Microbiology and Immunology

Rheumatoid arthritis (RA) is a chronic disease characterized by cycles of inflammation and resolution. Previously, it was believed that the resolution of inflammation is simply dissipation of pro-inflammatory signals, although current research indicates that resolution is an active process. Acute inflammation follows defined phases of induction, inflammation and resolution, and resolution occurs by an active process that requires COX-2 activity. This study aims to address whether this paradigm extends to a recognized model of chronic inflammation. We demonstrated in murine collageninduced arthritis that chronic inflammation follows the same sequential course. While there is the normal production of pro-inflammatory cytokines during inflammation and anti-inflammatory mediators such as 15-deoxyΔ12,14PGJ2 (15d-PGJ2) during resolution, interestingly there is sustained production of both COX-2 and the presumably proinflammatory PGE2 during both phases. Blocking COX-2 activity and therefore production of PGE2 during the resolution phase perpetuated instead of attenuated inflammation. Repletion with PGE2 analogs restored homeostasis, and this function is mediated by the pro-resolving lipoxygenase metabolite, lipoxin A4 (LXA4), which is a potent stop signal. Thus, the study provided in vivo evidence for a natural, endogenous link between the cyclooxygenase-lipoxygenase pathways and showed that PGE2 serves as a feedback inhibitor essential for limiting chronic inflammation in autoimmune arthritis. These findings may explain the enigma regarding why COX-2 inhibitors are palliative rather than curative in humans because blocking resolution may mitigate the benefit of preventing induction.

Temple University – Theses

Microbiology and Immunology

Chronic inflammation follows defined phases of induction, inflammation, and resolution. The resolution phase requires cycloxygenase-2 (COX-2) activity. This study aims to address what other molecules are required for a functional resolution phase. We demonstrated that in murine collagen-induced arthritis the transcription factor, PPARgamma plays a role in the resolution phase. Inhibition of COX-2 activity results in fewer PPARgamma positive cells in the arthritic synovium. Treatment with a PPARgamma antagonist, SR202, alone, also disrupts the process of resolution. PPARgamma antagonist treatment results in a decrease in eNOS phosphorylation within the arthritic synovium. These observations indicate that PPARgamma may function to regulate eNOS activity. The source of pro-resolving nitric oxide is eNOS but not, iNOS. The effect of COX-2 inhibition on the resolution phase is ameliorated by injection of a PGE2 analog. Restoration of PGE2 levels results in an increase in PPARgamma positive cells in the arthritic synovium which correlates with this restoration of resolution. Thus, this study provides in vivo evidence for the pro-resolving role of PPARgamma and its relationship with PGE2 and eNOS.

Temple University – Theses

Microbiology and Immunology

Cannabinoids are known to have anti-inflammatory and immunomodulatory properties. Cannabinoid receptor 2 (CB2) is expressed mainly on leukocytes and is the receptor implicated in mediating many of the effects of cannabinoids on immune processes. The capacity of delta-9-tetrahydrocannabinol (delta-9-THC) and of two CB2-selective agonists to inhibit the murine Mixed Lymphocyte Reaction (MLR), an in vitro correlate of graft rejection following skin and organ transplantation was tested. Both CB2-selective agonists and delta-9-THC significantly suppressed the MLR in a dose dependent fashion. The inhibition was via CB2, as suppression could be blocked by pretreatment with a CB2- selective antagonist, but not by a CB1 antagonist, and none of the compounds suppressed the MLR when splenocytes from CB2 deficient mice were used. The CB2 agonists were shown to act directly on T-cells, as exposure of CD3+ cells to these compounds completely inhibited their action in a reconstituted MLR and proliferation of purified T-cells by anti-CD3 and anti-CD28 antibodies was inhibited. Treatment of both CD4+ and CD8+ T-cells with a CB2-selective agonist inhibited the MLR, though significantly less than when both cell types were treated. T-cell function was decreased by CB2 agonists, as an ELISA of MLR culture supernatants revealed IL-2 release was significantly reduced in the cannabinoid treated cells. Further, treatment with O-1966 dose- dependently decreased levels of the active nuclear forms of the transcription factors NF- kappa-B and NFAT in wild-type T-cells, but not T-cells from CB2 knockout (CB2R k/o) mice. Additionally, a gene expression profile of purified T-cells from MLR cultures, generated using a PCR T-cell activation array, showed that O-1966 decreased mRNA expression of CD40 ligand and CyclinD3, and increased mRNA expression of Src-like-adaptor 2 (SLA2), Suppressor of Cytokine Signaling 5 (SOCS5), and IL-10. The increase in IL-10 was confirmed by measuring IL-10 protein levels in MLR culture supernatants. An increase in the percentage of regulatory T-cells (Tregs) was observed in MLR cultures and pretreatment with anti-IL-10 resulted in a partial reversal of the inhibition of proliferation and blocked the increase of Tregs. Additionally, O-1966 treatment caused a dose-dependent decrease in the expression of CD4 in MLR cultures from wild-type, but not CB2R k/o, mice. The ability of O-1966 treatment to block rejection of skin grafts in vivo was also tested. Mice received skin grafts from a histoincompatible donor, and the time to graft rejection was analyzed. Compared to mice that received the vehicle, mice that received O-1966 treatment had significantly prolonged graft survival and increased Tregs in the spleen. The spleen cells from O-1966-treated mice had reduced proliferation in an MLR and an increased percentage of Tregs. Together, these data support the potential of this class of compounds as useful therapies to prolong graft survival in transplant patients and possibly as a new class of…

