Glomerular Endothelial Cells Impairs Activation of Protein C in Human

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TNF Regulates Essential Alternative Complement Pathway Components and Impairs Activation of Protein C in Human Glomerular Endothelial Cells This information is current as of September 25, 2021. Sarah E. Sartain, Nancy A. Turner and Joel L. Moake J Immunol published online 16 December 2015 http://www.jimmunol.org/content/early/2015/12/15/jimmun ol.1500960 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2015 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published December 16, 2015, doi:10.4049/jimmunol.1500960 The Journal of Immunology

TNF Regulates Essential Alternative Complement Pathway Components and Impairs Activation of Protein C in Human Glomerular Endothelial Cells

Sarah E. Sartain,*,† Nancy A. Turner,‡ and Joel L. Moake‡

Atypical hemolytic uremic syndrome (aHUS) is a thrombotic microangiopathy with severe renal injury secondary to an overactive alternative complement pathway (AP). aHUS episodes are often initiated or recur during inflammation. We investigated gene expression of the surface complement regulatory proteins (CD55, CD59, CD46, and CD141 [thrombomodulin]) and AP components in human glomerular microvascular endothelial cells (GMVECs) and in HUVECs, a frequently used investigational model of endothelial cells. Surface complement regulatory proteins were also quantified by flow cytometry. All experiments were done with and without exposure to IL-1b or TNF. Without cytokine stimulation, we found that GMVECs had greater AP activation than did Downloaded from HUVECs. With TNF stimulation, THBD gene expression and corresponding CD141 surface presence in HUVECs and GMVECs were reduced, and gene expression of complement components C3 (C3) and factor B (CFB) was increased. Consequently, AP activation, measured by Ba production, was increased, and conversion of protein C (PC) to activated PC by CD141-bound thrombin was decreased, in GMVECs and HUVECs exposed to TNF. IL-1b had similar, albeit lesser, effects on HUVEC gene expression, and it only slightly affected GMVEC gene expression. To our knowledge, this is the first detailed study of the expression/display of AP components and surface regulatory proteins in GMVECs with and without cytokine stimulation. In http://www.jimmunol.org/ aHUS patients with an underlying overactive AP, additional stimulation of the AP and inhibition of activated PC–mediated anticoagulation in GMVECs by the inflammatory cytokine TNF are likely to provoke episodes of renal failure. The Journal of Immunology, 2016, 196: 000–000.

typical hemolytic uremic syndrome (aHUS) is a thrombotic convertase of the AP) (17), releasing the activation product Ba. The microangiopathy presenting with microangiopathic hemo- C3 convertase is stabilized by factor P (FP; properdin) (18–20). The A lytic anemia, thrombocytopenia, and renal failure secondary Bb in C3bBb cleaves C3 to generate additional C3b; as the ratio of to formation of platelet-ﬁbrin clots in the glomerular microvasculature C3btoBbincreases,C3bBbC3b(theC5convertase)formsand (1–3). aHUS is associated with heterozygous mutations in compo- cleaves C5 to C5b, releasing the soluble C5a fragment (17, 21). by guest on September 25, 2021 nents of the alternative complement pathway (AP) that result in ex- The AP is regulated by both soluble and cell surface–bound cessive AP activation. Defects include loss-of-function mutations in proteins. FH and FI are soluble inhibitory regulators of the AP: FH the genes for factor H (FH) (4, 5), factor I (FI) (6, 7), CD46 (8, 9), suppresses the formation or persistence of C3bBb (22, 23), and FI, and CD141 (thrombomodulin) (10), or gain of function mutations in along with FH, promotes the cleavage/inactivation of C3b (24). C3 (11) or factor B (FB) (12). CD46 and CD141 are cell surface membrane regulatory proteins The AP is initiated when C3b is cleaved from C3 and attaches to an that have functions supplementary to FH, that is, all three act as activating surface, releasing a soluble C3a fragment in the process (13, cofactors for FI-mediated proteolysis of C3b (10, 25). CD141 is 14). FB then combines with C3b to form C3bB (15, 16), and factor D found almost exclusively on endothelial cell (EC) surfaces (26) (FD) cleaves FB in this complex to form C3bBb (the active C3 and has AP regulatory function analogous to complement receptor 1 (CD35), found exclusively on human erythrocytes, polymorphonuclear leukocytes, monocytes, and B lymphocytes (27, 28). CD141 *Section of Hematology–Oncology, Department of Pediatrics, Texas Children’s Can- cer and Hematology Centers, Houston, TX 77030; †Baylor College of Medicine, also functions as a natural anticoagulant by binding thrombin and Houston, TX 77030; and ‡Department of Bioengineering, Rice University, Houston, diverting thrombin substrate speciﬁcity to the activation of protein C TX 77005 (PC). Activated PC, with bound protein S, cleaves and inactivates Received for publication April 24, 2015. Accepted for publication November 13, coagulation factors Va and VIIIa (29) (Fig. 1, Table I). 2015. CD55 and CD59 are two other negative complement surface This work was supported by grants from the Hemostasis and Thrombosis Research regulatory proteins. CD55 accelerates the decay of C3 convertase Society (sponsored by Baxalta US, Inc.), the Mary R. Gibson Foundation, and the Mabel and Everett Hinkson Memorial Fund. (30). CD59 prevents accumulation of additional C9 molecules into Address correspondence and reprint requests to Dr. Sarah E. Sartain, Baylor College the C5b-(9)(1) membrane attack complex (31) (Table I). of Medicine/Texas Children’s Hospital, 6701 Fannin Street, Suite 1580, Houston, TX Uncleaved ultra-large von Willebrand factor (ULVWF) multimeric 77004. E-mail address: [email protected] strings secreted by, and anchored to, stimulated HUVECs serve as The online version of this article contains supplemental material. activating surfaces for C3b binding and AP assembly and activation Abbreviations used in this article: ADAMTS-13, a disintegrin and metalloprotease (32–34). We have previously demonstrated that C3 (as C3b), FB (as with thrombospondin domains type 13; aHUS, atypical hemolytic uremic syndrome; AP, alternative complement pathway; DCT, change in cycle threshold; EC, endothe- Bb), FD, FP, and C5 (as C5b), as well as smaller quantities of FH and lial cell; FB, factor B; FD, factor D; FH, factor H; FI, factor I; FP, factor P; GMVEC, FI, attach to HUVEC-secreted and anchored ULVWF strings. In glomerular microvascular endothelial cell; PC, protein C; ULVWF, ultra-large von contrast, C4 (as C4b) does not attach to the ULVWF strings, indi- Willebrand factor; VWF, von Willebrand factor. cating that the classical and lectin pathways are not activated. The Copyright Ó 2015 by The American Association of Immunologists, Inc. 0022-1767/15/$30.00 attachment to EC-secreted/anchored ULVWF strings of C3b, Bb, and

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1500960 2 TNF AND GLOMERULAR ENDOTHELIAL CELLS

