DAMAGE-INDUCED INFLAMMATION AND NOCICEPTIVE HYPERSENSITIVITY IN DROSOPHILA LARVAE

Mounting an effective response to tissue damage requires a concerted effort from a number of systems, including both the immune and nervous systems. Immune-responsive blood cells fight infection and clear debris from damaged tissues, and specialized pain receptors become hypersensitive to promote behavior that protects the damaged area while it heals. To uncover the cellular and molecular mechanisms underlying these processes, we have developed a genetically tractable invertebrate model of damage-induced inflammation and pain hypersensitivity using Drosophila larvae. To study wound-induced inflammation, we generated transgenic larvae with fluorescent epidermal cells and blood cells (hemocytes). Using live imaging, we monitored the circulatory dynamics of hemocytes and the methods by which they accumulate at epidermal wounds. We found that circulating hemocytes attach to wound sites directly from circulation, a mechanism once thought to work exclusively in species with a closed circulatory system. To study damage-induced pain hypersensitivity, we developed a “sunburn assay” and found that larvae have a lowered pain threshold (allodynia) and an exaggerated response to noxious stimuli (hyperalgesia) following UV damage. We screened for genes required for hypersensitivity in pain receptors (nociceptors), and discovered a number of novel mediators that have well conserved mammalian homologs. Together, these results help us to understand how various cell types in the immune and nervous systems both detect and respond to tissue damage.

Type I hypersensitivity and its pharmacological modulation in {\it Trichinella\/}-induced intestinal fluid secretion and rapid rejection of the parasite in immunized rats

Studies were performed to test the hypothesis that type I hypersensitivity underlies worm induced intestinal fluid secretion and the rapid rejection of Trichinella spiralis from immunized rats, and the two events may be related in a cause-effect manner.^ Two approaches were taken. One was to determine whether inhibition of anaphylaxis-mediated Cl$\sp{-}$ and fluid secretion accompanying a secondary infection impedes worm rejection from immune hosts. The other was to determine whether induction of intestinal fluid secretion in nonimmune hosts interfered with worm establishment. In both studies, fluid secretion was measured volumetrically 30 min after a challenge infection and worms were counted.^ In immunized rats indomethacin did not affect the worm-induced fluid secretion when used alone, despite inhibiting mucosal prostaglandin synthesis. Fluid secretion was reduced by treatment with diphenhydramine and further reduced by the combination of diphenhydramine and indomethacin. The paradoxical effects of indomethacin when used alone compared with its coadministration with diphenhydramine is explained by the enhancing effect of indomethacin on histamine release. Abolishing net fluid secretion in these studies had no effect on rapid worm rejection in immune hosts.^ Worm establishment was reduced in recipients of immune serum containing IgE antibodies. Net intestinal fluid secretion induced in normal rats by PGE$\sb2$, cholera toxin, or hypertonic mannitol solution had no effect on worm establishment compared with untreated controls.^ In a related experiment, worm-induced intestinal fluid secretion and worm rejection in immune rats were partially blocked by concurrent injection with 5-HT$\sb2$ and 5-HT$\sb3$ blockers (Ketanserin and MDL-72222), suggesting that 5-HT is involved. This possible involvement was supported in that treatment of nonimmune rats with 5-HT significantly inhibited worm establishment in the intestine.^ Results indicate that anaphylaxis is the basis for both worm-induced intestinal fluid secretion and rapid rejection of T. spiralis in immune rats, but these events are independent of one another. 5-HT is a possible mediator of worm rejection, however, its mechanism of action is related to something other than fluid secretion. ^

DNA damage-induced Fas ligand expression by epidermal dendritic cells mediates UV-induced immune suppression of contact hypersensitivity

Exposure to UVB radiation induces local and systemic immune suppression, evidenced by inhibition of the contact hypersensitivity response (CHS). Epidermal dendritic cells, the primary antigen presenting cells responsible for the induction of CHS, are profoundly altered in phenotype and function by UVB exposure and possess UV-specific DNA damage upon migrating to skin-draining lymph nodes. Expression of the proapoptotic protein FasL has been demonstrated in both skin and lymph node cells following UVB exposure. Additionally, functional FasL expression has recently been demonstrated to be required in the phenomenon of UV-induced immune suppression. To test the hypothesis that FasL expression by DNA-damaged Langerhans cells migrating to the skin-draining lymph nodes is a crucial event in the generation of this phenomenon, mice were given a single 5KJ/m2 UV-B exposure and sensitized to 0.5% FITC through the exposed area. Dendritic cells (DC) harvested from skin-draining lymph nodes (DLN) 18 hours following sensitization by magnetic CD11c-conjugated microbeads expressed high levels of Iab, CD80 and CD86, DEC-205 and bore the FITC hapten, suggesting epidermal origin. Radioimmunoassay of UV-specific DNA damage showed that DC contained the vast majority of cyclobutane pyrimidine dimers (CPDs) found in the DLN after UVB and exhibited increased FasL mRNA expression, a result which correlated with greatly increased FasL-mediated cytotoxicity. The ability of DCs to transfer sensitization to naïve hosts was lost following UVB exposure, a phenomenon which required DC FasL expression, and was completely reversed by cutaneous DNA repair. Collectively, these results demonstrate the central importance of DNA damage-induced FasL expression on migrating dendritic cells in mediating UV-induced suppression of contact hypersensitivity. ^

TRPV1 Channels Contribute to Behavioral Hypersensitivity and Spontaneous Activity in Nociceptors After Spinal Cord Injury

A majority of persons who have sustained spinal cord injury (SCI) develop chronic pain. While most investigators have assumed that the critical mechanisms underlying neuropathic pain after SCI are restricted to the central nervous system (CNS), recent studies showed that contusive SCI results in a large increase in spontaneous activity in primary nociceptors, which is correlated significantly with mechanical allodynia and thermal hyperalgesia. Upregulation of ion channel transient receptor vanilloid 1 (TRPV1) has been observed in the dorsal horn of the spinal cord after SCI, and reduction of SCI-induced hyperalgesia by a TRPV1 antagonist has been claimed. However, the possibility that SCI enhances TRPV1 expression and function in nociceptors has not been tested. I produced contusive SCI at thoracic level T10 in adult, male rats and harvested lumbar (L4/L5) dorsal root ganglia (DRG) from sham-treated and SCI rats 3 days and 1 month after injury, as well as from age-matched naive control rats. Whole-cell patch clamp recordings were made from small (soma diameter <30 >μm) DRG neurons 18 hours after dissociation. Capsaicin-induced currents were significantly increased 1 month, but not 3 days, after SCI compared to neurons from control animals. In addition, Ca2+ transients imaged during capsaicin application were significantly greater 1 month after SCI. Western blot experiments indicated that expression of TRPV1 protein in DRG is also increased 1 month after SCI. A major role for TRPV1 channels in pain-related behavior was indicated by the ability of a specific TRPV1 antagonist, AMG9810, to reverse SCI-induced hypersensitivity of hindlimb withdrawal responses to heat and mechanical stimuli. Similar reversal of behavioral hypersensitivity was induced by intrathecal delivery of oligodeoxynucleotides antisense to TRPV1, which knocked down TRPV1 protein and reduced capsaicin-evoked currents. TRPV1 knockdown also decreased the incidence of spontaneous activity in dissociated nociceptors after SCI. Limited activation of TRPV1 was found to induce prolonged repetitive firing without accommodation or desensitization, and this effect was enhanced by SCI. These data suggest that SCI enhances TRPV1 expression and function in primary nociceptors, increasing the excitability and spontaneous activity of these neurons, thus contributing to chronic pain after SCI.