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

Atypical hemolytic uremic syndrome (aHUS) is a thrombotic microangiopathy presenting with microangiopathic hemolytic anemia, thrombocytopenia, and renal failure secondary to formation of platelet-fibrin clots in the glomerular microvasculature (1–3). aHUS is associated with heterozygous mutations in components of the alternative complement pathway (AP) that result in excessive AP activation. Defects include loss-of-function mutations in the genes for factor H (FH) (4, 5), factor I (FI) (6, 7), CD46 (8, 9), and CD141 (thrombomodulin) (10), or gain of function mutations in C3 (11) or factor B (FB) (12).

The AP is initiated when C3b is cleaved from C3 and attaches to an activating surface, releasing a soluble C3a fragment in the process (13, 14). FB then combines with C3b to form C3bB (15, 16), and factor D (FD) cleaves FB in this complex to form C3bBb (the active C3 convertase of the AP) (17), releasing the activation product Ba. The C3 convertase is stabilized by factor P (FP; properdin) (18–20). The Bb in C3bBb cleaves C3 to generate additional C3b; as the ratio of C3b to Bb increases, C3bBbC3b (the C5 convertase) forms and cleaves C5 to C5b, releasing the soluble C5a fragment (17, 21).

The AP is regulated by both soluble and cell surface–bound proteins. FH and FI are soluble inhibitory regulators of the AP: FH suppresses the formation or persistence of C3bBb (22, 23), and FI, along with FH, promotes the cleavage/inactivation of C3b (24). CD46 and CD141 are cell surface membrane regulatory proteins that have functions supplementary to FH, that is, all three act as cofactors for FI-mediated proteolysis of C3b (10, 25). CD141 is found almost exclusively on endothelial cell (EC) surfaces (26) 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 also functions as a natural anticoagulant by binding thrombin and diverting thrombin substrate specificity to the activation of protein C (PC). Activated PC, with bound protein S, cleaves and inactivates coagulation factors Va and VIIIa (29) (Fig. 1, Table I).

CD55 and CD59 are two other negative complement surface regulatory proteins. CD55 accelerates the decay of C3 convertase (30). CD59 prevents accumulation of additional C9 molecules into the C5b-(9)₍₁₎ membrane attack complex (31) (Table I).

Uncleaved ultra-large von Willebrand factor (ULVWF) multimeric strings secreted by, and anchored to, stimulated HUVECs serve as activating surfaces for C3b binding and AP assembly and activation (32–34). We have previously demonstrated that C3 (as C3b), FB (as Bb), FD, FP, and C5 (as C5b), as well as smaller quantities of FH and FI, attach to HUVEC-secreted and anchored ULVWF strings. In contrast, C4 (as C4b) does not attach to the ULVWF strings, indicating that the classical and lectin pathways are not activated. The attachment to EC-secreted/anchored ULVWF strings of C3b, Bb, and C5b occurs in quantitative and functional patterns consistent with the assembly of AP components into active complexes of C3 convertase (C3bBb) and C5 convertase (C3bBbC3b) (34).

In aHUS, the kidneys are affected more severely than other organs. The vulnerability of the kidney to AP-mediated injury in aHUS led us to investigate complement surface regulatory protein expression and membrane presence, as well as AP component expression, in glomerular microvascular ECs (GMVECs) and, for comparison, in HUVECs. We hypothesized that GMVECs have differences in AP regulation, compared with HUVECs, thereby explaining their susceptibility to injury in aHUS. Because infectious/inflammatory conditions may lead to initial or recurrent episodes of aHUS (35, 36), we also studied the effects of two proinflammatory cytokines, TNF and IL-1β, on complement parameters in both EC types. We further hypothesized that these inflammatory cytokines affect AP regulation in HUVECs and GMVECS by altering gene expression or surface presence of the AP components/regulators, thereby explaining the recurrences of aHUS during inflammatory or infectious events.

Pooled primary human GMVECs were purchased from Cell Systems (Kirkland, WA, ACBRI-128 vial). GMVECs were grown in complete media (CM131, MCDB-131 medium [Sigma-Aldrich], supplemented with penicillin/streptomycin/l-glutamine [Life Technologies], plus microvascular growth supplement [Life Technologies]).

Primary HUVECs were isolated from umbilical veins as previously described (37), and pooled from multiple human donor umbilical veins. HUVECs were grown in CM131 plus low-serum growth supplement for large vessel ECs (Life Technologies). The only difference between GMVEC media and HUVEC media was the type of growth supplement added as required by cell type; culture conditions were otherwise identical.

GMVECs and HUVECs were seeded in T-25 flasks (for flow cytometry experiments), T-75 flasks (for gene expression experiments and complement component/activated PC supernatant measurements), or on gelatin-coated coverslips (for immunofluorescence microscopy experiments). Cell type was confirmed by immunofluorescence showing that 95% of the GMVECs or HUVECs were positive for von Willebrand Factor (VWF) in Weibel–Palade bodies.