C5b occurs in quantitative and functional patterns consistent with the projection lens (Nikon, Garden City, NY), SensiCam QE CCD camera assembly of AP components into active complexes of C3 convertase (Cooke, Romulus, MI), motorized stage and dual filter wheels (Prior) with (C3bBb) and C5 convertase (C3bBbC3b) (34). single band excitation and emission filters for FITC/tetramethylrhodamine isothiocyanate/Cy5/DAPI (Chroma, Rockingham, VT). Image areas acquired In aHUS, the kidneys are affected more severely than other at original magnification 360 are 78 3 58 mm, and at 3100 are 41 3 30 mm. organs. The vulnerability of the kidney to AP-mediated injury in aHUS led us to investigate complement surface regulatory protein Flow cytometry expression and membrane presence, as well as AP component ex- Cytokine stimulation of HUVECs and GMVECs. Once confluent in T-25 pression, in glomerular microvascular ECs (GMVECs) and, for flasks, control cells were incubated for 24 h in serum-free media (MCDB- 131 plus insulin-transferrin-selenium, Life Technologies), and experimental comparison, in HUVECs. We hypothesized that GMVECs have cells were incubated for 24 h with CM131 plus TNF (10 ng/ml, Life Tech- differences in AP regulation, compared with HUVECs, thereby nologies)orIL-1b (3 ng/ml, Life Technologies), and then incubated for an explaining their susceptibility to injury in aHUS. Because additional 24 h in serum-free media plus 10 ng/ml TNF or 3 ng/ml IL-1b (total infectious/inflammatory conditions may lead to initial or recurrent cytokine exposure of 48 h). Both control and experimental flasks were incu- episodes of aHUS (35, 36), we also studied the effects of two bated in serum-free media for a total of 24 h to eliminate any proteins derived b from serum that might affect surface receptor detection. The specific con- proinflammatory cytokines, TNF and IL-1 , on complement pa- centrations and duration of exposure of TNF and IL-1b were chosen based on rameters in both EC types. We further hypothesized that these use of these cytokines in EC gene expression studies (40–42). inflammatory cytokines affect AP regulation in HUVECs and Cell surface labeling of CD55, CD46, CD59, and CD141. Control and GMVECS by altering gene expression or surface presence of the experimental cells were detached by 10 min incubation with 5 mM EDTA in +2 +2 AP components/regulators, thereby explaining the recurrences of Ca /Mg -free PBS (to retain surface proteins) and centrifuged (10 min at 400 3 g). Cells were counted using the TC10 automated cell counter (Bio- Downloaded from aHUS during inflammatory or infectious events. Rad, Hercules, CA) and resuspended in 1% BSA/PBS at 106 cells/ml. Samples of 2 3 104 GMVECs or HUVECs (20 ml) were labeled indi- Materials and Methods vidually with saturating amounts of each fluorescently conjugated mAb (see below, 1 ml each specific Ab per 20 ml cell suspension) or labeled with Cells saturating amounts of FITC-conjugated (CD55, CD59, CD46) or PE- GMVECs. Pooled primary human GMVECs were purchased from Cell conjugated (CD141) isotype controls (see below, 1 ml isotype control per Systems (Kirkland, WA, ACBRI-128 vial). GMVECs were grown in com- 20 ml cell suspension) to measure background fluorescence. Samples were plete media (CM131, MCDB-131 medium [Sigma-Aldrich], supplemented incubated for 20 min in the dark. Cells were then fixed with 0.5 ml 1% http://www.jimmunol.org/ with penicillin/streptomycin/L-glutamine [Life Technologies], plus micro- formaldehyde/PBS. Cells expressing higher fluorescence than background vascular growth supplement [Life Technologies]). (isotype control alone) were considered positive, and background fluo- HUVECs. Primary HUVECs were isolated from umbilical veins as pre- rescence was subtracted from positive fluorescence. Experiments were viously described (37), and pooled from multiple human donor umbilical repeated with increasing passage number (passages 5–6 for GMVECs and veins. HUVECs were grown in CM131 plus low-serum growth supplement passages 4–6 for HUVECs) to exclude the possibility that any differences for large vessel ECs (Life Technologies). The only difference between appreciated were related to passage differences. GMVEC media and HUVEC media was the type of growth supplement Abs to human complement regulatory proteins (flow cytometry). Abs included: added as required by cell type; culture conditions were otherwise identical. CD141, clone 1A4, mouse monoclonal IgG1,k conjugated to PE (BD Bio- k Seeding. GMVECs and HUVECs were seeded in T-25 flasks (for flow sciences, 559781); CD46, clone E4.3, mouse monoclonal IgG2a, conjugated to FITC (BD Biosciences, 555949); CD55, clone IA10, mouse monoclonal cytometry experiments), T-75 flasks (for gene expression experiments and by guest on September 25, 2021 k complement component/activated PC supernatant measurements), or on IgG2a, conjugated to FITC (BD Biosciences, 555693); and CD59, clone k gelatin-coated coverslips (for immunofluorescence microscopy experi- p282 (H19), mouse monoclonal IgG2a, conjugated to FITC (BD Biosciences, ments). Cell type was confirmed by immunofluorescence showing that 95% 555763). Isotype controls included PE mouse, clone IgG1 (BD Biosciences, k of the GMVECs or HUVECs were positive for von Willebrand Factor 349043) and FITC mouse, clone IgG2a, (BD Biosciences, 555573). (VWF) in Weibel–Palade bodies. Acquisition. Samples were acquired using FACScan (BD Biosciences), and the data were analyzed using CellQuest software (BD Biosciences). The Fluorescence microscopy instrument settings of the forward scatter and side scatter profiles were log mode. EC samples appeared as single populations and were gated based on Complement regulatory protein staining of GMVECs and HUVECs. their forward and side scatter profiles. Five thousand gated events were GMVECs and HUVECs, grown on gelatin-coated glass coverslips, were analyzed for each sample. stained for the external complement regulatory proteins CD55, CD59, CD46, and CD141, and for internal VWF (to confirm cell type). ECs were Gene expression fixed with 1% p-formaldehyde for 10 min and stained with 1 mg/ml anti- CD46, 10 mg/ml anti-CD55 or anti-CD59, or a 1:100 dilution of anti-CD141 Cytokine stimulation. Once confluent in T-75 flasks, control cells were (see details of each Ab below) plus 20 mg/ml fluorescent secondary goat incubated for 24 h in serum-free media, and experimental cells were incubated for 24 h with CM131 plus 10 ng/ml TNF or 3 ng/ml IL-1b, and then anti-mouse Alexa Fluor 647 F(ab9)2 fragment–IgG (Life Technologies, A21237), or stained with the secondary goat anti-mouse Ab alone to assess incubated for an additional 24 h in serum-free media plus 10 ng/ml TNF or background fluorescence (Supplemental Fig. 1A, 1D). The cells were fixed 3 ng/ml IL-1b (total cytokine exposure of 48 h). Both control and ex- again (to retain surface Abs). Cells were then treated with 0.2% Triton perimental flasks were incubated in serum-free media for a total of 24 h. X-100 in PBS for 5 min (to allow internal staining) and stained with 10 mg/ml RNA isolation of unstimulated and cytokine-stimulated GMVECs and polyclonal rabbit anti-human VWF (Ramco Laboratories, Sugarland, TX) HUVECs. ECs in T-75 flasks were washed with cold PBS, and RNA was plus 20 mg/ml secondary Ab chicken anti-rabbit Alexa Fluor 488 IgG (Life isolated using TRIzol (Invitrogen), chloroform extraction, and isopropanol Technologies, A21441) for 15 min, or stained with the secondary chicken precipitation. The RNA integrity was verified by 260/280 OD ratios and 1% anti-rabbit Ab alone to assess background fluorescence (Supplemental Fig. agarose/formaldehyde electrophoresis. 1B, 1E). Cell nuclei were detected with DAPI included in the mounting Real-time PCR. GMVEC and HUVEC RNA samples were reverse tran- medium (Fluoro-Gel II, Electron Microscopy Sciences). scribed using SuperScript III Supermix (Invitrogen). Samples (100 ng Abs to human complement regulatory proteins (immunostaining). Abs in- cDNA) were amplified in quadruplicate or triplicate by real-time PCR under cluded: CD141, clone QBEND/40, mouse monoclonal IgG2a (Thermo Fisher, the following conditions: 95˚C for 3 min, 40 cycles of 10 s at 95˚C, 10 s at MA1-35905); CD46, clone M177, mouse monoclonal IgG1 (Thermo Fisher, 55˚C, and 30 s at 72˚C, then 95˚ for 10 s, followed by melting curves from MA1-40183); CD55, clone BRIC-216, mouse monoclonal IgG1 (Thermo 65˚C to 95˚C (CFX96, Bio-Rad). Amplified products were detected using Fisher, MA1-91161); and CD59, clone MEM-43, mouse monoclonal IgG2a TaqMan gene expression assays (with FAM-labeled probes that span target (Thermo Fisher, MA1-82206). Verification of the specificity of these mAbs exon junctions; see Supplemental Table I for the list of assay probe IDs) has been previously published or stated by the manufacturer (38, 39). and fast advanced master mix (Life Technologies). Microscope instrumentation. Fluorescent images were acquired using IP Lab Relative quantification measurements. The relative quantification of gene software version 3.9.4r4 (Scanalytics, Fairfax, VA) on a Nikon Diaphot TE300 expression with/without cytokine stimulation in GMVECs and HUVECs was microscope equipped with CFI Plan Fluor 360 oil, numerical aperture 1.4 and calculated as described in the Applied Biosystems User Bulletin No. 2 (P/N CFI Plan Apo Lambda 3100 oil, numerical aperture 1.45 objectives plus 310 4303859) and by Livak and Schmittgen (43), as follows: change in cycle The Journal of Immunology 3 threshold (DCT) for each gene (in each cell type) = DCT (gene) 2 DCT of the standard curve, in addition to values between the lower limit and higher (GAPDH); DCT number relative to HUVECs (DDCT HUVECs)=DCT (gene limit of the standard curve (Supplemental Fig. 2). in HUVECS) 2 DCT (gene in HUVECs); and DCT number relative to FB, C3a, and C5a fluorescence immunoassays. FB, C3a, and C5a levels DD D 2 D GMVECs ( CT GMVECs)= CT (gene in GMVECs) CT (gene in were measured in unstimulated and TNF-stimulated GMVEC and HUVEC GMVECs).The fold changes relative to HUVECs were calculated by supernatant samples using the same protocol as for the C3 immunoassay. 