Scott A. Crist

Platelet-mediated modulation of adaptive immunity: unique delivery of CD154 signal by platelet-derived membrane vesicles

Although mounting evidence indicates that platelets participate in the modulation of both innate and adaptive immunity, the mechanisms by which platelets exert these effects have not been clearly defined. The study reported herein uses a previously documented adoptive transfer model to investigate the ability of platelet-derived membrane vesicles to communicate activation signals to the B-cell compartment. The findings demonstrate for the first time that platelet-derived membrane vesicles are sufficient to deliver CD154 to stimulate antigen-specific IgG production and modulate germinal center formation through cooperation with responses elicited by CD4(+) T cells. The data are consistent with the hypothesis that platelets modulate inflammation and adaptive immunity at sites distant from the location of activation and that platelet-derived membrane vesicles are sufficient to mediate the effect.

In vivo suppressive function of myeloid-derived suppressor cells is limited to the inflammatory site

Current paradigms suggest that, despite the heterogeneity of myeloid‐derived suppressor cells (MDSC), all Gr‐1+CD11b+ cells can exert suppressive function when exposed to inflammatory stimuli. In vitro evaluation shows that MDSC from multiple tissue sites have suppressive activity, and in vivo inhibition of MDSC enhances T‐cell function; however, the relative capacity of MDSC present at localized inflammatory sites or in peripheral tissues to suppress T‐cell responses in vivo has not been directly evaluated. In the current study, we observed that during a tissue‐specific inflammatory response, MDSC inhibition of CD8+ T‐cell proliferation and IFN‐γ production was restricted to the inflammatory site. Using a prostate‐specific inflammatory model and a heterotopic prostate tumor model, we showed that MDSC from inflammatory sites or from tumor tissue possess immediate capacity to inhibit T‐cell function, whereas those isolated from peripheral tissues (spleens and liver) were not suppressive without activation of iNOS by exposure to IFN‐γ. These data suggest that MDSC are important regulators of immune responses in the prostate during acute inflammation and the chronic inflammatory setting of tumor growth, and that regulation of T‐cell function by MDSC during a localized inflammatory response is restricted in vivo to the site of an ongoing immune response.

Platelet-derived CD154 enables T-cell priming and protection against Listeria monocytogenes challenge

Collagen exposure in tissue activates platelets, initiates wound healing, and modulates adaptive immunity. In this report, data are presented to demonstrate a requirement for platelet-derived CD154 for both collagen-induced augmentation of T-cell immunity and induction of pro-tective immunity to Listeria challenge. Specifically, we demonstrate that Ad5 encoding the membrane-bound form of ovalbumin (Ad5-mOVA) delivered in collagen induces higher ovalbumin-specific cytotoxic T lymphocyte (CTL) activity in a dose-dependent manner compared with Ad5-mOVA delivered in PBS. Increased CTL activity was dependent on the ability of platelets to respond to collagen and to express CD154. Furthermore, mice immunized with low-dose Ad5-mOVA in collagen were able to control a challenge of Listeria monocytogenes recombinant for ovalbumin expression (Lm-OVA), whereas mice immunized with low-dose Ad5-mOVA in PBS were not. These data indicate that in a physiologic setting that mimics wounding, platelets perform a sentinel function when antigen dose is too low to provoke an efficient immune response, and can enhance the generation of antigen-specific CD8 T cells that are functionally relevant to the host.

Platelet CD40L at the interface of adaptive immunity

Initiated by the finding that platelets express functional CD40 ligand (CD40L, CD154), many new roles for platelets have been discovered in unanticipated areas, including the immune response. When current literature is considered as a whole, the picture that is emerging begins to show that platelets are able to significantly affect, for better or worse, the overall health and condition of the mammalian host. Animal models have made significant contributions to our expanding knowledge of platelet function, much of which is anticipated to be clinically relevant. While still mostly circumstantial, the evidence supports a critical role for CD40L in many normal and disease processes.

Use of graphene as protection film in biological environments

Corrosion of metal in biomedical devices could cause serious health problems to patients. Currently ceramics coating materials used in metal implants can reduce corrosion to some extent with limitations. Here we proposed graphene as a biocompatible protective film for metal potentially for biomedical application. We confirmed graphene effectively inhibits Cu surface from corrosion in different biological aqueous environments. Results from cell viability tests suggested that graphene greatly eliminates the toxicity of Cu by inhibiting corrosion and reducing the concentration of Cu2+ ions produced. We demonstrated that additional thiol derivatives assembled on graphene coated Cu surface can prominently enhance durability of sole graphene protection limited by the defects in graphene film. We also demonstrated that graphene coating reduced the immune response to metal in a clinical setting for the first time through the lymphocyte transformation test. Finally, an animal experiment showed the effective protection of graphene to Cu under in vivo condition. Our results open up the potential for using graphene coating to protect metal surface in biomedical application.

