IT IS WELL KNOWN that the binding ofT-cell receptor with antigen/major histocom-
patibility complex (MHC) class II on antigen-presenting cells (APCs) alone is insufficient toactivate CD4-positive (CD4�) T cells. A secondor costimulatory signal is required for T-cellactivation. Failure to receive this signal afterantigen presentation may cause T-cell clonal an-ergy.1 CD80 (B7-1) and CD86 (B7-2) can pro-vide this costimulatory signal when they bind totheir CD28/cytotoxic T-lymphocyte–associatedantigen 4 (CTLA-4) receptors on T cells.
CD80 and CD86 are cell-surface glycopro-teins belonging to the B7 family, found only onsuch APCs as macrophages, dendritic cells, andactivated B cells.1,2 They generally are not ex-
pressed on unstimulated APCs, although onlyCD86 may be expressed at a low level.3,4 How-ever, they can be induced, and levels of expres-sion increase rapidly when APCs are activatedby a variety of stimuli.2-4 Structures of CD80 andCD86 are very similar in that they both have twoextracellular domains, a transmembrane domain,and a short cytoplasmic domain, although theiramino acid sequences have only approximately25% similarity.1
Some evidence suggests that CD80 and CD86contribute to T helper subtype 1 (TH1) and TH2immune responses, respectively.5,6 However,other reports indicate no difference betweenCD80 and CD86 in inducing TH1/TH2 cell–mediated immune reactions.7,8 It seems the rolesof CD80 and CD86 in activating CD4� T cellsdiffer according to the period of immune re-sponse. CD86 has a critical role before antigenexposure, whereas CD80 has an important role incontrol of the immune response after antigenexposure,9 although the exact role of these twomolecules is still unclear.
Crescentic glomerulonephritis (GN) is the mostsevere form of human GN, with a characteristicof rapid deterioration in renal function. It hasbeen shown that this disease is characterized byTH1 cell–directed delayed type hypersensitivity(DTH)-like response in both experimental cres-centic GN10,11 and human crescentic GN.12,13
From the Department of Internal Medicine, School ofMedicine, Tokai University, Isehara, Kanagawa, Japan.
Received July 29, 2002; accepted in revised form January3, 2003.
Supported in part by grants from Tokai University; theMinistry of Health, Labor and Welfare; and the Ministry ofEducation, Culture, Sports, Science, and Technology ofJapan.
Address reprint requests to Qiong Wu, MD, Departmentof Internal Medicine, School of Medicine, Tokai Univer-sity, Isehara, Kanagawa, 259-1193 Japan. E-mail:[email protected]
American Journal of Kidney Diseases, Vol 41, No 5 (May), 2003: pp 950-961950
However, how these CD4�� T cells are acti-vated presently is obscure.
Recent studies showed that both CD80 andCD86 can induce TH1-mediated injury, but theyhave distinct roles in different diseases. CD80 isrelated to clinical acute relapse in multiple scle-rosis,14 whereas CD86 is essential for T-cellactivation in insulin-dependent diabetes mod-els.15 CD86 also has a crucial role in the initia-tion and subsequent progression of Sjogren’ssyndrome.16 In an experimental model of crescen-tic GN, CD80 and CD86 appeared to be relatedto crescent formation and glomerular accumula-tion of CD4� T cells.17,18 It still is not knownwhether these two costimulatory molecules havea role in human crescentic GN. In the presentstudy, we investigated CD80 and CD86 expres-sion in human crescentic GN and identified thetype of APC.
PATIENTS AND METHODS
PatientsRenal tissues obtained from 12 patients were used in this
study. Eleven tissue specimens were from open renal biop-sies, and one tissue specimen was from an autopsy. Alltissues underwent light microscopic and direct immunofluo-rescence examination for diagnosis. Some tissues also weresubjected to electron-microscopic examination. CrescenticGN was diagnosed in all 12 patients according to WorldHealth Organization criteria.