2DDCT evaluating 2 for each gene. For each HUVEC gene, DDCT = 0 and The Abs and standard curves used were as follows: 1) FB: capture, 100 2˚ = 1. ng/well polyclonal goat anti-human FB (Complement Technologies, no. DD 2 SD calculations for CT were determined as the square root of (S1 + A235); standard, FB protein (Complement Technologies, no. A135) with a 2 S2 ), where S1 indicates the average SD of CT for each gene, and S2 in- range of 12.5–800 ng/ml; detection Abs, 0.1 mg/ml monoclonal mouse dicates the average of CT for GAPDH. The ranges for the fold changes anti–factor Ba (Quidel Corporation, no. A225) that was generated using 2DDCT relative to HUVECs were calculated by evaluating 2 plus the SD purified human FB as the Ag and is reactive with both FB and the Ba 2DDCT (low range value) and 2 minus the SD (high range value). fragment, and 0.1 mg/ml secondary donkey anti-mouse IgG-HRP (Pierce, Quantitative gene expression measurements. Changes in gene expression in no. PA1-28748). 2) C3a: capture, 50 ng/well polyclonal rabbit anti-human GMVECs and HUVECs in the presence of cytokines were calculated using the C3a (Complement Technologies, no. A218); standard, purified C3a desArg method developed by Pfaffl (44), which uses specific primer efficiencies (E) protein (Complement Technologies, no. A119) with a range of 61 pg/ml– to evaluate the amount of cDNA amplification per each PCR cycle (44): 3.9 ng/ml; detection Abs, 100 ng/ml mouse mAb to human C3a (Pierce, (21/slope) DCT(control 2 treated) DCT(control 2 treated) E =10 and Ratio = ETarget /ERef . Thermo Scientific, no. GAU 013-16-02) and 0.25 mg/ml secondary goat Genes quantified. mRNA levels were quantified for the complement reg- anti-mouse IgG-HRP (Rockland Immunochemicals, Limerick, PA). 3) ulatory proteins CD59, CD46, CD55, and THBD (gene for CD141), for the C5a: capture, 100 ng/well polyclonal rabbit anti-human C5a (Complement AP components C3, C5, CFB, CFD, CFP, CFH, and CFI, for the classical Technologies, no.A221); standard, C5a desArg protein (Complement complement component C4, and for VWF and ADAMTS13 (gene for the Technologies, no.A145) with a range of 0.156–10 ng/ml; detection Abs, VWF cleavage protein a disintegrin and metalloprotease with thrombo- 0.1 mg/ml mouse monoclonal anti-human C5a (Pierce, Thermo Scientific, spondin domains type 13 [ADAMTS-13]). VWF and ADAMTS13 were no. MA1-40162) and 0.25 mg/ml secondary goat anti-mouse IgG-HRP. Downloaded from chosen because these two genes are known to be biologically significant in Ba ELISA. Ba levels were measured in unstimulated and TNF-stimulated ECs (45, 46), and they were thought likely to be important in synthesis and GMVEC and HUVEC supernatant samples using an ELISA kit (Quidel, interpretation of the data. San Diego, CA, no. A033) with a standard curve range of 0.07–2.19 ng/ml p value calculations. The p values for the gene expression graphs (see (Supplemental Fig. 2). Figs. 2, 5, 6) were calculated with a Student t test, using the end-point gene copy numbers, after normalization to GAPDH.Ap value ,0.05 was Measurement of activated PC from EC supernatant considered statistically significant. In these experiments, activated PC was assayed by ELISA from the su- http://www.jimmunol.org/ pernatant of unstimulated or TNF-stimulated GMVECs and HUVECs after Measurement of complement components from EC supernatant the addition of PC and thrombin. Experiments were not performed on IL- 1b–stimulated GMVECs and HUVECs because this cytokine did not result In these experiments, AP components C3 and FB, as well as AP activation in appreciable changes in complement surface regulatory proteins, by flow products C3a, C5a, and Ba, were assayed by ELISA from the supernatant of cytometry, or complement component gene expression. unstimulated or TNF-stimulated GMVECs and HUVECs. Experiments were not performed on IL-1b–stimulated GMVECs and HUVECs because this PC/thrombin supplementation, supernatant collection, and TNF stimulation. cytokine did not result in appreciable changes in complement surface regula- HUVECs and GMVECs confluent in T-75 flasks were incubated for 24 h in tory proteins, by flow cytometry, or complement component gene expression. serum-free media. The cells were then washed with PBS and supplemented with 0.2 mM human PC (Haematologic Technologies, no. HCPC-0070) and Supernatant collection and TNF stimulation. Once confluent in T-75 flasks, 10 nM human a-thrombin (Haematologic Technologies, no. HCT-0020) in by guest on September 25, 2021 HUVECs and GMVECs were incubated for 24 h in 1 ml serum-free media per 1 ml activated PC buffer (0.1% BSA, 3 mM CaCl ,0.6mMMgCl in Ca+2 T-75 flask to concentrate the components secreted by the cells. The supernatant 2 2 /Mg+2-free PBS) (29) and incubated at 37˚C for 60 min (26, 29). Further was collected after 24 h and immediately frozen in liquid nitrogen for 20 s (to activation of PC to activated PC was inhibited with the addition of 10 nM prevent further activation of the AP) prior to storing at 280˚C until use. These hirudin (1.5 U/ml, Sigma-Aldrich, no. H7016) (29). Supernatants were col- samples were designated as the unstimulated controls. After at least 24–48 h lected and frozen in liquid nitrogen for 20 s prior to storage at 280˚C until of recovery in CM131, the same flasks were then incubated for 24 h in analysis. These samples were designated as the unstimulated controls. After CM131 plus 10 ng/ml TNF and incubated for an additional 24 h in 1 ml per allowing recovery for at least 24–48 h in complete media (CM131), the same T-75 flask of serum-free media plus 10 ng/ml TNF (total TNF exposure of flasks were incubated for 24 h in CM131 plus 10 ng/ml TNF, and then for an 48 h). The concentrated supernatant was again collected, flash frozen, and additional 24 h in serum-free media plus 10 ng/ml TNF. The flasks were stored at 280˚C until use. These samples were designated as the TNF- again washed with PBS and supplemented with 0.2 mM human PC and stimulated samples. HUVECs in these experiments were studied at pas- 10 nM human a-thrombin in 1 ml activated PC buffer and incubated at 37˚C sages 1–4 and GMVECs were used at passages 3 and 4. for 60 min. Supernatant was collected, flash frozen, and stored at 280˚C. Sample preparation. Samples were rapidly thawed and kept on ice prior to These samples were designated as the TNF-stimulated samples. The 24–48 h use to prevent spontaneous complement activation, and then analyzed for recovery time in CM131 after collection of control supernatant samples the various complement components and activation products. allowed the ECs to replenish nutrients after serum-free media incubation. Cell C3 fluorescence immunoassay. Black 96-well plates were coated with numbers were not affected by the presence of TNF (Supplemental Fig. 3). 50 ng/well polyclonal rabbit anti-human C3a (detects human C3a and C3, Sample preparation and assay of activated PC. Samples were rapidly Complement Technologies, no. A218) in 100 nM bicarbonate buffer (pH thawed and kept on ice prior to use. Activated PC levels were measured in 9.6) overnight at 4˚C. TBST-washed wells were blocked overnight with 1% unstimulated and TNF-stimulated GMVEC and HUVEC supernatant Ig-free BSA in PBS (BSA/PBS), followed by 50 min incubation with samples using an ELISA kit (Cloud-Clone, Houston, TX, no. SEA738Hu) m 100 l/well test samples (EC supernatant from unstimulated or TNF- with a standard curve range of 31.25–2000 pg/ml (Supplemental Fig. 2). stimulated cells, diluted by 10% with 10% BSA/PBS) or purified C3 protein (Complement Technologies, no. A113) for the standard curve (with Statistics a range of 9.4–600 ng/ml). TBST-washed wells were next incubated with 31 ng/ml goat polyclonal Ab to human C3 (Complement Technologies, no. The p values for statistical significance in flow cytometry and ELISA A213) for 25 min, followed by incubation with 100 ng/ml secondary experiments (see Figs. 4, 7, 9–11) were obtained by using two-tailed , donkey anti-goat IgG-HRP (Pierce, no. PA1-28664). Fluorescence was distribution, two-sample equal variance Student t tests. A p value 0.05 measured in a Tecan Infinite M200 Pro plate reader 25 min after the addition was considered statistically significant. of the HRP substrate 10-acetyl-3,7-dihydroxyphenoxazine (AnaSpec, Fremont, CA) with excitation of 530 nm and emission of 590 nm. Results The high range of sensitivity of the C3 immunoassay (as well as the others detailed below) is based on 10-acetyl-3,7-dihydroxyphenoxazine, a substrate for Expression and surface display of complement regulatory HRP that reacts with hydrogen peroxide to produce a highly fluorescent product. proteins CD55, CD59, CD46, and CD141 by unstimulated The raw fluorescent readings for the standards range from 1,000 to 45,000. GMVECs and HUVECs Reciprocal plots of standard dilutions (1/concentration) versus fluorescence intensity at 590 nm (1/590 intensity) produce linear equations that allow the To investigate our hypothesis that GMVECs and HUVECs have interpolation of complement component concentrations from 0 to the lower limit differences in their ability to regulate complement activation, we 4 TNF AND GLOMERULAR ENDOTHELIAL CELLS

