Joy A. Williams

Carbonyl assays for determination of oxidatively modified proteins.

Publisher Summary Enzymes and structural proteins may be attacked whenever free radicals are generated. As a consequence, oxidative modification of proteins may occur in a variety of physiologic and pathologic processes. Although the distinction is sometimes arbitrary, these modifications may be primary or secondary. Primary modifications occur in metal-catalyzed oxidation, radiation-mediated oxidation, and oxidation by ozone or oxides of nitrogen. Secondary modifications occur when proteins are modified by molecules generated by oxidation of other molecules. One important example is the covalent modification of proteins by hydroxynonenal produced by oxidation of lipids. Carbonyl groups (aldehydes and ketones) may be introduced into proteins by any of these reactions, and the appearance of such carbonyl groups is taken as presumptive evidence of oxidative modification. Assay of carbonyl groups in proteins provides a convenient technique for detecting and quantifying oxidative modification of proteins. This chapter presents new methods for determination of carbonyl content, which are based on the reaction of carbonyl groups with 2,4-dinitrophenylhydrazine to form a 2,4-dinitrophenylhydrazone. The assays provide substantial improvements in both sensitivity and specificity.

DIFFERENTIAL SUSCEPTIBILITY OF PLASMA PROTEINS TO OXIDATIVE MODIFICATION :EXAMINATION BY WESTERN BLOT IMMUNOASSAY

Plasma proteins are exposed to oxidants in a variety of circumstances in vivo, such as during tissue injury and inflammation. In this report, the relative susceptibility of each of the major plasma proteins to oxidative modification was assessed by exposing whole plasma to a metal-catalyzed radical generating system and detecting oxidation (protein carbonyl groups) using a novel Western blot immunoassay. Proteins were derivitized with dinitrophenylhydrazine, separated by SDS-gel electrophoresis, and screened with antibodies against dinitrophenyl groups. As little as 1 pmol of protein-associated carbonyls could be detected (100 ng of a 50 kD protein containing 0.5 mol carbonyl/mol protein). Individual plasma proteins were identified by their comigration with standards, crossreactivity with specific antibodies, and by comparison of plasma to serum. Using this approach, we found that plasma fibrinogen was much more susceptible to oxidative modification compared to the other major plasma proteins, albumin, immunoglobulins, and transferrin. The results emphasize the utility of this method for studying oxidation of proteins in cell extracts and tissues and indicate that experiments on the effects of oxidation on fibrinogen function are merited.

Serum-derived protein S binds to phosphatidylserine and stimulates the phagocytosis of apoptotic cells

Rapid phagocytosis of apoptotic cells is thought to limit the development of inflammation and autoimmune disease. Serum enhances macrophage phagocytosis of apoptotic cells. Here we identified protein S as the factor responsible for serum-stimulated phagocytosis of apoptotic cells. Protein S is best known for its anti-thrombotic activity, serving as a cofactor for protein C. Purified protein S was equivalent to serum in its ability to stimulate macrophage phagocytosis of apoptotic lymphoma cells, and immunodepletion of protein S eliminated the prophagocytic activity of serum. Protein S acted by binding to phosphatidylserine expressed on the apoptotic cell surface. Protein S is thus a multifunctional protein that can facilitate clearance of early apoptotic cells in addition to regulating blood coagulation.

Oxidative modification of fibrinogen inhibits thrombin-catalyzed clot formation

Plasma fibrinogen plays a central role in controlling hemostasis. In an earlier report, we found that fibrinogen is oxidized when whole plasma is treated with a metal-catalyzed oxidation system. These studies show that oxidative modification of purified human fibrinogen leads to an exposure-dependent loss of thrombin-induced clot formation. Inhibition of clotting occurred when either metal-catalyzed oxidation or gamma-irradiation was employed to generate oxidizing radicals. Both systems caused covalent modification of fibrinogen, assessed by measuring incorporation of protein carbonyls. Thrombin-catalyzed fibrinopeptide release was normal in irradiated fibrinogen and was only slightly diminished in protein exposed to metal-catalyzed oxidation, indicating that the inhibition of clotting activity was due to impaired fibrin monomer polymerization. Thus, oxidative modification of normal fibrinogen causes dysfibrinogenemia and constitutes a novel mechanism for inhibition of thrombosis.