Pharmacology

Amylin, a 37 amino acid peptide secreted from pancreatic beta cells upon stimulation by meal/glucose, belongs to the family of the calcitonin or calcitonin gene-related peptide (CGRP) and shares up to 50% homology with CGRP, which is a well-documented pain-related peptide. The amylin receptor is composed of a calcitonin receptor (CTR) and receptor activity modifying proteins (RAMPs). Numerous studies have shown that amylin plays an important role in glucose homeostasis and food intake. Few studies have been conducted with respect to the effect of amylin in the central or peripheral neuraxis. In this thesis, immunohistochemical study revealed a dense network of amylin-immunoreactive (irAMY) cell processes in the superficial dorsal horn of the mice. Numerous dorsal root ganglion and trigeminal ganglion cells expressed moderate to strong irAMY. Reverse transcriptase-polymerase chain reaction (RT-PCR) revealed amylin receptor mRNA in the mouse spinal cord, brain stem, cortex, hypothalamus and hippocampus. The nociceptive or antinociceptive effects of amylin were evaluated in the tail flick and acetic acid-induced writhing test. Amylin (1-10 µg, i.t.) reduced the number of writhing in a dose-dependent manner. Pretreatment of the mice with the amylin receptor antagonist salmon calcitonin (8-32) [sCT(8-32)]or AC187 by i.t. antagonized the effect of amylin on acetic acid-induced writhing test. Locomotor activity was not significantly modified by amylin injected either i.p. (0.01-1 mg/kg) or i.t. (1-10 µg). Measurement of c-fos mRNA by RT-PCR or proteins by Western blot showed that the levels were up-regulated in the spinal cord of mice in acetic acid-induced visceral pain model and the increase was attenuated by pretreatment with amylin. Pretreatment of sCT[8-32] or AC187 significantly reversed the effect of amylin on c-fos expression in the spinal cord. As the neuronal response to amylin is closely dependent on the molecular property of amylin receptor, the localization, internalization and regulation of the calcitonin and amylin receptor were examined in the second part of the thesis. Immunofluorescence microscopy demonstrated the surface expression of CTRa, and intracellular distribution of RAMP1. Moreover, co-expression of CTRa translocated the RAMP1 to the cell surface and generated the amylin receptor phenotype. Both immunocytochemistry and on cell western analysis showed the internalization of CTRa and amylin receptor (CTRa/RAMP1) stimulated by different agonists, which was partially ß-arrestin dependent. Moreover, RAMP1 did not change the surface expression pattern of CTRa, but co-localized with the receptor with and without agonist treatment. sCT and amylin activated the ERK1/2 in HEK293 cells stably expressing amylin receptors, indicating the involvement of MAPK in amylin receptor signaling cascade. Collectively, these results led us to conclude that 1) irAMY is expressed in dorsal root ganglion neurons with their cell processes projecting to the superficial layers of the dorsal horn, and that the…