THBD in Fig. 1 and Table I). CD59 expression was ∼1.7-fold higher in GMVECs than in HUVECs (p , 0.001). The differences in gene expression for CD46 and CD55 were only ∼0.3-fold higher in GMVECs than in HUVECs (Fig. 2). We next confirmed the presence of the surface complement regulatory proteins in both HUVECs and GMVECs by fluorescent microscopy and quantified the receptors by flow cytometry. Whereas it has been previously reported that HUVECs possess all four regulatory proteins (26, 47), little is known about these proteins on the surfaces of GMVECs. Fluorescent microscopy verified that each of the regulatory proteins is present on both GMVECs and HUVECs (Fig. 3, Supplemental Fig. 1). Internal staining of VWF in Weibel–Palade bodies confirmed that the cells studied were ECs. Fluorescence microscopy enabled us to determine surface complement regulatory protein presence; however, the technique did not give us quantitative information. This was obtained using flow cytometry. In both EC types, CD141 was present in ∼2-fold

greater concentrations than CD55 and CD46, whereas the pres- Downloaded from ence of CD59 was ∼13-fold higher than CD141. The relative amounts of the four surface regulatory proteins were similar on the surfaces of both EC types, although the quantities of each were statistically signiﬁcantly higher on GMVECs than on HUVECs: CD55 was present in ∼2.5-fold higher concentrations (p , 0.001),

CD46 in ∼1.5-fold higher concentrations (p , 0.001), CD141 in http://www.jimmunol.org/ ∼1.3-fold higher concentrations (p , 0.05), and CD59 in ∼1.4- fold higher concentrations (p , 0.05, Fig. 4). Gene expression of AP components in unstimulated GMVECs and HUVECs To investigate further our hypothesis that GMVECs and HUVECs have different capacities for complement regulation, we assessed gene expression levels of AP proteins by real-time PCR in both EC types. GMVEC gene expression levels of CFP, CFD, and CFH by guest on September 25, 2021 were ∼7-, ∼3-, and ∼3-fold higher, respectively, than levels in HUVECs (p , 0.05). The other complement genes studied (C3, C5, CFB, CFI, and C4), as well as VWF and ADAMTS13, were expressed at similar levels in GMVECs and HUVECs (Fig. 5). Expression and surface display of complement regulatory proteins CD55, CD59, CD46, and CD141 by cytokine- FIGURE 1. Cartoons showing two prominent functions of CD141 stimulated GMVECs and HUVECs (thrombomodulin). CD141 is a surface membrane protein present primarily on ECs (26). (A) CD141 participates in the FH- and FI-mediated inactivation of To investigate our secondary hypothesis that inflammatory cyto- C3b (to iC3b), thereby contributing to negative regulation of the AP (10). Be- kines alter complement regulation, GMVECs and HUVECs were cause the host cell is a nonactivating surface, FH does not bind to the EC surface stimulated with IL-1b or TNF. We used real-time PCR to quantify during this inactivation. (B) CD141 binds thrombin and enables it to convert PC gene expression of the complement regulatory genes, and flow into activated PC. Activated PC, in the presence of protein S, then inactivates cytometry to quantify the relative numbers of each complement clotting factors VIIIa and Va (to iVIIIa and iVa) and impairs coagulation. regulatory protein on the surfaces of GMVECs and HUVECs, with and without IL-1b or TNF stimulation. first assessed the gene expression levels of the surface regulatory IL-1b. We quantified changes in gene expression levels of proteins by real-time PCR. GMVECs expressed higher mRNA the surface complement regulatory proteins in GMVECs and levels than did HUVECs for each of the complement regulatory HUVECS after stimulation with IL-1b. The expression levels of genes tested. Although not significant (p = 0.06), THBD expression CD46 and CD55 were slightly higher (1.15- and 1.2-fold, re- was ∼2-fold higher in GMVECs than in HUVECs (see function of spectively) in IL-1b–stimulated versus unstimulated HUVECs.