Platelet influence on T- and B-cell responses

Understanding the adaptive immune response is an area of research critically important in medicine. Several positive regulators of B- and T-cell activation exist to eliminate pathogens, in which CD40 ligand (CD154) plays a fundamental role. It is well documented that CD154 expressed by CD4 T helper cells can be critical in the proper activation of dendritic cells for the productive stimulation of CD8 T cells and is required for proper T-dependent B-cell immunity. However, platelets are an abundant and systemic source of CD154. While classically known to be important for hemostasis and inflammation, several lines of evidence suggest that platelet-derived ligands can modulate the adaptive immune compartment.

Nuclear factor of activated T cells (NFAT) mediates CD154 expression in megakaryocytes

Platelets are an abundant source of CD40 ligand (CD154), an immunomodulatory and proinflammatory molecule implicated in the onset and progression of several inflammatory diseases, including systemic lupus erythematosus (SLE), diabetes, and cardiovascular disease. Heretofore considered largely restricted to activated T cells, we initiated studies to investigate the source and regulation of platelet-associated CD154. We found that CD154 is abundantly expressed in platelet precursor cells, megakaryocytes. We show that CD154 is expressed in primary human CD34+ and murine hematopoietic precursor cells only after cytokine-driven megakaryocyte differentiation. Furthermore, using several established megakaryocyte-like cells lines, we performed promoter analysis of the CD154 gene and found that NFAT, a calcium-dependent transcriptional regulator associated with activated T cells, mediated both differentiation-dependent and inducible megakaryocyte-specific CD154 expression. Overall, these data represent the first investigation of the regulation of a novel source of CD154 and suggests that platelet-associated CD154 can be biochemically modulated.

An inducible model of abacterial prostatitis induces antigen specific inflammatory and proliferative changes in the murine prostate.

Prostatitis is a poorly understood disease and increasing evidence suggests inflammation is involved in other prostatic diseases including prostate cancer.

Interleukin-1 Induces Growth Arrest by Hypophosphorylation of the Retinoblastoma Susceptibility Gene Product RB

Interleukin-1 (IL-1) causes G/G phase growth arrest in human melanoma cells, A375-C6. Because hypophosphorylation of the retinoblastoma susceptibility gene product, RB, is one of the key events responsible for G/G phase growth arrest, we investigated whether IL-1 altered the phosphorylation status of RB protein in these cells. Exposure to IL-1 caused a time-dependent increase in hypophosphorylated RB that correlated with an accumulation of cells arrested in the G/G phase. The ability of IL-1 to cause hypophosphorylation of RB and growth arrest was abrogated by the SV40 large T antigen, which binds preferentially to hypophosphorylated RB, but not by the K1 mutant of the T antigen, which is defective in binding to RB. Furthermore, the cells were protected from IL-1-inducible growth inhibition by ectopic expression of dominant-negative mutants of the Rb gene, or the transcription factor E2F-1, which is a downstream target of RB. These results suggest that hypophosphorylated RB mediates the growth arrest induced by IL-1.

AUTOCRINE IL-6 PRODUCTION BY HUMAN TRANSITIONAL CARCINOMA CELLS UPREGULATES EXPRESSION OF THE α5β1 FIBRONECTIN RECEPTOR

Purpose: Studies have demonstrated elevated expression and secretion of IL-6 by transitional cell carcinomas (TCC) following bacillus Calmette-Guerin (BCG) therapy. At present, the role of IL-6 on the biology of TCC is poorly understood. This study evaluated the influence of IL-6 expression on a critical variable regulating BCG-tumor interaction, the tumor expression of α5β1 integrin. Materials and Methods: A human TCC cell line (253J) was transfected with an expression vector containing the full-length IL-6 cDNA sequence. Overexpression of IL-6 mRNA and protein was confirmed by Northern analysis and ELISA, respectively. Clones found to overexpress IL-6 were then assayed for α5β1 expression using Northern analysis and flow cytometry. The effect of alterations in α5β1 expression on tumor adherence to fibronectin (FN), and BCG adherence to tumor cells was determined using specific adherence assays. Results: mRNAs for both the α5 and β1 subunits of the FN receptor were increased an average of 9.4 fold and 125.7 fold respectively in the IL-6 overexpressing transfectants relative to the parental 253J cells. Increased mRNA of α5 and β1 was associated with increased cell surface expression of both proteins. Increased protein expression resulted in greater FN substrate binding affinity and increased adherence of BCG to tumor cells. Conclusions: Autocrine expression of IL-6 upregulates the expression of FN receptor subunits in TCC. Increased α5β1 expression increases cellular adherence to FN, and BCG adherence to tumor cells. These results suggest a role for IL-6 in mediating the antitumor activity of BCG by influencing BCGs adherence to TCC.