GMVECs and HUVECs, grown on gelatin-coated glass coverslips, were stained for the external complement regulatory proteins CD55, CD59, CD46, and CD141, and for internal VWF (to confirm cell type). ECs were fixed with 1% p-formaldehyde for 10 min and stained with 1 μg/ml anti-CD46, 10 μg/ml anti-CD55 or anti-CD59, or a 1:100 dilution of anti-CD141 (see details of each Ab below) plus 20 μg/ml fluorescent secondary goat anti-mouse Alexa Fluor 647 F(ab′)₂ fragment–IgG (Life Technologies, A21237), or stained with the secondary goat anti-mouse Ab alone to assess background fluorescence (Supplemental Fig. 1A, 1D). The cells were fixed again (to retain surface Abs). Cells were then treated with 0.2% Triton X-100 in PBS for 5 min (to allow internal staining) and stained with 10 μg/ml polyclonal rabbit anti-human VWF (Ramco Laboratories, Sugarland, TX) plus 20 μg/ml secondary Ab chicken anti-rabbit Alexa Fluor 488 IgG (Life Technologies, A21441) for 15 min, or stained with the secondary chicken anti-rabbit Ab alone to assess background fluorescence (Supplemental Fig. 1B, 1E). Cell nuclei were detected with DAPI included in the mounting medium (Fluoro-Gel II, Electron Microscopy Sciences).

Abs included: CD141, clone QBEND/40, mouse monoclonal IgG2a (Thermo Fisher, MA1-35905); CD46, clone M177, mouse monoclonal IgG1 (Thermo Fisher, MA1-40183); CD55, clone BRIC-216, mouse monoclonal IgG1 (Thermo Fisher, MA1-91161); and CD59, clone MEM-43, mouse monoclonal IgG2a (Thermo Fisher, MA1-82206). Verification of the specificity of these mAbs has been previously published or stated by the manufacturer (38, 39).

Fluorescent images were acquired using IP Lab software version 3.9.4r4 (Scanalytics, Fairfax, VA) on a Nikon Diaphot TE300 microscope equipped with CFI Plan Fluor ×60 oil, numerical aperture 1.4 and CFI Plan Apo Lambda ×100 oil, numerical aperture 1.45 objectives plus ×10 projection lens (Nikon, Garden City, NY), SensiCam QE CCD camera (Cooke, Romulus, MI), motorized stage and dual filter wheels (Prior) with single band excitation and emission filters for FITC/tetramethylrhodamine isothiocyanate/Cy5/DAPI (Chroma, Rockingham, VT). Image areas acquired at original magnification ×60 are 78 × 58 μm, and at ×100 are 41 × 30 μm.

Once confluent in T-25 flasks, control cells were incubated for 24 h in serum-free media (MCDB-131 plus insulin-transferrin-selenium, Life Technologies), and experimental cells were incubated for 24 h with CM131 plus TNF (10 ng/ml, Life Technologies) or IL-1β (3 ng/ml, Life Technologies), and then incubated for an additional 24 h in serum-free media plus 10 ng/ml TNF or 3 ng/ml IL-1β (total cytokine exposure of 48 h). Both control and experimental flasks were incubated in serum-free media for a total of 24 h to eliminate any proteins derived from serum that might affect surface receptor detection. The specific concentrations and duration of exposure of TNF and IL-1β were chosen based on use of these cytokines in EC gene expression studies (40–42).

Control and experimental cells were detached by 10 min incubation with 5 mM EDTA in Ca⁺²/Mg⁺²-free PBS (to retain surface proteins) and centrifuged (10 min at 400 × g). Cells were counted using the TC10 automated cell counter (Bio-Rad, Hercules, CA) and resuspended in 1% BSA/PBS at 10⁶ cells/ml. Samples of 2 × 10⁴ GMVECs or HUVECs (20 μl) were labeled individually with saturating amounts of each fluorescently conjugated mAb (see below, 1 μl each specific Ab per 20 μl cell suspension) or labeled with saturating amounts of FITC-conjugated (CD55, CD59, CD46) or PE-conjugated (CD141) isotype controls (see below, 1 μl isotype control per 20 μl cell suspension) to measure background fluorescence. Samples were incubated for 20 min in the dark. Cells were then fixed with 0.5 ml 1% formaldehyde/PBS. Cells expressing higher fluorescence than background (isotype control alone) were considered positive, and background fluorescence was subtracted from positive fluorescence. Experiments were repeated with increasing passage number (passages 5–6 for GMVECs and passages 4–6 for HUVECs) to exclude the possibility that any differences appreciated were related to passage differences.

Abs included: CD141, clone 1A4, mouse monoclonal IgG1,κ conjugated to PE (BD Biosciences, 559781); CD46, clone E4.3, mouse monoclonal IgG2a,κ conjugated to FITC (BD Biosciences, 555949); CD55, clone IA10, mouse monoclonal IgG2a,κ conjugated to FITC (BD Biosciences, 555693); and CD59, clone p282 (H19), mouse monoclonal IgG2a,κ conjugated to FITC (BD Biosciences, 555763). Isotype controls included PE mouse, clone IgG1 (BD Biosciences, 349043) and FITC mouse, clone IgG2a,κ (BD Biosciences, 555573).