Table I. Functions of EC surface complement regulatory proteins for the AP

Gene Protein Function CD55 CD55 Displaces Bb from membrane-bound C3bBb and C3bBbC3b (30) CD46 CD46 Cofactor for FH and FI-mediated cleavage/inactivation of C3b (25) CD59 CD59 Blocks additional attachment of C9 proteins to C5b-9(1) on EC membranes (31) THBD CD141 Cofactor for FH and FI-mediated cleavage/inactivation of C3b (10) The Journal of Immunology 5

face complement regulatory proteins at both the gene and protein levels. The greatest change in the gene expression levels of surface complement regulatory proteins after TNF stimulation in both EC types was in THBD expression. It has been previously described that TNF induces downregulation of THBD in HUVECs, bovine arterial ECs, human coronary artery ECs, lung microvascular ECs, and human microvascular ECs (42, 48); however, this phenomenon has not been analyzed before in GMVECs. THBD gene expression was re- duced2.8-foldinHUVECs(p , 0.05) and 6.7-fold in GMVECs (p , 0.05) after TNF exposure (Fig. 6). Because GMVECs had ∼2- fold higher THBD mRNA level prior to TNF stimulation (Fig. 2), the 6.7-fold reduction with TNF stimulation represents a more extensive reduction of THBD in GMVECs. Gene expression levels of CD59, FIGURE 2. Relative transcript levels of complement regulatory protein genes in unstimulated GMVECs and HUVECs. RNA was extracted from CD46,andCD55 were modestly higher (1.52-, 1.45-, and 1.62-fold, unstimulated GMVECs and HUVECs maintained in serum-free medium for respectively) in TNF-stimulated versus unstimulated HUVECs 24 h. The mRNA levels of CD59, CD46, THBD,andCD55 were quantified (Fig. 6A, dark gray bars). Expression levels of CD59 and CD46 were relative to the mRNA levels in HUVECs after normalization to GAPDH in also increased modestly (1.29- and 1.45-fold) in TNF-stimulated each PCR analysis with triplicate measurements. Data shown are means 6 SD GMVECs. Only CD55 expression levels in GMVECs were unaf- from three to five separate RNA extractions from each EC type. **p , 0.001. fected by exposure to TNF (Fig. 6B, dark gray bars). Downloaded from The reduced gene expression levels of THBD induced by TNF in THBD expression was slightly lower (down 1.35-fold) in IL- both EC types were associated with reduced amounts of CD141 1b–stimulated versus unstimulated HUVECs, and CD59 gene ex- protein on EC surfaces: TNF-stimulated GMVECs had a 20-fold pression levels were unchanged in IL-1b–stimulated versus decrease in surface CD141 protein (p , 0.001), and TNF- unstimulated HUVECs (Fig. 6A, light gray bars). The gene ex- stimulated HUVECs had a 14-fold CD141 reduction (p , pression levels of each complement regulatory protein were slightly 0.001, Fig. 7C–E). Surface CD55 protein increased by ∼1.4-fold http://www.jimmunol.org/ reduced in IL-1b–stimulated versus unstimulated GMVECs: CD59 (p , 0.001), and surface CD46 protein increased by ∼1.6-fold and CD46, each down 1.18-fold; THBD, down 1.45-fold; and (p , 0.001), on TNF-stimulated versus unstimulated HUVECs. CD55, down 1.3-fold (Fig. 6B, light gray bars). Surface CD59 protein was unchanged in HUVECs exposed to We also quantified the complement regulatory proteins present TNF (Fig. 7F). CD46 protein on surfaces of TNF-stimulated on surfaces of GMVECs and HUVECs, with and without IL-1b GMVECs was increased ∼1.5-fold (p , 0.05), whereas surface stimulation, by flow cytometry. IL-1b induced only small changes CD55 and CD59 protein quantities were both unaltered on in CD46, CD55, CD59, and CD141 proteins on the surfaces of GMVECs after TNF stimulation (Fig. 7G). both EC types. Although there were statistically significant changes in CD55 on HUVECs (∼1.1-fold higher on IL-1b–stimu- Gene expression of AP components by cytokine-stimulated by guest on September 25, 2021 lated versus unstimulated HUVECs, p , 0.05) and in CD141 on GMVECs and HUVECs GMVECs (∼1.4-fold higher on IL-1b–stimulated versus unstimu- To investigate our hypothesis that the inflammatory cytokines lated GMVECs, p , 0.05), the changes were minimal (Fig. 7A, 7B). affect production of AP components, HUVECs and GMVECs were TNF. In contrast to the modest changes observed in both EC types stimulated with IL-1b or TNF, and gene expression of the AP with IL-1b stimulation, TNF induced substantial changes in sur- proteins was quantified by real-time PCR.

FIGURE 3. Fluorescence microscopic images of the complement regulatory proteins on the surfaces of unstimulated GMVECs and HUVECs. GMVECs (A–D) and HUVECs (E–H) grown on glass coverslips were stained with mouse mAbs to each of the human complement regulatory proteins plus secondary Ab goat anti-mouse Alexa Fluor 647-IgG (red). The cells were then treated with Triton X-100 to enable internal staining with polyclonal rabbit anti-human VWF plus secondary Ab chicken anti-rabbit Alexa Fluor 488-IgG (green) to conﬁrm cell type. Cell nuclei were detected with DAPI (blue). Images shown in (A) and (E) (CD59), (B) and (F) (CD46), (C) and (G) (CD141), and (D) and (H) CD55 were acquired at original magniﬁcation 3100 and were chosen from a selection of 4–10 images for each complement regulatory protein. Scale bars, 10 mm. 6 TNF AND GLOMERULAR ENDOTHELIAL CELLS Downloaded from http://www.jimmunol.org/ by guest on September 25, 2021

FIGURE 4. Complement regulatory proteins on the surfaces of unstimulated HUVECs and GMVECs, as measured by flow cytometry. Samples of 2 3 104 unstimulated HUVECs and GMVECs were labeled with saturating amounts of FITC-conjugated mAbs to CD55, CD46, or CD59, or with FITC- conjugated isotype control; or with saturating amounts of PE-conjugated mAb to CD141, or with PE-conjugated isotype control. The cells were fixed and analyzed by flow cytometry. (A) CD55, CD46, and CD141 on the surface of HUVECs (n = 11) and GMVECs (n = 8), as measured by mean fluorescence, after subtracting the background fluorescence (isotype control alone). (B) CD59 on the surface of HUVECs (n = 11) and GMVECs (n = 8), as measured by mean fluorescence, after subtracting the background fluorescence. CD59 is present in considerably greater quantities and, therefore, is shown on a separate graph. (C–J) Histograms of the surface complement regulatory proteins on unstimulated HUVECs and GMVECs. (C and D) CD55 on HUVECs and GMVECs (black lines) compared with FITC isotype controls (gray lines). (E and F) CD46 on HUVECs and GMVECs (black lines) compared with FITC isotype controls (gray lines). (G and H) CD141 on HUVECs and GMVECs (black lines) compared with PE isotype controls (gray lines). (I and J) CD59 on HUVECs and GMVECs (black lines) compared with FITC isotype controls (gray lines). The data were normalized by counting the same number of ECs (5000) for each experiment. *p , 0.05, **p , 0.001.