Clinical parameters of all patients at the time of renalbiopsy were collected, except for data from one patient, whodied within 48 hours after referral to our hospital and someclinical examinations were not performed when she enteredthe hospital. Anti–glomerular basement membrane (anti-GBM) crescentic GN was diagnosed in two patients, andpolyangiitis with positive myeloperoxidase (MPO)-antineu-trophil cytoplasmic autoantibody (ANCA), in three patients.The others were all MPO-ANCA associated, except one casewas proteinase-3–ANCA associated. Ten open renal biopsyspecimens from patients with non–immunoglobulin A mes-angial proliferative GN (PGN) with minor tissue damagewere used as controls, because patients who had kidneycarcinoma were of advanced age and their uninvolved por-tions of removed kidneys had global glomerulosclerosiswith tubulointerstitial damage.
MethodsImmunoperoxidase staining. All renal tissues were fro-
zen in ornithine carbamoyletransferase compound in dryice-acetone and stored at �80°C until the study. Serial andmirror sections were cut at 3 to 4 �m, air-dried for 30minutes, and fixed with 100% acetone at 4°C for 10 minutes.After preincubation with 10% normal goat serum in phos-phate-buffered saline (PBS)/1% bovine serum albumin atroom temperature for 20 minutes, sections were incubated
with primary antibodies at the optimal dilution for 1 hour orovernight at 4°C. Endogenous peroxidase activity wasblocked by 0.3% hydrogen peroxide in methanol for 30minutes. Next, sections were incubated with Envision�
(Dako, Tokyo, Japan) for 30 minutes. Slides were rinsed inPBS after each step. Reaction products were developed with3,3�-diaminobenzidine and hydrogen peroxide. After stain-ing with periodic acid–Schiff (PAS) solution and counter-staining with Mayer’s hematoxylin, slides were mounted forlight microscopic examination.
Mirror sections were used to stain antibody pairs: CD86and CD68, CD86 and CD14, and CD80 and HLA-DR.
Scoring technique. CD86�, CD4�, CD14�, and CD68�
cells are defined as cells with brown around the nuclei.Periglomerular area is defined as the area between Bow-man’s capsule and adjacent tubules. Peritubular interstitiumindicates the interstitial area, excluding periglomerular andperivascular areas from interstitium. Partially ruptured Bow-man’s capsule is defined as that with divided intraglomerularand periglomerular cells. Completely ruptured Bowman’scapsule is defined as that with undivided intraglomerular andperiglomerular cells.
Intraglomerular, periglomerular, and peritubular intersti-tial CD86�, CD4�, CD14�, and CD68� cells were counted.All glomeruli in sections excluding sclerotic ones werecalculated. Five to 37 glomeruli were counted in eachsection (average, 14 glomeruli). Intraglomerular positivecells were positive cells in the glomerulus and crescent withunruptured or partially ruptured Bowman’s capsules, as-sessed as mean number of cells per glomerular cross-section. Periglomerular positive cells were positive cells inthe periglomerular area of the glomerulus with unruptured orpartially ruptured Bowman’s capsule, assessed as meannumber of cells per glomerulus. To evaluate correctly therole of infiltrating cells in glomerular damage, intraglomeru-lar and periglomerular positive cells of all glomeruli, includ-ing those with completely ruptured Bowman’s capsules,were counted, and results are shown as mean number ofpositive cells per intraglomerular plus periglomerular area.All peritubular interstitial positive cells in each section werecounted using an eyepiece, and results are expressed asmean number of cells per 0.0625 mm2. A CD80� tubulus aredefined as a tubulus with one or more positive cells. Num-bers of CD80� tubuli and total tubuli were counted, andresults are expressed as percentage of positive tubuli.
Statistical analysis. Pearson’s correlation analysis wasused in this study with SPSS software (SPSS Inc, Chicago,IL) to observe relationships between number of CD86� cellsand CD80� tubular values with renal function; number ofCD86� cells with CD4� T cells, CD14� cells, and CD68�
cells; and CD80� tubular value and peritubular interstitialCD4� cells. Mann-Whitney test was used to compare clini-cal differences and differences in numbers of intraglomeru-lar, periglomerular, intraglomerular plus periglomerular, andperitubular interstitial positive cells between patients with
CD80 AND CD86 IN HUMAN CRESCENTIC GN 951
crescentic GN and PGN. P less than 0.05 is consideredsignificant.