Regulation of macrophage interleukin-6 (IL-6) and IL-10 expression by prostaglandin E2: the role of p38 mitogen-activated protein kinase.

Prostaglandin E2 (PGE2) regulates production of a wide array of cytokines. We have found that PGE2 can upregulate the levels of both interleukin-10 (IL-10) and IL-6 produced by activated murine macrophages, but the molecular pathways leading to their augmentation differ. Synthesis of IL-10 in response to PGE2 is dependent on p38 MAP kinase activity, whereas synthesis of IL-6 is not. Evidence to support this derives from two experimental approaches. First, we established that PGE2 is effective in elevating IL-10 levels only when it is added to cells in which p38 kinase has been activated. In contrast, PGE2 can augment IL-6 levels regardless of whether or not p38 kinase is active. Second, we showed that inhibitors that are selective for p38 kinase prevent the IL-10 response to PGE2 but not the IL-6 response. We found that p38 kinase inhibitors are able to inhibit IL-6 production in activated macrophages, but this occurs primarily as a result of their concurrent inhibition of cyclooxygenase-2 and endogenous PGE2 synthesis. These results indicate that macrophage IL-10 and IL-6 expression is differentially regulated by PGE2 and p38 MAP kinase in murine inflammatory macrophages.

Differential Requirements for Expression of CD80/86 and CD40 on B Cells for T-Dependent Antibody Responses In Vivo

The CD80/86-CD28 and CD40-CD40 ligand costimulatory pathways are essential for Th cell-dependent B cell responses that generate high-affinity, class-switched Ab in vivo. Disruption of either costimulatory pathway results in defective in vivo humoral immune responses, but it remains unclear to what extent this is due to deficient activation of Th cells and/or of B cells. To address this issue, we generated mixed chimeras in which CD80/86- or CD40-deficient bone marrow-derived cells coexist with wild-type (WT) cells, thereby providing the functional T cell help and accessory cell functions required for fully competent B cell responses. We were then able to assess the requirement for CD80/86 or CD40 expression on B cells producing class-switched Ig in response to a T-dependent Ag. In CD80/86 WT plus CD80/86 double-knockout mixed chimeras, both WT- and CD80/86-deficient B cells produced IgG1 and IgE responses, indicating that direct signaling by CD80/86 is not essential for efficient B cell activation. In marked contrast, only WT IgG1 and IgE responses were detected in the chimeras containing CD40-deficient cells, demonstrating that CD40 expression on B cells is essential for class switching by those B cells. Thus, while disrupting either the CD80/86-CD28 or the CD40-CD40 ligand costimulatory pathway abrogates T-dependent B cell immune responses, the two pathways are nonredundant and mediated by distinct mechanisms.

Regulation of thymic NKT cell development by the B7-CD28 costimulatory pathway.

Invariant NKT (iNKT) cells are a population of TCRαβ-expressing cells that are unique in several respects. In contrast to conventional T cells, iNKT cells are selected in the thymus for recognition of CD1, rather than conventional MHC class I or II, and are selected by CD1-expressing double-positive thymocytes, rather than by the thymic stromal cells responsible for positive selection of conventional T cells. We have probed further the requirements for thymic iNKT cell development and find that these cells are highly sensitive to B7-CD28 costimulatory interactions, as evidenced by the substantially decreased numbers of thymic iNKT cells in CD28 and in B7 knockout mice. In contrast to the requirement for CD1, B7-CD28 signaling does not affect early iNKT cell lineage commitment, but exerts its influence on the subsequent intrathymic expansion and differentiation of iNKT cells. CD28 wild-type/CD28-deficient mixed bone marrow chimeras provided evidence of both cell-autonomous and non-cell-autonomous roles for CD28 during iNKT cell development. Paradoxically, transgenic mice in which thymic expression of B7 is elevated have essentially no measurable thymic iNKT cells. Taken together, these results demonstrate that the unique pathway involved in iNKT cell development is marked by a critical role of B7-CD28 interactions and that disruption or augmentation of this costimulatory interaction has substantial effects on iNKT cell development in the thymus.