IL-1b. We assessed gene expression of the AP components C3, an ∼16-fold increase in C3 and an ∼5-fold increase in CFB gene CFB, CFD, CFP, C5, CFH, CFI, C4, VWF,andADAMTS13 in expression. Expression of the other AP genes in HUVECs, as IL-1b–stimulated HUVECs and GMVECs, compared with well as C4, VWF,andADAMTS13,waschangedonlyslightlyby unstimulated ECs. Incubation of HUVECs with IL-1b induced IL-1b incubation (Fig. 8A). The expression of GMVEC AP The Journal of Immunology 7

increases in C3 and CFB mRNA levels with TNF stimulation were consistent with protein levels. Incubation of HUVECs with TNF induced increases in C3 and FB protein levels by ∼14- (p , 0.05) and ∼5-fold (p = 0.08), respectively (Fig. 10A, 10B). Incubation of GMVECs with TNF also induced increases in C3 and FB protein levels: C3 protein increased by ∼7-fold (p , 0.05), and FB protein increased to 1646 pg/ml. The fold change in FB protein in TNF-stimulated GMVECs cannot be mathematically calculated because comparator amounts of FB in the supernatant of unstimulated GMVECs were below detection limits of the assay (Fig. 10E, 10F). We compared the levels of the AP activation product C3a, C5a, and Ba in the supernatants of TNF-stimulated and unstimulated FIGURE 5. Relative transcript levels of complement components in HUVECs and GMVECs to assess the capacity of TNF to activate unstimulated GMVECs and HUVECs. RNA was extracted from un- the AP. There was no significant change in C3a protein levels in the stimulated GMVECs and HUVECs maintained in serum-free medium for supernatant of TNF-stimulated HUVECs compared with unstim- 24 h. The mRNA levels of AP components, plus control genes C4 of the ulated HUVECs (Fig. 10C); however, C5a and Ba proteins in- classical pathway, VWF, and ADAMTS13 (A13), were quantified relative to creased. The fold change and significance of the increase in C5a in the mRNA levels in HUVECs after normalization to GAPDH in each PCR Downloaded from analysis. Data were collected from five to seven separate RNA extractions TNF-stimulated HUVEC supernatant could not be determined from each EC type. *p , 0.05. because comparator levels in unstimulated HUVECs were below the detection limit of the assay (Fig. 10C). Ba levels increased by ∼5-fold in TNF-stimulated versus unstimulated HUVEC super- genes, as well as C4, VWF,andADAMTS13, was only slightly natant (p , 0.05, Fig. 10D). In the supernatant of TNF-stimulated affected by IL-1b (Fig. 8B). versus unstimulated GMVECs, there was no significant change in

TNF. We quantified gene expression of the same AP components in C3a and C5a protein levels (Fig. 10G), but there was a significant http://www.jimmunol.org/ TNF-stimulated HUVECs and GMVECs. In HUVECs, TNF in- increase in Ba protein levels by ∼1.5-fold (p , 0.05, Fig. 10H, duced increases of ∼32-fold in C3 and ∼16-fold in CFB gene Table II). expression (Fig. 8C). These values were 2- and 3-fold greater, Effect of TNF on CD141/thrombin-mediated generation of b respectively, than the upregulation of C3 and CFB in IL-1 – activated PC by GMVECs and HUVECs stimulated HUVECs. Incubation of GMVECs with TNF induced even more substantial increases in gene expression of C3 and Because of the TNF-induced decrease in THBD gene expression CFB: ∼153-fold for C3 and ∼59-fold for CFB. TNF reduced and CD141 surface protein in both GMVECs and HUVECs, we GMVEC expression of other genes to a lesser extent: CFP by compared the CD141/thrombin-mediated generation of activated ∼10-fold and VWF by ∼14-fold (Fig. 8D). The expression of CFP PC by unstimulated and TNF-stimulated GMVECs and HUVECs by guest on September 25, 2021 and VWF in HUVECs, along with the expression of C5, CFH, (Fig. 11). We added PC and thrombin to unstimulated and TNF- CFD, CFI, C4, and ADAMTS13 in both GMVECs and HUVECs, stimulated GMVECs and HUVECs, and then measured activated was altered only slightly by incubation with TNF. PC by ELISA in the cell supernatants. Activated PC levels in the supernatants of unstimulated HUVECs and GMVECs were 3.1 Levels of complement components from supernatants of and 11.2 pg/ml, respectively (i.e., ∼3.6-fold higher in unstimu- GMVECs and HUVECs stimulated with and without TNF lated GMVECs, Fig. 11A). Stimulation with TNF reduced acti- Because of our experimental results showing that TNF stimulation vated PC to levels below the lower detection limit of the assay in increased C3 and CFB expression levels and decreased THBD the supernatant of HUVECs, and to 1.81 pg/ml in GMVECs (∼6- expression/CD141 surface presence in both cell types, we con- fold decrease compared with unstimulated GMVECs, Fig. 11B, ducted experiments to determine whether TNF induced AP acti- Table II). vation. We measured release of C3 and FB proteins (by ELISA) from HUVECs and GMVECs without TNF stimulation to deter- Discussion mine a baseline of C3 and FB protein release from unstimulated In this study, we assessed the activation and regulation of the AP and cells. Without TNF stimulation, GMVECs had ∼1.6-fold higher coagulation in GMVECs and HUVECs to elucidate the renal vul- levels of C3 as compared with HUVECs (Fig. 9A). FB levels in nerability to injury in aHUS. We also investigated the effects on the supernatant of unstimulated GMVECs were below the detec- these processes by the inflammatory cytokines IL-1b and TNF, with tion limit of the assay, but were detected at ∼4000 pg/ml in the the hypothesis that our studies could provide an explanation for supernatant of unstimulated HUVECs (Fig. 9B). the provocation of aHUS episodes during infectious/inflammatory We also measured the AP activation products C3a, C5a, and Ba conditions. from unstimulated HUVECs and GMVECs to assess AP activation By evaluating AP regulatory components in unstimulated in both EC types at baseline. Unstimulated GMVECs had ∼5.7-fold GMVECs and HUVECs, we confirmed that the surface comple- higher amounts of C3a (p , 0.05) and ∼1.6-fold higher amounts ment regulatory proteins are present on both EC types at baseline. of Ba, compared with unstimulated HUVECs. C5a levels were 56 We additionally found that gene expression levels of CD55, CD59, pg/ml in the supernatant of unstimulated GMVECs, and were CD46,andTHBD were higher in GMVECs than in HUVECs, below the detection limit of the assay in unstimulated HUVECs accounting for the slightly increased amounts of CD55, CD59, (Fig. 9C). These results suggest that unstimulated GMVECs have CD46, and CD141 proteins on GMVEC surface membranes. increased AP activation compared with unstimulated HUVECs. Similarly, GMVECs expressed the negative regulatory protein We measured the AP components, C3 and FB, in the supernatant CFH in 3-fold higher quantities than did HUVECs. These results of TNF-stimulated HUVECs and GMVECs and compared these suggest that GMVECs may require increased self-protection component levels to unstimulated levels to determine whether the against complement-mediated injury. 8 TNF AND GLOMERULAR ENDOTHELIAL CELLS

FIGURE 6. Fold change in gene expression of surface complement regulatory proteins in HUVECs and GMVECs after incubation with TNF and IL-1b.The mRNA levels of surface regulatory protein genes CD59, CD46, THBD, and CD55 were quantiﬁed in (A) TNF- and IL-1b–incubated

HUVECs relative to the mRNA levels in Downloaded from unstimulated HUVECs, and in (B) TNF- and IL-1b–incubated GMVECs relative to the mRNA levels in unstimulated GMVECs. The values in both (A) and (B) were normalized to GAPDH. Data are means 6 SD from three to ﬁve real-time

PCR runs with triplicate measurements. http://www.jimmunol.org/ RNA was extracted in three to ﬁve separate experiments using each EC type, with and without either IL-1b (3 ng/ml for 48 h, light gray bars) or TNF (10 ng/ml for 48 h, dark gray bars). *p , 0.05. by guest on September 25, 2021

