Abstract. The rapid and reversible upregulation of the functional activity of integrin receptors on T lymphocytes is a vital step in the adhesive interactions that occur during successful T cell recognition of foreign antigen and transendothelial migration. Although the ligation of several different cell surface receptors, including the antigen-specific CD3/T cell receptor complex, the CD2, CD7, and CD28 antigens, as well as several chemokine receptors, has been shown to rapidly upregulate integrin function, the intracellular signaling events that initiate this increase in adhesion remain poorly defined. In this study, we have used DNA-mediated gene transfer to explore the role of phosphatidylinositol 3-kinase (PI 3-K) in the upregulation of 131 integrin functional activity mediated by the CD2 antigen. CD2 was expressed in the myelomonocytic cell line HL60, which expresses 131 integrins that mediate adhesion to fibronectin and VCAM-1 in an activationdependent manner. Antibody stimulation of CD2 expressed on HL60 transfectants resulted within minutes in increased 131-mediated adhesion to fibronectin and VCAM-1 at levels comparable to that obtained upon stimulation with the phorbol ester PMA. A role for PI 3-K in CD2-mediated increases in 131 integrin function is suggested by: (a) the ability of the PI 3-K inhibitor wortmannin to completely inhibit CD2-induced increases in 131 integrin activity; (b) the association of PI 3-K with CD2; and (c) induced PI 3-K activity upon CD2 stimulation. The mode of association of PI 3-K with CD2 is not mediated by tyrosine phosphorylationdependent binding of PI 3-K via SH2 domains, since: (a) PI 3-K is associated with CD2 in unstimulated cells; (b) CD2 stimulation fails to increase the amount of associated PI 3-K; and (c) the CD2 cytoplasmic domain lacks tyrosine residues. A role for both protein kinase C and cytoskeletal rearrangements in CD2 regulation of integrin activity is also suggested, since a PKC inhibitor partially inhibits CD2-induced increases in 131 integrin function, and CD2 stimulation increases F-actin content in a wortmannin-sensitive manner. Analysis of human peripheral T cells indicated that CD2 stimulation also results in PI 3-K-dependent upregulation of 131 integrin activity. Thus, these results demonstrate that CD2 can function as an adhesion regulator in the absence of expression of the CD3/T cell receptor complex; and directly implicate PI 3-K as a critical intracellular mediator involved in the regulation of 131 integrin functional activity by the CD2 antigen.

Current biomarkers for sepsis are limited by their non-specificity, short half-life, and insensitive response to therapy. Recently, we have demonstrated that citrullinated histone H3(CitH3) is released into the blood from neutrophil extracellular traps(NETs) in response to severe infection, and CitH3 may be a potential biomarker for sepsis. In the present study, we found that NET components were released in mouse models of both lipopolysaccharide(LPS)-induced shock (LPSS) and hemorrhagic shock (HS). To further quantify CitH3 in the NETs, we established a CitH3 specific enzyme-linked immunosorbent assay. Circulating CitH3 was found to be elevated only in LPSS but not in HS. Importantly, blood CitH3 was detected 30 minutes after LPS insult, and remained elevated for 24 hours (period of the highest mortality). Treatment of endotoxic mice with YW3-56, a peptidylarginine deiminase-2/4 inhibitor, significantly diminished levels of CitH3 in the blood. Interleukin-1β did not respond to LPS early, and interleukin-1β and interleukin-6 fluctuated although they responded to treatment. Procalcitonin reacted to LPS insult late. Compared to CitH3, these biomarkers were non-specifically induced in LPSS and HS. Collectively, our results demonstrate that YW3-56 protects animals from LPSS, and CitH3 is a reliable biomarker due to its early appearance, specificity, duration, and response to therapeutic intervention.

Objective. To evaluate the role of the MEK/ERK MAP kinase pathway in murine collagen-induced arthritis (CIA) using the selective MEK inhibitor PD184352. We examined the effects of the inhibitor in cytokine-stimulated synovial fibroblasts and in cytokine-induced arthritis in rabbits to investigate its antiinflammatory mechanisms.Methods. Murine CIA was used to assess the effects of the selective MEK inhibitor on paw edema, clinical scores, weight loss, histopathologic features, and joint levels of p-ERK. Western blotting and immunohistochemistry techniques were used to assess p-ERK in human and rabbit synovial fibroblasts and synovial tissue from rheumatoid arthritis (RA) patients. Interleukin-1␣ (IL-1␣)-stimulated stromelysin production in rabbit synovial fibroblasts was assessed by enzyme-linked immunosorbent assay. A rabbit IL-1␣-induced arthritis model was used to assess the effects of the inhibitor on IL-1␣-induced MEK activity, stromelysin production, and cartilage degradation.Results. In the CIA model, PD184352 inhibited paw edema and clinical arthritis scores in a dosedependent manner. Disease-induced weight loss and histopathologic changes were also significantly improved by treatment. Inhibition of disease-induced p-ERK levels in the joints was seen with the inhibitor. Levels of p-ERK in the synovium were higher in RA patients than in normal individuals. PD184352 reduced IL-1␣-induced p-ERK levels in human RA synovial fibroblasts. The production of p-ERK and stromelysin was also inhibited in IL-1␣-stimulated rabbit synovial fibroblasts. We observed IL-1␣-induced p-ERK in the synovial lining, subsynovial vasculature, and articular chondrocytes. IL-1␣-induced stromelysin production and proteoglycan loss from the articular cartilage were reduced by PD184352.Conclusion. These data demonstrate the inhibition of murine CIA by PD184352, support the hypothesis that antiinflammatory activity contributes to the mechanism of action of the inhibitor, and suggest that a selective inhibitor may effectively treat RA and other inflammatory disorders.