Samples were acquired using FACScan (BD Biosciences), and the data were analyzed using CellQuest software (BD Biosciences). The instrument settings of the forward scatter and side scatter profiles were log mode. EC samples appeared as single populations and were gated based on their forward and side scatter profiles. Five thousand gated events were analyzed for each sample.

Once confluent in T-75 flasks, control cells were 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-1β, and then incubated for an additional 24 h in serum-free media plus 10 ng/ml TNF or 3 ng/ml IL-1β (total cytokine exposure of 48 h). Both control and experimental flasks were incubated in serum-free media for a total of 24 h.

ECs in T-75 flasks were washed with cold PBS, and RNA was isolated using TRIzol (Invitrogen), chloroform extraction, and isopropanol precipitation. The RNA integrity was verified by 260/280 OD ratios and 1% agarose/formaldehyde electrophoresis.

GMVEC and HUVEC RNA samples were reverse transcribed using SuperScript III Supermix (Invitrogen). Samples (100 ng cDNA) were amplified in quadruplicate or triplicate by real-time PCR under the following conditions: 95°C for 3 min, 40 cycles of 10 s at 95°C, 10 s at 55°C, and 30 s at 72°C, then 95° for 10 s, followed by melting curves from 65°C to 95°C (CFX96, Bio-Rad). Amplified products were detected using TaqMan gene expression assays (with FAM-labeled probes that span target exon junctions; see Supplemental Table I for the list of assay probe IDs) and fast advanced master mix (Life Technologies).

The relative quantification of gene expression with/without cytokine stimulation in GMVECs and HUVECs was calculated as described in the Applied Biosystems User Bulletin No. 2 (P/N 4303859) and by Livak and Schmittgen (43), as follows: change in cycle threshold (ΔCT) for each gene (in each cell type) = ΔCT (gene) − ΔCT (GAPDH); ΔCT number relative to HUVECs (ΔΔCT _HUVECs) = ΔCT (gene in HUVECS) − ΔCT (gene in HUVECs); and ΔCT number relative to GMVECs (ΔΔCT _GMVECs) = ΔCT (gene in GMVECs) − ΔCT (gene in GMVECs).The fold changes relative to HUVECs were calculated by evaluating 2^−ΔΔCT for each gene. For each HUVEC gene, ΔΔCT = 0 and 2° = 1.

SD calculations for ΔΔCT were determined as the square root of (S₁² + S₂²), where S₁ indicates the average SD of CT for each gene, and S₂ indicates the average of CT for GAPDH. The ranges for the fold changes relative to HUVECs were calculated by evaluating 2^−ΔΔCT plus the SD (low range value) and 2^−ΔΔCT minus the SD (high range value).

Changes in gene expression in GMVECs and HUVECs in the presence of cytokines were calculated using the method developed by Pfaffl (44), which uses specific primer efficiencies (E) to evaluate the amount of cDNA amplification per each PCR cycle (44): E = 10^(−1/slope) and Ratio = E_Target^{ΔCT(control − treated)}/E_Ref^{ΔCT(control − treated)}.

mRNA levels were quantified for the complement regulatory proteins CD59, CD46, CD55, and THBD (gene for CD141), for the AP components C3, C5, CFB, CFD, CFP, CFH, and CFI, for the classical complement component C4, and for VWF and ADAMTS13 (gene for the VWF cleavage protein a disintegrin and metalloprotease with thrombospondin domains type 13 [ADAMTS-13]). VWF and ADAMTS13 were chosen because these two genes are known to be biologically significant in ECs (45, 46), and they were thought likely to be important in synthesis and interpretation of the data.

The p values for the gene expression graphs (see Figs. 2, 5, 6) were calculated with a Student t test, using the end-point gene copy numbers, after normalization to GAPDH. A p value <0.05 was considered statistically significant.

In these experiments, AP components C3 and FB, as well as AP activation products C3a, C5a, and Ba, were assayed by ELISA from the supernatant of unstimulated or TNF-stimulated GMVECs and HUVECs. Experiments were not performed on IL-1β–stimulated GMVECs and HUVECs because this cytokine did not result in appreciable changes in complement surface regulatory proteins, by flow cytometry, or complement component gene expression.

Once confluent in T-75 flasks, HUVECs and GMVECs were incubated for 24 h in 1 ml serum-free media per T-75 flask to concentrate the components secreted by the cells. The supernatant was collected after 24 h and immediately frozen in liquid nitrogen for 20 s (to prevent further activation of the AP) prior to storing at −80°C until use. These samples were designated as the unstimulated controls. After at least 24–48 h of recovery in CM131, the same flasks were then incubated for 24 h in CM131 plus 10 ng/ml TNF and incubated for an additional 24 h in 1 ml per T-75 flask of serum-free media plus 10 ng/ml TNF (total TNF exposure of 48 h). The concentrated supernatant was again collected, flash frozen, and stored at −80°C until use. These samples were designated as the TNF-stimulated samples. HUVECs in these experiments were studied at passages 1–4 and GMVECs were used at passages 3 and 4.

Samples were rapidly thawed and kept on ice prior to use to prevent spontaneous complement activation, and then analyzed for the various complement components and activation products.

