On study days 0 and 7, mice are anesthetized with Isoflurane and sensitized with a 0.1 mL intradermal injection at the base of the tail consisting of an equal mixture of 4 mg/mL Mycobacterium tuberculosis (MTB) in Freund’s complete adjuvant (CFA) and 4 mg/mL methylated bovine serum albumin (mBSA) in water. On study day 10, mice are injected into the right hind footpad with 20 µL of 10 mg/mL mBSA.
The Type IV DTH reaction is an acute inflammation driven by cell-mediated immunity.1 Typically, a DTH response develops as a result of a 1 to 2 week sensitization period following primary antigen contact in which dendritic cells and macrophages interact with an antigen and present to T cells in the regional lymph nodes. During this time TH cell recruitment, activation and clonal expansion of predominantly CD4+ Th1 cells occurs. Collectively, this process as known as the Sensitization Phase.2 A follow-on exposure to the antigen initiates the Effector Phase of the DTH response, in which Th1 cells secrete a variety of cytokines that recruit and activate macrophages and other non-specific inflammatory cells. This results in a visible inflammatory response within 24 to 48 hours post antigen challenge. The delayed response is a direct result of the time needed for cytokine secretion and recruitment/activation of macrophages. More specifically, the DTH response is mediated by T helper cells secreting predominantly interferon-g and IL-2 with production of inflammatory mediators and subsequent leukocyte recruitment. The mechanism by which leukocytes are attracted to the site of antigen re-introduction is not clear, but multiple cytokines and chemokines are thought to be involved in the DTH reaction.
Measurement of the DTH reaction can be done grossly by comparing the paw weight difference between normal versus antigen-injected paws. Right and left hind paws are collected, transected at the medial and lateral malleolus, and weighed.
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Chemokines such as IL-8, monocyte chemoattractant protein 1 (MCP-1), macrophage inflammatory protein 1a (MIP-1a), and macrophage migration inhibitory factor (MIF) have been found to be involved in the recruitment of leukocytes to the DTH reaction site. Cytokine inhibitors, such as anti-IL-16, have been shown to minimize the DTH response. Other compounds that effectively inhibit the DTH reaction are dexamethasone (a potent steroid that induces lympholysis) and cyclosporine-A (CsA), which inhibit the action and growth of T-lymphocytes.