Mast Cell Activation Contributes to Neurogenic Inflammation and Pain in Sickle Cell Disease

Abstract 374

Sickle cell disease (SCD) is associated with inflammation, endothelial dysfunction and pain. We observed increased immunoreactivity (ir) of pro-inflammatory and vasoactive neuropeptides, substance P (SP) and calcitonin-gene related peptide (CGRP) accompanied by decreased mu opioid receptor (MOR)-ir in the skin of sickle as compared to control mice (Kohli et al., Blood 2010). SP activates mast cells (MC), which are tissue resident leukocytes, leading to the release of inflammatory cytokines, tryptase and neuropeptides. SP also stimulates vascular permeability resulting in plasma extravasation and neurogenic inflammation. We hypothesized that pain in SCD is associated with a persistent feed-forward cycle of mast cell degranulation and neurogenic inflammation characterized by increased release of SP and CGRP from activated nociceptors in the skin leading to neuroinflammation, plasma extravasation and pain. We examined this hypothesis using sickle (HbSS-BERK) and control (HbAA-BERK) mice expressing sickle and normal human hemoglobin, respectively; and MOR-knockout (MOR-KO) mice with their wild type 129S6 controls. We developed an ex-vivo system to analyze the release of inflammatory cytokines, mast cell degranulation markers (tryptase and beta-hexosaminidase) and neuropeptides in skin biopsies. Neurogenic inflammation was studied in vivo using the Miles' assay. Evans blue was injected into the tail vein and its extravasation in skin evoked by stimulation with SP and capsaicin was quantified. Skin biopsies from sickle mice exhibited constitutively enhanced release of several cytokines (IL6, MCP-1, TNFalpha, MIP-1alpha, GM-CSF, RANTES, etc), tryptase and the neuropeptides SP, and CGRP as compared to control mouse skin (p<0.05 for each). Increased RANTES and GM-CSF are suggestive of mast cell recruitment. Mast cell tryptase-ir was increased 2-fold while MOR-ir (but not delta- or kappa-OR-ir), was reduced by ∼50% in the skin of sickle as compared to control mice, suggestive of enhanced MC degranulation in sickle. In MC cultures prepared from sickle skin increased c-kit/CD117-, FCeR- and tryptase-ir were observed as compared to control mouse MCs. The plasma of sickle exhibited a ∼60–80% increase in MC degranulation markers, tryptase and beta-hexosaminidase, acute phase protein, serum amyloid protein, and neuropeptides, SP and CGRP, as compared to control mice (p<0.01 for each). These correlative molecular changes in the plasma and skin were accompanied by increased SP- and capsaicin-induced Evans blue dye leakage in the skin of sickle mice suggestive of neurogenic inflammation as compared to control (p<0.001 for each). MOR-KO mice also exhibited increased SP- and CGRP-ir in the skin and neurogenic inflammation, indicative of a contribution by MOR to the neuroinflamamtory process. In sickle mice treated with the mast cell stabilizer cromolyn sodium (CS), or the c-kit inhibitor, Imatinib, for 5 days, the inflammatory cytokine and neuropeptide release from the skin and the neurogenic inflammation were ameliorated as compared to vehicle (p<0.01). Additionally, morphine at a dose of 10 mg/Kg was ineffective in treating tonic cutaneous and thermal hyperalgesia, but effectively reduced hyperalgesia in CS and Imatinib treated sickle mice. Thus, MC degranulation contributes to neurogenic inflammation and pain in sickle mice. Imatinib treatment by itself reduced tonic hyperalgesia and significantly decreased GM-CSF release from the skin (p<0.05) correlative to the reduced white blood cell (WBC) count in sickle mice vs vehicle. In addition to inhibiting MC activity, Imatinib may be inhibiting protein tyrosine kinases involved in cytokine processing, vascular function and nociception. Together, our observations demonstrate that MCs contribute to a vicious cycle of pain and neurogenic inflammation mediated by increased neuropeptides in SCD. It is likely that mast cell inhibitors such as Imatinib may have a therapeutic effect on pain, inflammation and vascular dysfunction in SCD by reducing mast cell activation and neurogenic inflammation. Since, Imatinib decreased GM-CSF levels and WBC, it may even increase HbF levels, which are negatively regulated by GM-CSF in SCD. We therefore speculate that therapies targeting mast cells may potentiate therapeutic outcomes of analgesics, anti-inflammatory agents and Hydroxyurea® in SCD.