Abstract

Activation of complement (C) with consequent generation of the C5-derived peptides C5a and C5a des Arg is a primary chemotactic stimulus at sites of infection or injury. Excessive C5a- mediated leukocyte recruitment into various tissues is known to be a critical step in the pathogenesis of many inflammatory disorders. C5-derived peptides are very potent chemoattractants for almost the entire cellular repertoire of the immune system, and consequently are an attractive target for therapeutic intervention. But the regulation of C5a chemotactic activity in a physiological setting is poorly understood. Previously, several groups have shown that the vitamin D binding protein (DBP), also known as Gc-globulin, can significantly enhance the chemotactic activity of C5-derived peptides, i.e., function as a chemotactic cofactor. This activity of DBP is specific for the C5-derived peptides. However, its role in vivo has not been determined and the mechanism of chemotaxis enhancement is not known. It is our HYPOTHESIS that DBP functions as a physiologically important regulator of the chemotactic activity for C5a, and that this activity is initiated by formation of a novel multi-faceted DBP binding/signaling complex on the cell surface. The objective of this proposal is two- fold. First, the in vivo function of DBP as a C5a chemotactic cofactor will be determined in murine models of C-dependent inflammation using DBP-/- and DBP+/+ mice.
This aim will investigate the role of DBP in immune complex mediated (I) peritonitis, (II) alveolitis, (III) alveolitis induced by C-activated serum and purified C5a, and (IV) in vitro using murine leukocytes and purified proteins in a chemotaxis assay. The second specific aim will investigate how DBP enhances chemotaxis to C5a using human neutrophils and U937 cells in vitro.
This aim will employ a reverse genetic approach to dissect the process by utilizing (I) mutant DBPs generated by selective deletion of the co-chemotactic and cell binding sequences and (II) RNAi directed at individual members of the DBP binding/signaling complex to silence their expression in U937 cells. Both will be used to determine the effect on DBP-cell binding, calcium signaling, chemotaxis and shedding of the binding/signaling complex. Results of this study will provide a better understanding of how C5a recruits leukocytes in a physiological setting, and also may reveal that DBP could be a target for therapeutic intervention to reduce the leukocyte burden at sites of C activation. ? ?

Public Health Relevance

Complement activation peptide C5a is one of the most potent and physiologically important molecules that induce accumulation of white blood cells in tissues (chemotaxis). Excessive or aberrant production of C5a is strongly associated with tissue injury in numerous inflammatory diseases. The vitamin D binding protein (DBP) is a blood protein that has been shown to significantly enhance the chemotactic activity of C5a. This proposal will investigate the mechanisms by DBP regulates the chemotactic activity of C5a in a physiological setting. ? ? ?

  Funding Agency

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM063769-06A2
Application #
7580733
Study Section
Innate Immunity and Inflammation Study Section (III)
Program Officer
Dunsmore, Sarah
Project Start
2001-07-01
Project End
2012-08-31
Budget Start
2008-09-25
Budget End
2009-08-31
Support Year
6
Fiscal Year
2008
Total Cost
$327,600
Indirect Cost

  Institution

Name
State University New York Stony Brook
Department
Pathology
Type
Schools of Medicine
DUNS #
804878247
City
Stony Brook
State
NY
Country
United States
Zip Code
11794

  Publications

Kew, Richard R; Tabrizian, Tahmineh; Vosswinkel, James A et al. (2018) Vitamin D-binding protein deficiency in mice decreases systemic and select tissue levels of inflammatory cytokines in a murine model of acute muscle injury. J Trauma Acute Care Surg 84:847-854
Ramadass, Mahalakshmi; Ghebrehiwet, Berhane; Kew, Richard R (2015) Enhanced recognition of plasma proteins in a non-native state by complement C3b. A possible clearance mechanism for damaged proteins in blood. Mol Immunol 64:55-62
Ge, Lingyin; Trujillo, Glenda; Miller, Edmund J et al. (2014) Circulating complexes of the vitamin D binding protein with G-actin induce lung inflammation by targeting endothelial cells. Immunobiology 219:198-207
Ghebrehiwet, Berhane; Ji, Yan; Valentino, Alisa et al. (2014) Soluble gC1qR is an autocrine signal that induces B1R expression on endothelial cells. J Immunol 192:377-84
Penzo, Marianna; Habiel, David M; Ramadass, Mahalakshmi et al. (2014) Cell migration to CXCL12 requires simultaneous IKK? and IKK?-dependent NF-?B signaling. Biochim Biophys Acta 1843:1796-1804
Ramadass, Mahalakshmi; Ghebrehiwet, Berhane; Smith, Richard J et al. (2014) Generation of multiple fluid-phase C3b:plasma protein complexes during complement activation: possible implications in C3 glomerulopathies. J Immunol 192:1220-30
Trujillo, Glenda; Habiel, David M; Ge, Lingyin et al. (2013) Neutrophil recruitment to the lung in both C5a- and CXCL1-induced alveolitis is impaired in vitamin D-binding protein-deficient mice. J Immunol 191:848-56
Kew, Richard R; Penzo, Marianna; Habiel, David M et al. (2012) The IKK?-dependent NF-?B p52/RelB noncanonical pathway is essential to sustain a CXCL12 autocrine loop in cells migrating in response to HMGB1. J Immunol 188:2380-6
Trujillo, Glenda; Zhang, Jianhua; Habiel, David M et al. (2011) Cofactor regulation of C5a chemotactic activity in physiological fluids. Requirement for the vitamin D binding protein, thrombospondin-1 and its receptors. Mol Immunol 49:495-503
Guerriero, Jennifer L; Ditsworth, Dara; Catanzaro, Joseph M et al. (2011) DNA alkylating therapy induces tumor regression through an HMGB1-mediated activation of innate immunity. J Immunol 186:3517-26

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