Black 96-well plates were coated with 50 ng/well polyclonal rabbit anti-human C3a (detects human C3a and C3, Complement Technologies, no. A218) in 100 nM bicarbonate buffer (pH 9.6) overnight at 4°C. TBST-washed wells were blocked overnight with 1% Ig-free BSA in PBS (BSA/PBS), followed by 50 min incubation with 100 μl/well test samples (EC supernatant from unstimulated or TNF-stimulated cells, diluted by 10% with 10% BSA/PBS) or purified C3 protein (Complement Technologies, no. A113) for the standard curve (with 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. A213) for 25 min, followed by incubation with 100 ng/ml secondary donkey anti-goat IgG-HRP (Pierce, no. PA1-28664). Fluorescence was measured in a Tecan Infinite M200 Pro plate reader 25 min after the addition of the HRP substrate 10-acetyl-3,7-dihydroxyphenoxazine (AnaSpec, Fremont, CA) with excitation of 530 nm and emission of 590 nm.

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 HRP that reacts with hydrogen peroxide to produce a highly fluorescent product. The raw fluorescent readings for the standards range from 1,000 to 45,000. Reciprocal plots of standard dilutions (1/concentration) versus fluorescence intensity at 590 nm (1/590 intensity) produce linear equations that allow the interpolation of complement component concentrations from 0 to the lower limit of the standard curve, in addition to values between the lower limit and higher limit of the standard curve (Supplemental Fig. 2).

FB, C3a, and C5a levels were measured in unstimulated and TNF-stimulated GMVEC and HUVEC supernatant samples using the same protocol as for the C3 immunoassay. The Abs and standard curves used were as follows: 1) FB: capture, 100 ng/well polyclonal goat anti-human FB (Complement Technologies, no. A235); standard, FB protein (Complement Technologies, no. A135) with a range of 12.5–800 ng/ml; detection Abs, 0.1 μg/ml monoclonal mouse anti–factor Ba (Quidel Corporation, no. A225) that was generated using purified human FB as the Ag and is reactive with both FB and the Ba fragment, and 0.1 μg/ml secondary donkey anti-mouse IgG-HRP (Pierce, no. PA1-28748). 2) C3a: capture, 50 ng/well polyclonal rabbit anti-human C3a (Complement Technologies, no. A218); standard, purified C3a desArg protein (Complement Technologies, no. A119) with a range of 61 pg/ml–3.9 ng/ml; detection Abs, 100 ng/ml mouse mAb to human C3a (Pierce, Thermo Scientific, no. GAU 013-16-02) and 0.25 μg/ml secondary goat anti-mouse IgG-HRP (Rockland Immunochemicals, Limerick, PA). 3) C5a: capture, 100 ng/well polyclonal rabbit anti-human C5a (Complement Technologies, no.A221); standard, C5a desArg protein (Complement Technologies, no.A145) with a range of 0.156–10 ng/ml; detection Abs, 0.1 μg/ml mouse monoclonal anti-human C5a (Pierce, Thermo Scientific, no. MA1-40162) and 0.25 μg/ml secondary goat anti-mouse IgG-HRP.

Ba levels were measured in unstimulated and TNF-stimulated GMVEC and HUVEC supernatant samples using an ELISA kit (Quidel, San Diego, CA, no. A033) with a standard curve range of 0.07–2.19 ng/ml (Supplemental Fig. 2).

In these experiments, activated PC was assayed by ELISA from the supernatant of unstimulated or TNF-stimulated GMVECs and HUVECs after the addition of PC and thrombin. Experiments were not performed on IL-1β–stimulated GMVECs and HUVECs because this cytokine did not result in appreciable changes in complement surface regulatory proteins, by flow cytometry, or complement component gene expression.

HUVECs and GMVECs confluent in T-75 flasks were incubated for 24 h in serum-free media. The cells were then washed with PBS and supplemented with 0.2 μM human PC (Haematologic Technologies, no. HCPC-0070) and 10 nM human α-thrombin (Haematologic Technologies, no. HCT-0020) in 1 ml activated PC buffer (0.1% BSA, 3 mM CaCl₂, 0.6 mM MgCl₂ in Ca⁺²/Mg⁺²-free PBS) (29) and incubated at 37°C for 60 min (26, 29). Further activation of PC to activated PC was inhibited with the addition of 10 nM hirudin (1.5 U/ml, Sigma-Aldrich, no. H7016) (29). Supernatants were collected and frozen in liquid nitrogen for 20 s prior to storage at −80°C until analysis. These samples were designated as the unstimulated controls. After allowing recovery for at least 24–48 h in complete media (CM131), the same flasks were incubated for 24 h in CM131 plus 10 ng/ml TNF, and then for an additional 24 h in serum-free media plus 10 ng/ml TNF. The flasks were again washed with PBS and supplemented with 0.2 μM human PC and 10 nM human α-thrombin in 1 ml activated PC buffer and incubated at 37°C for 60 min. Supernatant was collected, flash frozen, and stored at −80°C. These samples were designated as the TNF-stimulated samples. The 24–48 h recovery time in CM131 after collection of control supernatant samples allowed the ECs to replenish nutrients after serum-free media incubation. Cell numbers were not affected by the presence of TNF (Supplemental Fig. 3).