It was our hypothesis that the increased amount of CD141 on the specific AP activation product Ba, in the supernatant of unstim- surface of unstimulated GMVECs compared with HUVECs would ulated GMVECs compared with HUVECs, support this conclu- make GMVECs more anticoagulant at baseline. This is because sion and may explain the particular vulnerability of the kidneys to more thrombin can bind to CD141 on GMVECs, leading to in- AP-mediated injury in aHUS. creased activation of PC to activated PC, with subsequent inacti- In contrast to CFP and CFD expression, the gene expression of vation of coagulation factors Va and VIIIa (Fig. 1B). This CFB was similar in both unstimulated HUVECs and GMVECs; explanation was confirmed by our data demonstrating that acti- however, FB levels in the supernatant of unstimulated GMVECs vated PC levels in unstimulated GMVECs were ∼3.6-fold higher were below the lower detection limit of the assay. This low value than HUVECs. may be because of the underlying increased AP activation in Our evaluation of AP component expression in unstimulated GMVECs, which may secrete and anchor some ULVWF multimeric GMVECs and HUVECs demonstrated that expression of CFP and strings in response to minor experimental manipulation. In this case, CFD was notably different between the two EC types. GMVECs most of the FB produced would attach to C3b on the ULVWF strings expressed CFP and CFD in relatively greater quantities (7- and 3- and be cleaved to Bb by the increased FD produced, releasing the Ba fold, respectively) than did HUVECs. AP components FP and FD fragment. Bb would remain attached to ULVWF strings as part of the potentiate AP activation. Assuming that the AP is activated on C3 convertase and be stabilized by the increased FP protein released. GMVEC ULVWF strings in the same manner as on HUVEC The putative result is FB protein levels that are very low in the su- ULVWF strings (32–34), the greater GMVEC CFD and CFP pernatant of unstimulated GMVECs. expression may promote AP C3bBb (C3 convertase) formation The propensity for aHUS relapses in association with infection/ (FD) and stability (FP) on GMVEC-secreted/anchored ULVWF inflammation (35, 36) led us to evaluate complement parameters strings. These results suggest that GMVECs may have increased in both GMVECs and HUVECs during cytokine stimulation using AP activation at baseline. Our experiments showing higher levels TNF and IL-1b. Whereas IL-1b affected AP production and of the complement activation products C3a and C5a, as well as the regulation by GMVECs and HUVECs only mildly, TNF caused The Journal of Immunology 9 Downloaded from http://www.jimmunol.org/ by guest on September 25, 2021

FIGURE 7. Effects of IL-1b or TNF on surface complement regulatory proteins, as measured by flow cytometry. GMVECs (n = 8) and HUVECs (n = 11) were incubated with or without IL-1b (3 ng/ml)– or TNF (10 ng/ml)-supplemented media for 48 h. Samples of 2 3 104 HUVECs or GMVECs were labeled with saturating amounts of FITC- or PE-conjugated mAbs to CD55, CD59, CD46, and CD141 or with FITC-conjugated or PE-conjugated isotype controls alone (to measure background fluorescence), fixed, and analyzed by flow cytometry. (A) and (B) show mean fluorescence values of the four surface complement regulatory proteins on HUVECs (A) and GMVECs (B) with and without IL-1b.(C) Mean fluorescence of CD141 on unstimulated and TNF- stimulated HUVECs and GMVECs. (D) and (E) are histograms of CD141 on unstimulated and TNF-stimulated HUVECs and GMVECs. (D) CD141 on HUVECs without TNF (gray line, bottom panel) and with TNF (black line, bottom panel) compared with PE isotype control (top panel). (E) CD141 on GMVECs without TNF (gray line) and with TNF (black line) compared with the PE isotype control (light gray, filled). (F) and (G) show mean fluorescence values of CD55, CD46, and CD59 on HUVECs (F) and GMVECs (G) with and without TNF. Values presented are those obtained after subtracting the background fluorescence. The data were normalized by counting the same number of ECs (5000) for each experiment. *p , 0.05, **p , 0.001. 10 TNF AND GLOMERULAR ENDOTHELIAL CELLS Downloaded from http://www.jimmunol.org/

FIGURE 8. Fold change in gene expression of AP components in HUVECs and GMVECs after treatment with IL-1b or TNF. The mRNA levels of AP components, as well as of the classical component C4, VWF, and ADAMTS13 (A13), were quantified in (A) IL-1b–incubated HUVECs (3 ng/ml) relative to by guest on September 25, 2021 mRNA levels in unstimulated HUVECs, (B) IL-1b–incubated GMVECs (3 ng/ml) relative to mRNA levels in unstimulated GMVECs, (C) TNF-incubated HUVECs (10 ng/ml) relative to the mRNA levels in unstimulated HUVECs, and (D) TNF-incubated GMVECs (10 ng/ml) relative to the mRNA levels in unstimulated GMVECs. The values in (A)–(D) were normalized to GAPDH. Data were from three to four experiments using both cell types and each condition (unstimulated or cytokine-stimulated for 48 h). Each gene was evaluated from three to four real-time PCR runs with triplicate measurements. significant changes in these parameters in both EC types. The tokines may stimulate EC secretion/anchorage of ULVWF multi- major receptor for TNF, TNFR-1, is present at similar densities on meric strings in excess of the capacity of EC-produced ADAMTS- both HUVECs and human microvascular ECs (49, 50). TNF 13 (37) to cleave the strings rapidly and completely, therefore binding to TNFR-1 stimulates signaling pathways that include promoting the initiation, assembly, and activation of the AP on these NF-kB (51). Our data indicate that TNF/TNFR-1 interaction and strings. This latter process will be magnified in aHUS patients with signaling also occur on GMVECs, as well as on HUVECs. a chronically overactive AP. Inflammatory cytokines, including TNF, initiate EC stimulation We found that both GMVECs and HUVECs incubated with TNF and secretion/anchorage of ULVWF multimeric strings (52). Cy- had extensive decreases in the expression of THBD and corre-

FIGURE 9. Levels of complement components C3 and FB, as well as levels of complement activation products C3a, C5a, and Ba, in the supernatant of unstimulated GMVECs compared with unstimulated HUVECs. Supernatant from unstimulated HUVECs (n = 3) and GMVECs (n = 6) in T-75 ﬂasks was collected and assayed for complement components C3 (A) and FB (B) and complement activation products C3a, C5a, and Ba (C). FB levels in unstimulated GMVECs, as well as C5a levels in unstimulated HUVECs, were below the lower detection limit (LDL) of the assay. C3 and FB proteins were measured in considerably greater quantities and, therefore, are shown on separate graphs from C3a, C5a, and Ba. *p , 0.05. The Journal of Immunology 11 Downloaded from http://www.jimmunol.org/ by guest on September 25, 2021

FIGURE 10. Levels of complement components C3 and FB, and levels of complement activation products C3a, C5a, and Ba, in the supernatant of TNF- stimulated versus unstimulated HUVECs and GMVECs. Supernatant from unstimulated HUVECs (n = 3) and GMVECs (n = 6) in T-75 ﬂasks was collected and assayed for complement components C3 (A and E) and FB (B and F) and complement activation products C3a, C5a (C and G), and Ba (D and H). The same ﬂasks were then stimulated for 48 h with 10 ng/ml TNF, and the supernatant was again collected and assayed for the same components. (A)–(D)show comparisons of the components in the supernatant of TNF-stimulated HUVECs as compared with unstimulated HUVECs, and (E)–(H) show comparisons of components in the supernatant of TNF-stimulated GMVECs as compared with unstimulated GMVECs. C5a levels in unstimulated HUVECs, and FB levels in unstimulated GMVECs, were less than the lower detection limit (LDL) of the assay. C3 and FB proteins were measured in considerably greater quantities and, therefore, are shown on separate graphs from C3a, C5a, and Ba. *p , 0.05, †precise p value was unable to be calculated because of values ,LDL of the assay. sponding decreases in surface CD141. Although others have TNF on GMVECs demonstrated in our study were previously previously found that TNF downregulates THBD in HUVECs, unknown. bovine arterial ECs, human coronary artery ECs, lung microvas- In addition to THBD downregulation, we found that TNF in- cular ECs, and human microvascular ECs (42, 48), the effects of creased gene expression of C3 and CFB in GMVECs and 12 TNF AND GLOMERULAR ENDOTHELIAL CELLS

