Faculty Bibliography

RATIONALE: Biomarkers of sepsis severity and mortality are an important area of investigation. Our group has shown that increased serum levels of soluble(s)CD28 and sCD40L are predictive of mortality from sepsis. This has more recently been validated by other groups. We hypothesize that the release of microparticles(MPs) bound to co-stimulatory molecules into the circulation is one of the causes of disseminated inflammation. The mechanism producing sCD28 and sCD40L is unknown. We have previously shown that MP activity is induced in murine sepsis. Our current work is focused on using flow-cytometry to identify the characteristics of MPs and determine if they express CD28 and CD40L in sepsis models. METHODS: in vivo: Polymicrobial sepsis was induced in C57BL/6 mice using 19-Gauge cecal ligation and puncture(CLP) and citrated plasma was collected by cardiac puncture 18 hours later. in vitro: RAW264.7 cells 2x106 cells/mL in a 6 well plate were exposed to LPS(40 ng/mL) or media alone and incubated overnight. MP Isolation: MP containing supernatants were centrifuged at 1,500gX15 min and ultracentrifugation at 70,000gX30 min. Flow Cytometric Analysis was performed to quantify MP populations in the in vivo and in vitro models (AccuriC6). Forward scatter(FSC) and side scatter(SSC) of light was set in logarithmic scale, and the threshold was set at the FSC parameter(FS=1). The fluorescence channels were also set at logarithmic gain. Standard fluorescent microbeads(1 mum) were used to set the MP gate. In order to distinguish true events from electronic noise and increase the specificity of microparticle detection, events in the MP gate were further discriminated by labeling with PE-Annexin-V. MP expression of CD28/CD40L was determined using APC-labeled CD28 and PerCP-labeled CD40L. RESULTS: MPs were imaged using TEM, Figure 1. MPs were identified and characterized using flow cytometry, Figure 2A-B. Plasma from operated septic mice had 2.5-fold greater Annexin-V+ MPs compared to unoperated non-septic controls, Figure 2C. Annexin-V+ MPs were 3.5-fold greater in RAW cells exposed to LPS as compared to control. We have observed MPs expressing CD28 and CD40L in the serum of septic mice. CONCLUSIONS: MP counts are higher in both in vivo and in vitro models of sepsis as compared to unoperated non-septic controls. Preliminary data shows that MPs may be carriers of biologically active CD28 and CD40L. Future experiments will further delineate inflammatory mediators expressed on the surface of MPs and their role in sepsis. (Figure Presented)

OBJECTIVES: Particulate matter (PM) exposure causes adverse health effects. The WTC collapse led to significant PM exposure and lung injury (Weiden et al. Chest 2009). The mechanism by which WTC PM causes pulmonary morbidity is not understood. We are investigating the differential cytokine effects on human alveolar cells, comparing ambient PM of WTC to ambient PM from NYC, South Bronx (SB) and Sterling Forest (SF), a rural area northwest of NYC. METHODS AND POPULATION: AM were obtained from Bronchoalveolar lavage (BAL) by adherence overnight. AM were exposed to 50mug/mL suspensions of WTC, SB, and SF PM2.5. Media alone was the negative control and 40 ng/mL of LPS was the positive control. After 24hrs, supernatants were collected and analyzed in duplicate using Human Cytokine Panel I (Millipore) on a Luminex-200. RESULTS: Fold induction of mediators was expressed as ratios of PM exposure/media alone. Exposure to WTC PM was markedly more inflammatory than SB and SF. The most significant inductions were of the leukocyte growth factors (GM-CSF, G-CSF), a promoter of angiogenesis (VEGF), the chemokine (RANTES) and the potent multifunctional cytokine IL-6. LPS caused a greater induction for all of the analytes when compared to WTC PM except for IL-1ra. SIGNIFICANCE OF STUDY: WTC PM2.5 produces a marked inflammatory effect in comparison to PM2.5 from both NYC, SB and rural sites. The large number of cytokines induced by WTC PM may drive airway injury and may be biomarkers for lung injury. WTC PM has been observed in induced sputum obtained 9 months after 9/11/2001 and so the elaboration of cytokines may underlie the severe and long lasting health effects produced by exposure to WTC PM