RESULTS
Clinical Findings
Clinical data for patients with crescentic GNand PGN are listed in Table 1, with mean valuesand SDs for normally distributed data and medi-ans and interquartile ranges for data not normallydistributed. Except for sex and serum total pro-tein level, all clinical parameters showed signifi-cant differences at P less than 0.05 betweenpatients with crescentic GN and those with PGN.
CD4, CD14, and CD68 Expression
Similar distributions of CD4�, CD14�, andCD68� cells were observed in patients withcrescentic GN and PGN. These cells were inglomeruli and interstitium. In PGN, intraglomeru-lar positive cells were almost in the glomerularcapillaries, whereas in crescentic GN, these cellsalso were observed in adhesion to Bowman’scapsule, segmental sclerosis, most cellular cres-cents, and some cellular-fibrosis crescents.
In crescentic GN, monocyte/macrophages weremore common in glomeruli, and CD4� T cellswere more prevalent in the interstitium. Figure1A shows CD4� T cells in an MPO-ANCA–positive patient. Many CD4� T cells and macro-phages were found in the local interstitial cellinfiltration area and often surrounded atrophic
tubuli, which showed thickened basement mem-brane in PAS staining (Fig 2A and B). In mostcases, monocyte/macrophages and CD4� T cellsappeared among tubular epithelial cells (tubuli-tis) (Fig 2C and D) and appeared in the tubularlumen.
Mean numbers of intraglomerular, periglo-merular, intraglomerular plus periglomerular, andperitubular interstitial CD4�, CD14�, and CD68�
cells in patients with crescentic GN and PGN arelisted in Table 2. The number of these cells inpatients with crescentic GN was larger than inpatients with PGN (P � 0.001). Intraglomerular,periglomerular, intraglomerular plus periglomeru-lar, and peritubular interstitial CD14� and CD68�
cells showed positive correlations with corre-sponding CD4� T cells, except for periglomeru-lar CD14� cells and CD4� T cells (data notshown).
CD80 Expression
In crescentic GN, CD80 expression was thesame as in PGN with only a wider range. CD80was expressed only in some tubular epithelialcells, but not in the glomerulus or interstitium(Fig 2E).
No difference was found in CD80 distributionin tubuli among 12 crescentic GN sections. StrongCD80 expression was observed in mild atrophictubuli, which had mild thickened or windingbasement membrane in PAS staining, and tubuli
Table 1. Clinical Parameters of Patients With Crescentic GN and PGN
NOTE. Normally distributed data are shown as mean values and SDs only. To convert blood urea nitrogen from mg/dL tommol/L, multiply by 0.357; to convert serum creatinine in mg/dL to �mol/L, multiply by 88.4; to convert total protein andalbumin from g/dL to g/L, multiply by 10.
Fig 1. In an MPO-ANCA–positive patient, (A, B) serial sections show similar distributions of (A) CD4 and (B)CD86; and (C) CD86 was expressed in glomeruli, periglomerular area, and peritubular interstitium, but not tubularepithelial cells. Glomerulus 1 shows intense CD86 expression in the crescent and corresponding periglomerulararea. Glomerulus 2 shows fewer intraglomerular and periglomerular positive cells. Glomerulus 3 shows a smallcrescent with intense CD86 expression in the periglomerular area. Immunostaining of CD86 shows (D) intense CD86expression in cellular crescents and corresponding periglomerular area, (E) less CD86 expression in fibrocellularcrescents, and (F) reduced CD86 expression in global sclerotic glomeruli. Serial sections of (G) CD86 with (H) CD68show intense CD86 expression in (G) the area of adhesion to Bowman’s capsule, and (H) macrophages appeared inthe same area. (I) CD86� cells in the tubular lumen and (J) many CD86� cells in Bowman’s space and the connectedtubular lumen. (K, L) Mirror sections show that most CD86� cells in (K) the glomerulus also were (L) CD68�. Thesame arrow or arrowhead shows the same cell.
CD80 AND CD86 IN HUMAN CRESCENTIC GN 953
with such surrounding infiltrating cells as T cellsand monocyte/macrophages (Fig 2G through J).Completely atrophic tubuli, which had markedthickened or thinned and sometimes broken base-ment membrane in PAS staining, had no CD80expression (Fig 2F). An MPO-ANCA–positivepatient had a large number of completely atro-phic tubuli, and his CD80� tubular values were
very low. Mean percentages of CD80� tubuli inpatients with crescentic GN and PGN are listedin Table 2. A significant difference was foundbetween these two groups. No correlation wasfound between CD80� tubular value and numberof peritubular interstitial CD4� T cells in patientswith crescentic GN (Table 3) and those withPGN (data not shown).