Table II. Regulation of the AP and coagulation in TNF-stimulated GMVECs

Experimental Result Consequences Suppression of THBD mRNA synthesis, 1) Decreased thrombin binding, reduced activation resulting in decrease of cell surface CD141 of PC to activated PC (29) (increased coagulation); 2) Decreased FI cleavage of C3b (10) (accelerated activation of the AP) Increased gene expression levels of C3 and Increased formation of the C3 convertase (C3bBb) CFB, resulting in increased C3 and FB and release of activation fragment Ba (15–17) protein production (accelerated activation of the AP)

HUVECs. This resulted in increased levels of C3 and FB proteins ceptor is either absent on ECs or present in small amounts; oth- in the supernatant of TNF-stimulated GMVECs and HUVECs erwise, we would not have been able to detect Ba in the compared with unstimulated ECs. C3 and FB in the supernatant of supernatant of TNF-stimulated ECs. TNF-stimulated HUVECs increased ∼14- and ∼5-fold, respec- Furthermore, we demonstrated the functional relevance of TNF- tively. Although the ratio of this increase was consistent with gene induced downregulation of THBD as related to its anticoagulant expression levels of C3 and CFB in the presence of TNF (C3 and activity. In addition to participating in regulation of the C3 con- CFB increased ∼32- and ∼16-fold, respectively), the increases in vertase, CD141 also functions to bind thrombin and activate PC

C3 and FB protein levels were not as great. This may be partially (Fig. 1B). In this study, we demonstrated that the decreases in Downloaded from the result of C3 (as C3b) binding to the increased quantity of THBD gene expression and surface presence of CD141 protein on HUVEC-secreted/anchored ULVWF strings during TNF stimula- TNF-stimulated GMVECs and HUVECs are associated with de- tion, as well as the attachment of FB to the ULVWF-bound C3b creased PC activation to activated PC. The fold decrease in acti- (34). vated PC levels in TNF-stimulated HUVEC supernatant relative to There were similar GMVEC responses to TNF: ∼7-fold increase unstimulated supernatant could not be calculated because TNF-

in C3 protein levels in the supernatant of TNF-stimulated stimulated HUVEC supernatant had activated PC levels below the http://www.jimmunol.org/ GMVECs in association with ∼153-fold increase in C3 gene ex- lower detection limit of the assay. In GMVECs, however, there pression. As described above for HUVECs, this disparity is likely was an ∼6-fold decrease in supernatant activated PC levels with to be because of C3 (as C3b) binding to GMVEC-secreted/ TNF stimulation compared with unstimulated GMVECs. These anchored ULVWF strings. CFB mRNA levels in TNF-stimulated results are consistent with our gene expression data where THBD GMVECs were also increased (∼59-fold), but the FB protein fold gene expression levels decreased 6.7-fold, and the amount of increase in the TNF-stimulated GMVEC supernatant could not be surface CD141 protein decreased by ∼20-fold, in TNF-stimulated determined because comparator FB levels in unstimulated versus unstimulated GMVECs. The considerable reduction in GMVECs were below assay detection limits. We hypothesize, activation to activated PC likely promotes increased microthrombi however, that (as described for HUVECs) the FB protein levels in formation in GMVECs during infection/inflammation. by guest on September 25, 2021 the supernatant of TNF-stimulated GMVECs are likely below expected values because of its avid binding to C3b on the TNF- induced secretion/anchorage of ULVWF strings. In our experiments, we demonstrated the functional relevance of both TNF-induced downregulation of THBD and upregulation of C3 and CFB in GMVECs and HUVECs. CD141 participates in the negative regulation of C3 convertase (Fig. 1A). Ba, the activation product of FB, is unique to the AP, whereas C3a and C5a are activation products of the alternative, lectin, and classical complement pathways. The significant increase in Ba in the supernatant of TNF-stimulated GMVECs and HUVECs demonstrates, therefore, that the AP is activated as a consequence of the elevated C3 and FB proteins, decreased surface presence of CD141 protein, and augmented EC-secreted/anchored ULVWF strings. In contrast to the elevation in Ba, C3a and C5a levels did not change significantly in TNF-stimulated GMVEC and HUVEC supernatant compared with unstimulated EC supernatant. C3a and C5a are anaphylatoxins, that is, proinflammatory polypeptides that FIGURE 11. Levels of activated PC in the supernatant of unstimulated bind to their respective cell receptors and cause chemotaxis, his- and TNF-stimulated HUVECs and GMVECs after addition of PC and tamine release, and increased vascular permeability. The C3a thrombin. One milliliter 0.2 mM PC and 10 nM thrombin was added to and C5a receptors, C3a-R and C5a-R, are present on HUVECs and unstimulated HUVECs (n = 3) and GMVECs (n = 5) in T-75 flasks. The human microvascular ECs (53–56). It is likely that any C3a and supernatant was collected after 60 min incubation and assayed for acti- C5a proteins generated during TNF stimulation bind to GMVEC vated PC by an ELISA kit. The flasks were then allowed to recover for 24– and HUVEC C3a-R and C5a-R receptors and, therefore, remain in 48 h in complete media and then stimulated for 48 h with 10 ng/ml TNF, followed by the addition of 1 ml 0.2 mM PC and 10 nM thrombin for the supernatants of TNF-stimulated ECs only at levels below the 60 min. The supernatant was collected again and assayed for activated lower detection limits of the assays. Less is known about the Ba PC. (A) Comparison of activated PC levels in unstimulated HUVECs fragment and its functions, if any. Ba is chemotactic for human and GMVECs. (B) Activated PC levels in TNF-stimulated HUVECs and peripheral blood leukocytes and guinea pig neutrophils, but is less GMVECs compared with activated PC levels in unstimulated HUVECs potent than C5a (57–59). It is not known whether there is a Ba and GMVECs. Activated PC levels in TNF-stimulated HUVECs were less receptor on human ECs. Our data suggest that this putative re- than the lower detection limit (LDL) of the assay. The Journal of Immunology 13

The extensive downregulation of THBD and surface CD141, Disclosures along with extreme upregulation of C3/C3 and CFB/FB, in TNF- The authors have no financial conflicts of interest. stimulated GMVECs explains the particular vulnerability of the kidneys to C3 convertase generation (and injury) and micro- thrombus formation in aHUS patients during infectious/ References 1. Benz, K., and K. Amann. 2009. Pathological aspects of membranoproliferative inflammatory events (Table II). The upregulation of the AP in glomerulonephritis (MPGN) and haemolytic uraemic syndrome (HUS)/thrombocytic TNF-stimulated GMVECs may be especially dangerous in aHUS thrombopenic purpura (TTP). Thromb. Haemost. 101: 265–270. patients who have heterozygous gain-of-function mutations in C3 2. Noris, M., and G. Remuzzi. 2009. Atypical hemolytic-uremic syndrome. N. Engl. J. Med. 361: 1676–1687. and CFB. Further upregulation of the C3 and FB proteins would 3. Ruggenenti, P., M. Noris, and G. Remuzzi. 2001. Thrombotic microangiopathy, be expected to intensify activation of the already overactive AP hemolytic uremic syndrome, and thrombotic thrombocytopenic purpura. Kidney in these individuals. By analogy, the extensive downregulation Int. 60: 831–846. 4. Caprioli, J., F. Castelletti, S. Bucchioni, P. Bettinaglio, E. Bresin, G. Pianetti, of THBD in TNF-stimulated GMVECs may be most threatening S. Gamba, S. Brioschi, E. Daina, G. Remuzzi, and M. Noris, International to aHUS patients with heterozygous loss-of-function mutations Registry of Recurrent and Familial HUS/TTP. 2003. Complement factor H mutations and gene polymorphisms in haemolytic uraemic syndrome: the C- in THBD. Further loss of surface CD141 during infection/ 257T, the A2089G and the G2881T polymorphisms are strongly associated inflammation will accentuate the underlying increased AP with the disease. Hum. Mol. Genet. 12: 3385–3395. overactivation and coagulation. Gene mutations in THBD have 5. Pe´rez-Caballero, D., C. 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