Samples were rapidly thawed and kept on ice prior to use. Activated PC levels were measured in unstimulated and TNF-stimulated GMVEC and HUVEC supernatant samples using an ELISA kit (Cloud-Clone, Houston, TX, no. SEA738Hu) with a standard curve range of 31.25–2000 pg/ml (Supplemental Fig. 2).

The p values for statistical significance in flow cytometry and ELISA experiments (see Figs. 4, 7, 9–11) were obtained by using two-tailed distribution, two-sample equal variance Student t tests. A p value <0.05 was considered statistically significant.

To investigate our hypothesis that GMVECs and HUVECs have differences in their ability to regulate complement activation, we first assessed the gene expression levels of the surface regulatory proteins by real-time PCR. GMVECs expressed higher mRNA levels than did HUVECs for each of the complement regulatory genes tested. Although not significant (p = 0.06), THBD expression was ∼2-fold higher in GMVECs than in HUVECs (see function of 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 presence 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 ∼1.3-fold higher concentrations (p < 0.05), and CD59 in ∼1.4-fold higher concentrations (p < 0.05, Fig. 4).

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 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).

To investigate our secondary hypothesis that inflammatory cytokines alter complement regulation, GMVECs and HUVECs were stimulated with IL-1β or TNF. We used real-time PCR to quantify gene expression of the complement regulatory genes, and flow cytometry to quantify the relative numbers of each complement regulatory protein on the surfaces of GMVECs and HUVECs, with and without IL-1β or TNF stimulation.

We quantified changes in gene expression levels of the surface complement regulatory proteins in GMVECs and HUVECS after stimulation with IL-1β. The expression levels of CD46 and CD55 were slightly higher (1.15- and 1.2-fold, respectively) in IL-1β–stimulated versus unstimulated HUVECs. THBD expression was slightly lower (down 1.35-fold) in IL-1β–stimulated versus unstimulated HUVECs, and CD59 gene expression levels were unchanged in IL-1β–stimulated versus unstimulated HUVECs (Fig. 6A, light gray bars). The gene expression levels of each complement regulatory protein were slightly reduced in IL-1β–stimulated versus unstimulated GMVECs: CD59 and CD46, each down 1.18-fold; THBD, down 1.45-fold; and CD55, down 1.3-fold (Fig. 6B, light gray bars).

We also quantified the complement regulatory proteins present on surfaces of GMVECs and HUVECs, with and without IL-1β stimulation, by flow cytometry. IL-1β induced only small changes in CD46, CD55, CD59, and CD141 proteins on the surfaces of both EC types. Although there were statistically significant changes in CD55 on HUVECs (∼1.1-fold higher on IL-1β–stimulated versus unstimulated HUVECs, p < 0.05) and in CD141 on GMVECs (∼1.4-fold higher on IL-1β–stimulated versus unstimulated GMVECs, p < 0.05), the changes were minimal (Fig. 7A, 7B).

In contrast to the modest changes observed in both EC types with IL-1β stimulation, TNF induced substantial changes in surface 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 reduced 2.8-fold in HUVECs (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, CD46, and CD55 were modestly higher (1.52-, 1.45-, and 1.62-fold, respectively) in TNF-stimulated versus unstimulated HUVECs (Fig. 6A, dark gray bars). Expression levels of CD59 and CD46 were also increased modestly (1.29- and 1.45-fold) in TNF-stimulated GMVECs. Only CD55 expression levels in GMVECs were unaffected by exposure to TNF (Fig. 6B, dark gray bars).

The reduced gene expression levels of THBD induced by TNF in both EC types were associated with reduced amounts of CD141 protein on EC surfaces: TNF-stimulated GMVECs had a 20-fold decrease in surface CD141 protein (p < 0.001), and TNF-stimulated HUVECs had a 14-fold CD141 reduction (p < 0.001, Fig. 7C–E). Surface CD55 protein increased by ∼1.4-fold (p < 0.001), and surface CD46 protein increased by ∼1.6-fold (p < 0.001), on TNF-stimulated versus unstimulated HUVECs. Surface CD59 protein was unchanged in HUVECs exposed to TNF (Fig. 7F). CD46 protein on surfaces of TNF-stimulated GMVECs was increased ∼1.5-fold (p < 0.05), whereas surface CD55 and CD59 protein quantities were both unaltered on GMVECs after TNF stimulation (Fig. 7G).

To investigate our hypothesis that the inflammatory cytokines affect production of AP components, HUVECs and GMVECs were stimulated with IL-1β or TNF, and gene expression of the AP proteins was quantified by real-time PCR.

We assessed gene expression of the AP components C3, CFB, CFD, CFP, C5, CFH, CFI, C4, VWF, and ADAMTS13 in IL-1β–stimulated HUVECs and GMVECs, compared with unstimulated ECs. Incubation of HUVECs with IL-1β induced an ∼16-fold increase in C3 and an ∼5-fold increase in CFB gene expression. Expression of the other AP genes in HUVECs, as well as C4, VWF, and ADAMTS13, was changed only slightly by IL-1β incubation (Fig. 8A). The expression of GMVEC AP genes, as well as C4, VWF, and ADAMTS13, was only slightly affected by IL-1β (Fig. 8B).