Fig 1 (continued).
WU ET AL954
Fig 2.
Fig 2 (continued).
4™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™Fig 2. Immunostaining of CD4 and CD68 shows many (A) peritubular interstitial infi ltrating T cells and (B)
macrophages surrounded by atrophic tubuli, which show thickened basement membrane in PAS staining. (C) TCells and (D) macrophages among tubular epithelial cells induce tubulitis. (E) Immunostaining of CD80 in anMPO-ANCA–positive patient. CD80 was expressed only on tubular epithelial cells, but not in the glomerulus(asterisk) and interstitium. (F) Completely atrophic tubuli (asterisk) had no CD80 expression. Serial sections of (G)CD80 with (H) CD4 and (I) CD80 with (J) CD68 show that T cells and macrophages surrounded some CD80� tubuli.Serial sections of (K) CD80 with (L) HLA-DR and (M) CD80 with (N) ICAM-1 show that some CD80� tubular epithelialcells expressed HLA-DR and some expressed ICAM-1 simultaneously. The same mark shows the same tubulus.
WU ET AL956
On serial sections, some atrophic tubular epi-thelial cells expressed CD80 and HLA-DR simul-taneously (Fig 2K and L), and some expressedCD80 and ICAM-1 simultaneously (Fig 2M andN). Many CD80� tubuli did not express HLA-DRor ICAM-1.
CD86 Expression
CD86 was expressed in glomerulus and inter-stitium (Fig 1B through G). Tubular epithelialcells showed no CD86 expression.
In PGN, CD86 was rarely observed. Intraglo-merular CD86� cells were present in glomerularcapillaries.
In crescentic GN, no difference in CD86 ex-pression was found among 12 sections with orwithout vasculitis, ANCA, or anti-GBM anti-body. Although a small number of normal glo-meruli, which refers to those with no adhesion,sclerosis, or crescent and no Bowman’s capsuleinjury, were surrounded by many CD86� cells,most normal glomeruli showed only a few CD86�
cells in the glomerulus, periglomerular area,and/or hilar area. Intense CD86 expression at thesite of adhesion to Bowman’s capsule was shown(Fig 1G), in which monocyte/macrophages some-times also were observed (Fig 1H), and some ofthese glomeruli were associated with intensecorresponding periglomerular CD86 expression.Intense CD86 expression also was shown inmost cellular crescents (Fig 1D), and the inten-sity of periglomerular CD86 expression rangedfrom mild to moderate when Bowman’s capsulewas intact (Fig 1C and J). When Bowman’scapsule was partially ruptured, CD86 stainingwas seen clearly from inside glomeruli to theperiglomerular area through the ruptured Bow-man’s capsule (Fig 1B and C), and when Bow-man’s capsule was completely ruptured, CD86staining showed a relatively higher intensity inthe periglomerular area than in glomeruli (Fig1K). Fewer CD86� cells were observed in somefibrocellular crescents (Fig 1E). In global scle-rotic glomeruli, reduced CD86 staining was ob-served in glomeruli, and staining of mild tomoderate intensity in the periglomerular areasurrounding the glomeruli (Fig 1F). In the tubu-lointerstitium, some CD86� cells were observedin the peritubular area, and most CD86� cellswere identified at sites of local cell infiltration.CD86� cells were found among tubular epithe-
Tab
le2.
Nu
mb
ers
ofC
D4�
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14�
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and
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319
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4.19
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8.81
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223
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0116
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28.3
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0.27
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CD80 AND CD86 IN HUMAN CRESCENTIC GN 957
lial cells in rare cases and occasionally in thetubular lumen (Fig 1I) that were considered tooriginate from the glomerulus (Fig 1J).