Regulated Costimulation in the Thymus Is Critical for T Cell Development: Dysregulated CD28 Costimulation Can Bypass the Pre-TCR Checkpoint

Expression of CD28 is highly regulated during thymic development, with CD28 levels extremely low on immature thymocytes but increasing dramatically as CD4−CD8− cells initiate expression of TCRβ. B7-1 and B7-2, the ligands for CD28, have a restricted distribution in the thymic cortex where immature thymocytes reside and are more highly expressed in the medulla where the most mature thymocytes are located. To determine the importance of this regulated CD28/B7 expression for T cell development, we examined the effect of induced CD28 signaling of immature thymocytes in CD28/B7-2 double-transgenic mice. Strikingly, we found that differentiation to the CD4+CD8+ stage in CD28/B7-2 transgenics proceeds independent of the requirement for TCRβ expression manifest in wild-type thymocytes, occurring even in Rag− or CD3ε− knockouts. These findings indicate that signaling of immature thymocytes through CD28 in the absence of TCR- or pre-TCR-derived signals can promote an aberrant pathway of T cell differentiation and highlight the importance of finely regulated physiologic expression of CD28 and B7 in maintaining integrity of the “β” checkpoint for pre-TCR/TCR-dependent thymic differentiation.

CD40 Ligand Functions Non-Cell Autonomously to Promote Deletion of Self-Reactive Thymocytes

CD40 ligand (CD40L)-deficient mice have been shown to have a defect in negative selection of self-reactive T cells during thymic development. However, the mechanism by which CD40L promotes deletion of autoreactive thymocytes has not yet been elucidated. We have studied negative selection in response to endogenous superantigens in CD40L-deficient mice and, consistent with previous reports, have found a defect in negative selection in these mice. To test the requirement for expression of CD40L on T cells undergoing negative selection, we have generated chimeric mice in which CD40L wild-type and CD40L-deficient thymocytes coexist. We find that both CD40L wild-type and CD40L-deficient thymocytes undergo equivalent and efficient negative selection when these populations coexist in chimeric mice. These results indicate that CD40L can function in a non-cell-autonomous manner during negative selection. Deletion of superantigen-reactive thymocytes was normal in B7-1/B7-2 double-knockout mice, indicating that CD40-CD40L-dependent negative selection is not solely mediated by B7 up-regulation and facilitation of B7-dependent T cell signaling. Finally, although the absence of CD40-CD40L interactions impairs negative selection of autoreactive CD4+ and CD8+ cells during thymic development, we find that self-reactive T cells are deleted in the mature CD4+ population through a CD40L-independent pathway.

A novel role for CD28 in thymic selection : elimination of CD28/B7 interactions increases positive selection

While the importance of the CD28/B7 costimulation pathway is well established for mature T cells, the role of CD28 in thymocyte selection is less well defined. The role of CD28 in both negative and positive selection was assessed using H‐Y‐specific TCR‐transgenic (Tg) RAG‐2‐deficient (H‐Yrag) mice. Negative selection in male H‐Yrag mice was not affected by deficiency in CD28 or B7. Surprisingly, absence of CD28 or B7 in H‐Yrag females resulted in increased numbers of CD8 single‐positive (SP) thymocytes. The CD8 SP thymocytes found in these females were mature and functionally competent. Furthermore, double‐positive (DP) thymocytes from CD28‐knockout (CD28KO) or B7.1/B7.2 double‐KO (B7DKO) females had higher levels of both CD5 and TCR than those from WT females, consistent with a stronger selecting signal. CD28KO H‐Yrag fetal thymic organ cultures also had elevated numbers of thymic CD8 SP cells, reflecting increased thymic differentiation and not recirculation of peripheral T cells. Finally, increased selection of mature CD4 and CD8 SP T cells was observed in non‐TCR‐Tg CD28KO and B7DKO mice, indicating that this function of CD28‐B7 interaction is not unique to a TCR‐Tg model. Together these findings demonstrate a novel negative regulatory role for CD28 in inhibiting differentiation of SP thymocytes, probably through inhibition of thymic selection.