We quantified gene expression of the same AP components in TNF-stimulated HUVECs and GMVECs. In HUVECs, TNF induced increases of ∼32-fold in C3 and ∼16-fold in CFB gene expression (Fig. 8C). These values were 2- and 3-fold greater, respectively, than the upregulation of C3 and CFB in IL-1β–stimulated HUVECs. Incubation of GMVECs with TNF induced even more substantial increases in gene expression of C3 and CFB: ∼153-fold for C3 and ∼59-fold for CFB. TNF reduced GMVEC expression of other genes to a lesser extent: CFP by ∼10-fold and VWF by ∼14-fold (Fig. 8D). The expression of CFP and VWF in HUVECs, along with the expression of C5, CFH, CFD, CFI, C4, and ADAMTS13 in both GMVECs and HUVECs, was altered only slightly by incubation with TNF.

Because of our experimental results showing that TNF stimulation increased C3 and CFB expression levels and decreased THBD expression/CD141 surface presence in both cell types, we conducted experiments to determine whether TNF induced AP activation. We measured release of C3 and FB proteins (by ELISA) from HUVECs and GMVECs without TNF stimulation to determine a baseline of C3 and FB protein release from unstimulated cells. Without TNF stimulation, GMVECs had ∼1.6-fold higher levels of C3 as compared with HUVECs (Fig. 9A). FB levels in the supernatant of unstimulated GMVECs were below the detection limit of the assay, but were detected at ∼4000 pg/ml in the supernatant of unstimulated HUVECs (Fig. 9B).

We also measured the AP activation products C3a, C5a, and Ba from unstimulated HUVECs and GMVECs to assess AP activation in both EC types at baseline. Unstimulated GMVECs had ∼5.7-fold higher amounts of C3a (p < 0.05) and ∼1.6-fold higher amounts of Ba, compared with unstimulated HUVECs. C5a levels were 56 pg/ml in the supernatant of unstimulated GMVECs, and were below the detection limit of the assay in unstimulated HUVECs (Fig. 9C). These results suggest that unstimulated GMVECs have increased AP activation compared with unstimulated HUVECs.

We measured the AP components, C3 and FB, in the supernatant of TNF-stimulated HUVECs and GMVECs and compared these component levels to unstimulated levels to determine whether the 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 HUVECs and GMVECs to assess the capacity of TNF to activate the AP. There was no significant change in C3a protein levels in the supernatant of TNF-stimulated HUVECs compared with unstimulated HUVECs (Fig. 10C); however, C5a and Ba proteins increased. The fold change and significance of the increase in C5a in TNF-stimulated HUVEC supernatant could not be determined 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 supernatant (p < 0.05, Fig. 10D). In the supernatant of TNF-stimulated versus unstimulated GMVECs, there was no significant change in C3a and C5a protein levels (Fig. 10G), but there was a significant increase in Ba protein levels by ∼1.5-fold (p < 0.05, Fig. 10H, Table II).

Because of the TNF-induced decrease in THBD gene expression and CD141 surface protein in both GMVECs and HUVECs, we compared the CD141/thrombin-mediated generation of activated PC by unstimulated and TNF-stimulated GMVECs and HUVECs (Fig. 11). We added PC and thrombin to unstimulated and TNF-stimulated GMVECs and HUVECs, and then measured activated PC by ELISA in the cell supernatants. Activated PC levels in the supernatants of unstimulated HUVECs and GMVECs were 3.1 and 11.2 pg/ml, respectively (i.e., ∼3.6-fold higher in unstimulated GMVECs, Fig. 11A). Stimulation with TNF reduced activated PC to levels below the lower detection limit of the assay in the supernatant of HUVECs, and to 1.81 pg/ml in GMVECs (∼6-fold decrease compared with unstimulated GMVECs, Fig. 11B, Table II).

In this study, we assessed the activation and regulation of the AP and coagulation in GMVECs and HUVECs to elucidate the renal vulnerability to injury in aHUS. We also investigated the effects on these processes by the inflammatory cytokines IL-1β and TNF, with the hypothesis that our studies could provide an explanation for the provocation of aHUS episodes during infectious/inflammatory conditions.

By evaluating AP regulatory components in unstimulated GMVECs and HUVECs, we confirmed that the surface complement regulatory proteins are present on both EC types at baseline. We additionally found that gene expression levels of CD55, CD59, CD46, and THBD were higher in GMVECs than in HUVECs, accounting for the slightly increased amounts of CD55, CD59, CD46, and CD141 proteins on GMVEC surface membranes. Similarly, GMVECs expressed the negative regulatory protein CFH in 3-fold higher quantities than did HUVECs. These results suggest that GMVECs may require increased self-protection against complement-mediated injury.