Mean numbers of intraglomerular, periglo-merular, intraglomerular plus periglomerular, andperitubular interstitial CD86� cells in patientswith crescentic GN and PGN are listed in Table2. There were more of these cells in patients withcrescentic GN than in patients with PGN (P �0.001). Similar distributions of CD86 and CD4were observed (Fig 1A and B), and positivecorrelations were shown between all numbers ofintraglomerular, periglomerular, intraglomerularplus periglomerular, and peritubular interstitialCD86� cells with corresponding CD4� T cells,CD14� cells, and CD68� cells, except for peri-glomerular CD86� cells with CD14� cells inpatients with crescentic GN (Table 3). In patientswith PGN, positive correlations also were ob-served between CD86� cells and correspondingCD4� T cells, CD14� cells, and CD68� cells,except for intraglomerular cells (data not shown).
In mirror sections, most CD86� cells in renal
tissues were monocyte/macrophages (Fig 1K andL), but some were other cells because of negativestaining for CD14 and CD68 molecules.
Clinical Correlation
No correlations were found between CD80�
tubular values and clinical parameters at the timeof renal biopsy. Intraglomerular and periglomeru-lar CD86� cells also showed no correlations withclinical parameters at the time of renal biopsy.Intraglomerular plus periglomerular and peritu-bular interstitial CD86� cells showed a negativecorrelation with serum albumin concentration(R � �0.650; P � 0.030 and R � �0.606; P �0.048). Only peritubular interstitial CD86� cellsshowed a positive correlation with deteriorationof renal function (Table 4).
DISCUSSION
It now is accepted that CD80/CD86 can sup-ply costimulatory signals to activate T cells bybinding to their CD28/CTLA-4 receptors on Tcells. Crescentic GN is considered a CD4� T-cell–
Table 3. Correlations Between CD80�, CD86�, and CD4�, CD68�, and CD14� Values
Abbreviations: G, intraglomerular; P, periglomerular; G � P, intraglomerular plus periglomerular; IS, peritubular interstitial.
WU ET AL958
directed DTH injury. It still is not clear whetherCD80 and CD86 are connected to human crescen-tic GN, relate with renal CD4� T-cell accumula-tion, and are involved in crescent formation. Inthis study, we investigated the expression ofthese two costimulatory molecules in humancrescentic GN.
Findings from experimental crescentic GNindicated that both the surface molecules CD80and CD86 are involved in glomerular accumula-tion of CD4� T cells and crescent formation.17,18
Based on our results, we found that CD86 hasimportant roles in the accumulation of CD4� Tcells in human crescentic GN. CD86 was ex-pressed strongly and widely in the glomerulusand interstitium in crescentic GN tissues, butonly slightly in control tissues. Moreover, posi-tive correlation between number of CD86� andCD4� T cells also was shown. CD80 was ex-pressed on tubular epithelial cells in all 12 sec-tions of crescentic GN, and the positive ratio washigher than that in PGN tissues. Partially atro-phic tubuli showed strong CD80 expression andwere surrounded by many infiltrating CD4� Tcells and monocyte/macrophages.
It appears in this study that these two costimu-latory molecules did not contribute equally tocrescentic GN. We found no expression of CD80on monocyte/macrophages in either the intraglo-merular or periglomerular area of glomeruli withor without crescent formation or in the peritubu-lar interstitium. CD80 expression was only shownon tubular epithelial cells. This finding suggeststhat CD80 does not have a dominant role increscent formation and CD4� T cell accumula-tion. Conversely, CD86 seems more importantthan CD80 because the CD86 molecule wasexpressed on monocyte/macrophages, which areprofessional APCs, and showed extensive distri-bution and intensive expression in the kidneysand positive correlation with CD4� T cells.
Macrophages appeared in this disease to act asthe main APC. Most of them showed CD86expression, and the number of macrophages washigher than that in controls with a positive corre-lation with CD4� T cells. Sainio-Pollanen et al21
showed that in the pancreas of nonobese diabeticmice, many of the CD80/CD86� cells were mac-rophages, and these macrophages accumulatedaround islets,21 suggesting a role of these cells inthe local regulation of immune responses. Both
monocyte/macrophages and glomerular epithe-lial cells are considered major components of thecellular crescent.22,23 Glomerular parietal epithe-lial cells form early-stage crescents,24,25 and whenBowman’s capsule is ruptured, macrophages andT cells become the main components.24 We foundin human crescentic GN that CD86� cells, mono-cyte/macrophages, and CD4� T cells increasedin cellular crescents when Bowman’s capsuleruptured. Experimental crescentic GN has shownthat CD28-B7 blockade inhibited the formationof crescents,17,18 suggesting that the CD86 mole-cule has an important role in the initiation andprogression of cellular crescents, probablythrough monocyte/macrophages. Adhesion is con-sidered to be related to damage of podocytes,which can induce adhesion when detached fromthe basement membrane.26 In our study, strongCD86 expression was observed in adhesion, andmost of these CD86� cells were shown to bemonocyte/macrophages, suggesting that mono-cyte/macrophages with CD86 expression alsoare related to podocyte damage.