Human peripheral blood monocytes are found as two distinct populations based upon differential expression of chemokine receptors, adhesion molecules, Fc receptors, and cytokines. cDNA microarray analysis now reveals additional differences between these subsets that suggest dramatically diverse functions. One monocyte subset (CD14++CD16-) appears to be closely paired with neutrophils, and may have as its primary function the removal and recycling of apoptotic neutrophils at sites of inflammation. The other monocyte subset (CD14+CD16+) expresses numerous genes encoding proteins with antimicrobial activity and thus may be more directly involved in peripheral host defense. The production of monocytes capable of efficiently removing dying neutrophils may be necessary to prevent host tissue damage and autoimmune response induction. Therefore, species like humans that produce relatively high levels of circulating neutrophils must also produce relatively high numbers of the recycling monocytes. Conversely, species such as mice and rats that maintain relatively lower levels of circulating neutrophils require fewer recycling monocytes.

The CD2 molecule is one of several lymphocyte receptors that rapidly initiates signaling events regulating integrin-mediated cell adhesion. CD2 stimulation of resting human T cells results within minutes in an increase in ␤1-integrin-mediated adhesion to fibronectin. We have utilized the HL60 cell line to map critical residues within the CD2 cytoplasmic domain involved in CD2 regulation of integrin function. A panel of CD2 cytoplasmic domain mutants was constructed and analyzed for their ability to upregulate integrin-mediated adhesion to fibronectin. Mutations in the CD2 cytoplasmic domain implicated in CD2-mediated interleukin-2 production or CD2 avidity do not affect CD2 regulation of integrin activity. A proline-rich sequence, K-G-P-P-L-P (amino acids 299 to 305), is essential for CD2-mediated regulation of ␤1 integrin activity. CD2-induced increases in ␤1 integrin activity could be blocked by two phosphoinositide 3-kinase (PI 3-K) inhibitors or by overexpression of a dominant negative form of the p85 subunit of PI 3-K. In addition, CD2 cytoplasmic domain mutations that abrogate CD2-induced increases in integrin-mediated adhesion also ablate CD2-induced increases in PI 3-K enzymatic activity. Surprisingly, CD2 cytoplasmic domain mutations that inhibit CD2 regulation of adhesion do not affect the constitutive association of the p85 subunit of PI 3-K association with CD2. Mutation of the proline residues in the K-G-P-P-L-P motif to alanines prevented CD2-mediated activation of integrin function and PI 3-K activity but not mitogen-activated protein (MAP) kinase activity. Furthermore, the MEK inhibitor PD 098059 blocked CD2-mediated activation of MAP kinase but had no effect on CD2-induced adhesion. These studies identify a proline-rich sequence in CD2 critical for PI 3-K-dependent regulation of ␤1 integrin adhesion by CD2. In addition, these studies suggest that CD2-mediated activation of MAP kinase is not involved in CD2 regulation of integrin adhesion.T lymphocytes continuously migrate throughout the body, mediating immune responses to foreign antigens. The capacity for normal T cells to function, develop, and migrate depends upon adhesive contacts with other cells as well as with extracellular matrix (ECM) components (76). These adhesive contacts are regulated by and initiate a complex series of elegantly controlled molecular signaling events. Members of the integrin superfamily of adhesion molecules are involved in the cell-cell and cell-ECM interactions that are essential for T-lymphocyte function and migration. The integrins are a family of ␣␤ heterodimeric cell surface receptors with a vast tissue distribution (67). There are at least 20 different integrin heterodimers, with each heterodimer containing 1 of 8 ␤ chains and 1 of 14 ␣ chains. Members of the ␤1 integrin subfamily bind to components of the ECM, such as fibronectin (FN), as well as cell surface counter-receptors, such as VCAM-1 (34). The functional activity of integrins on circulating leukocytes is dynamically regulated by signaling events (21...