFN-
). (B) Secondary MLR. After 3-d primary MLR, primed CD8+ T cells were harvested and cultured again in a 2-d secondary stimulation with DC previously pulsed with or without ALB1-24. As positive control, unpulsed DC were cultured in secondary MLR with allogeneic LW T cells recovered at the end of primary MLR (
) or without (