It was our hypothesis that the increased amount of CD141 on the surface of unstimulated GMVECs compared with HUVECs would make GMVECs more anticoagulant at baseline. This is because more thrombin can bind to CD141 on GMVECs, leading to increased activation of PC to activated PC, with subsequent inactivation of coagulation factors Va and VIIIa (Fig. 1B). This explanation was confirmed by our data demonstrating that activated PC levels in unstimulated GMVECs were ∼3.6-fold higher than HUVECs.

Our evaluation of AP component expression in unstimulated GMVECs and HUVECs demonstrated that expression of CFP and CFD was notably different between the two EC types. GMVECs expressed CFP and CFD in relatively greater quantities (7- and 3-fold, respectively) than did HUVECs. AP components FP and FD potentiate AP activation. Assuming that the AP is activated on GMVEC ULVWF strings in the same manner as on HUVEC ULVWF strings (32–34), the greater GMVEC CFD and CFP expression may promote AP C3bBb (C3 convertase) formation (FD) and stability (FP) on GMVEC-secreted/anchored ULVWF strings. These results suggest that GMVECs may have increased AP activation at baseline. Our experiments showing higher levels of the complement activation products C3a and C5a, as well as the specific AP activation product Ba, in the supernatant of unstimulated GMVECs compared with HUVECs, support this conclusion and may explain the particular vulnerability of the kidneys to AP-mediated injury in aHUS.

In contrast to CFP and CFD expression, the gene expression of CFB was similar in both unstimulated HUVECs and GMVECs; however, FB levels in the supernatant of unstimulated GMVECs were below the lower detection limit of the assay. This low value may be because of the underlying increased AP activation in GMVECs, which may secrete and anchor some ULVWF multimeric strings in response to minor experimental manipulation. In this case, most of the FB produced would attach to C3b on the ULVWF strings and be cleaved to Bb by the increased FD produced, releasing the Ba fragment. Bb would remain attached to ULVWF strings as part of the C3 convertase and be stabilized by the increased FP protein released. The putative result is FB protein levels that are very low in the supernatant of unstimulated GMVECs.

The propensity for aHUS relapses in association with infection/inflammation (35, 36) led us to evaluate complement parameters in both GMVECs and HUVECs during cytokine stimulation using TNF and IL-1β. Whereas IL-1β affected AP production and regulation by GMVECs and HUVECs only mildly, TNF caused significant changes in these parameters in both EC types. The major receptor for TNF, TNFR-1, is present at similar densities on both HUVECs and human microvascular ECs (49, 50). TNF binding to TNFR-1 stimulates signaling pathways that include NF-κB (51). Our data indicate that TNF/TNFR-1 interaction and signaling also occur on GMVECs, as well as on HUVECs.

Inflammatory cytokines, including TNF, initiate EC stimulation and secretion/anchorage of ULVWF multimeric strings (52). Cytokines may stimulate EC secretion/anchorage of ULVWF multimeric strings in excess of the capacity of EC-produced ADAMTS-13 (37) to cleave the strings rapidly and completely, therefore promoting the initiation, assembly, and activation of the AP on these strings. This latter process will be magnified in aHUS patients with a chronically overactive AP.

We found that both GMVECs and HUVECs incubated with TNF had extensive decreases in the expression of THBD and corresponding decreases in surface CD141. Although others have previously found that TNF downregulates THBD in HUVECs, bovine arterial ECs, human coronary artery ECs, lung microvascular ECs, and human microvascular ECs (42, 48), the effects of TNF on GMVECs demonstrated in our study were previously unknown.

In addition to THBD downregulation, we found that TNF increased gene expression of C3 and CFB in GMVECs and HUVECs. This resulted in increased levels of C3 and FB proteins in the supernatant of TNF-stimulated GMVECs and HUVECs compared with unstimulated ECs. C3 and FB in the supernatant of TNF-stimulated HUVECs increased ∼14- and ∼5-fold, respectively. Although the ratio of this increase was consistent with gene expression levels of C3 and CFB in the presence of TNF (C3 and CFB increased ∼32- and ∼16-fold, respectively), the increases in C3 and FB protein levels were not as great. This may be partially the result of C3 (as C3b) binding to the increased quantity of HUVEC-secreted/anchored ULVWF strings during TNF stimulation, as well as the attachment of FB to the ULVWF-bound C3b (34).

There were similar GMVEC responses to TNF: ∼7-fold increase in C3 protein levels in the supernatant of TNF-stimulated GMVECs in association with ∼153-fold increase in C3 gene expression. As described above for HUVECs, this disparity is likely to be because of C3 (as C3b) binding to GMVEC-secreted/anchored ULVWF strings. CFB mRNA levels in TNF-stimulated GMVECs were also increased (∼59-fold), but the FB protein fold increase in the TNF-stimulated GMVEC supernatant could not be determined because comparator FB levels in unstimulated GMVECs were below assay detection limits. We hypothesize, however, that (as described for HUVECs) the FB protein levels in 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 bind to their respective cell receptors and cause chemotaxis, histamine release, and increased vascular permeability. The C3a and C5a receptors, C3a-R and C5a-R, are present on HUVECs and human microvascular ECs (53–56). It is likely that any C3a and C5a proteins generated during TNF stimulation bind to GMVEC and HUVEC C3a-R and C5a-R receptors and, therefore, remain in the supernatants of TNF-stimulated ECs only at levels below the lower detection limits of the assays. Less is known about the Ba fragment and its functions, if any. Ba is chemotactic for human peripheral blood leukocytes and guinea pig neutrophils, but is less potent than C5a (57–59). It is not known whether there is a Ba receptor on human ECs. Our data suggest that this putative receptor is either absent on ECs or present in small amounts; otherwise, we would not have been able to detect Ba in the supernatant of TNF-stimulated ECs.

