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 Supplementary http://www.jimmunol.org/content/suppl/2015/12/15/jimmunol.150096 Material 0.DCSupplemental http://www.jimmunol.org/ Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! 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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 ex- pression 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 endo- thelial 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, polymorpho- nuclear 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