Periglomerular mononuclear cells were foundto be involved in the disruption of Bowman’scapsule through a DTH mechanism.22 In theperiglomerular area, many molecules were iden-tified, including tumor necrosis factor-�, interleu-kin-1�, and interleukin-2 receptor in ANCA-positive GN27; transforming growth factor-�messenger RNA in murine lupus nephritis28;monocyte chemoattractant protein-4 in humanacute renal allograft rejection and vasculitic GN29;and osteopontin in human crescentic GN.30 Inthis study, strong CD86 expression was observedin the periglomerular area associated with signifi-cant CD4� T-cell and monocyte/macrophage ac-cumulation, similar to experimental lupus nephri-tis.31 We also found that glomeruli with rupturedBowman’s capsule showed stronger periglomeru-lar CD86 expression and greater CD4� T-celland monocyte/macrophage accumulation thanthose with intact Bowman’s capsule. We consid-ered that the coaction of periglomerular macro-phages with CD4� T cells may increase theirnumber and finally cause disruption of Bow-man’s capsule through a DTH response andparticipate in the progression of cellular cres-cents, as well as glomerular structural collapse.
Recent evidence has indicated an accessoryrole for epithelial cells in autoimmune disease.
CD80 AND CD86 IN HUMAN CRESCENTIC GN 959
Bronchiolar epithelial cells with aberrant CD80expression might be involved in pulmonary fibro-sis.32 Biliary epithelial cells with CD86 expres-sion might induce bile duct destruction in pri-mary biliary cirrhosis and primary sclerosingcholangitis.33 As in the case of these epithelialcells, renal tubular epithelial cells also couldacquire an antigen-presenting function for infil-trating T cells through the expression of CD80molecules. Immunostaining using serial sectionsshowed that some tubular epithelial cells, espe-cially those that were mildly atrophic and sur-rounded by infiltrating cells, expressed HLA-DRsimultaneously. Hagerty et al34 found that whenmurine kidney tubular epithelial cells were trans-fected with complementary DNA for B7, theystimulated T-cell proliferation in response to an-tigens. Recently, Banu et al35 showed that murinerenal tubular epithelial cells expressed MHCclass II and costimulatory molecule CD80 whenthey were stimulated. These findings indicatethat tubular epithelial cells can act as APCs. Webelieve that when tubular epithelial cells expressCD80 and MHC class II molecules simulta-neously, they acquire an antigen-presenting func-tion.
The existence of the costimulatory moleculesCD80 and CD86 in crescentic GN tissues sug-gests that antigens may be involved in this dis-ease. We failed to find differences in renal CD80/CD86 distribution in patients with perinuclearANCA, cytoplasmic ANCA, or anti-GBM anti-body. There also was no difference in the actionof monocyte/macrophages as the main APC.These findings suggest that although antigens increscentic GN are different, APCs (monocyte/macrophages) and the process of antigen presen-tation, as well as activation of CD4� T cells, arethe same, even if there may be some differencesin rates of antigen processing, antigen presenta-tion, T-cell proliferation, and cytokine release.They also suggest that CD80 and CD86 expres-sion is not specific in one or two renal diseases.It’s necessary to investigate CD80/CD86 expres-sion in other types of renal diseases.
In this study, we show that the costimulatorymolecules CD80 and CD86 have different distri-butions in human crescentic GN. CD86 is con-cerned with crescent formation and renal CD4�
T-cell accumulation. Monocyte/macrophages actas the main APC, and tubular epithelial cells also
can act as APCs when they express CD80 andMHC class II molecules. Increased expression ofCD86 in the peritubular interstitium correlateswith the progression of renal dysfunction.
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