Furthermore, we demonstrated the functional relevance of TNF-induced downregulation of THBD as related to its anticoagulant activity. In addition to participating in regulation of the C3 convertase, CD141 also functions to bind thrombin and activate PC (Fig. 1B). In this study, we demonstrated that the decreases in THBD gene expression and surface presence of CD141 protein on TNF-stimulated GMVECs and HUVECs are associated with decreased PC activation to activated PC. The fold decrease in activated PC levels in TNF-stimulated HUVEC supernatant relative to unstimulated supernatant could not be calculated because TNF-stimulated HUVEC supernatant had activated PC levels below the lower detection limit of the assay. In GMVECs, however, there was an ∼6-fold decrease in supernatant activated PC levels with TNF stimulation compared with unstimulated GMVECs. These results are consistent with our gene expression data where THBD gene expression levels decreased 6.7-fold, and the amount of surface CD141 protein decreased by ∼20-fold, in TNF-stimulated versus unstimulated GMVECs. The considerable reduction in activation to activated PC likely promotes increased microthrombi formation in GMVECs during infection/inflammation.

The extensive downregulation of THBD and surface CD141, along with extreme upregulation of C3/C3 and CFB/FB, in TNF-stimulated GMVECs explains the particular vulnerability of the kidneys to C3 convertase generation (and injury) and microthrombus formation in aHUS patients during infectious/inflammatory events (Table II). The upregulation of the AP in TNF-stimulated GMVECs may be especially dangerous in aHUS patients who have heterozygous gain-of-function mutations in C3 and CFB. Further upregulation of the C3 and FB proteins would be expected to intensify activation of the already overactive AP in these individuals. By analogy, the extensive downregulation of THBD in TNF-stimulated GMVECs may be most threatening to aHUS patients with heterozygous loss-of-function mutations in THBD. Further loss of surface CD141 during infection/inflammation will accentuate the underlying increased AP overactivation and coagulation. Gene mutations in THBD have a prevalence of ∼3–5% in total aHUS cases (10, 60, 61). Infections trigger aHUS episodes (35, 36), and case reports suggest that some patients with THBD mutations do not have clinical signs of disease until they have a provocative infectious/inflammatory event (10). Our TNF/GMVEC data provide a plausible molecular explanation for these clinical observations in patients with loss-of-function mutations in THBD, or gain-of-function mutations in C3 and CFB, and they may also explain the linkage between infection/inflammation in any type of overactivation of the AP in aHUS.

During the evaluation of the gene expression of AP components (along with VWF and ADAMTS13) in cytokine-stimulated ECs, we found that TNF incubation reduced the expression levels of CFP and VWF in GMVECs by ∼10- and ∼ 14-fold, respectively. The resulting decrease in FP protein would be expected to partially impede AP activation by decreasing C3 convertase stability. This may explain why Ba in the supernatant of TNF-stimulated GMVECs increased only ∼1.5-fold with TNF stimulation (in contrast to the ∼7-fold increase in the supernatant of TNF-stimulated HUVECs). The reduced mRNA levels for VWF are unlikely to affect the number of secreted/anchored ULVWF strings (that serve as C3b binding/amplifying surfaces) because of the previously stored VWF in EC Weibel–Palade bodies.

GMVEC and HUVEC AP component and complement regulatory protein expression was less affected by IL-1β compared with TNF. IL-1β resulted in upregulation of C3 and CFB in HUVECs to a lesser degree than did TNF. IL-1β had no effect, however, on GMVEC C3 and CFB expression, and it only modestly affected expression of other AP components and complement regulatory proteins in both cell types. IL-1β is therefore likely to be less perturbing than TNF to the renal microvasculature during inflammation. The receptor for IL-1β, IL-1R, is present on HUVECs (62, 63); however, evidence is lacking about its presence on GMVECs. The expression changes in AP components and surface regulators demonstrated in our study by IL-1β–stimulated GMVECs suggests that IL-1R is present on GMVECs at a lower density than on HUVECs.

In conclusion, to our knowledge, our study is the first detailed analysis of AP component expression and surface complement regulatory protein expression/display in human GMVECs, and it is the basis for additional study of other microvascular EC types. Our data demonstrate activation of the AP and inhibition of PC-mediated anticoagulation in GMVECs and HUVECs exposed to the inflammatory cytokine TNF. The results provide insight into the pathophysiology of kidney injury and microthrombi formation during infection/inflammation, especially in aHUS patients with genetic overactivity of the AP.

The authors have no financial conflicts of interest.