Increased expression of MAIL, a cytokine‐associated nuclear protein, in the prodromal stage of black walnut‐induced laminitis

Abstract

Summary Reasons for performing study: The mediators and signalling cascades important in the initiation of laminitis remain unclear. We therefore wanted to explore the genes and overall signalling mechanisms that play an important role in the developmental stage of laminitis. Objective: To use a broad genomic screening technique to identify novel genes that are differentially regulated in the equine lamellae during the developmental period of laminitis. Methods: Differential mRNA display (DRD) was performed to discover regulated genes, and real‐time quantitative polymerase chain reaction (RT‐qPCR) was then used to evaluate lamellar mRNA levels of a regulated gene (MAIL) and mediators related to that gene (IL‐1β and IL‐6) in control horses (n = 5) and horses administered black walnut extract (BWE; n = 5). Results: Using DRD, MAIL was identified as a regulated gene. RT‐qPCR indicated a 4‐fold increase in expression of the MAIL mRNA in BWE lamellae compared to controls. A 30‐fold increase in IL‐1β, and a 160‐fold difference in IL‐6 mRNA expression was present in BWE lamellae. Differences in MAIL, IL‐1β and IL‐6 mRNA expression were statistically significant between groups (P<0.05). Conclusions and potential relevance: The data strongly support a role for inflammatory cytokines in the developmental stages of laminitis, possibly inducing the vascular and metabolic alterations reported to occur in the affected digit. These results potentially support the use of anti‐inflammatory drugs in horses at risk of laminitis, and warrant further investigation of the link between systemic disease processes associated with laminitis and the reported digital inflammation.

Immunolocalization of pro-inflammatory cytokines in normal and inflamed hoof of buffalo calves.

“…Cytokines play a vital role in leukocyte activation and emigration that reportedly plays a central role in laminar injury in equine laminitis . There is up-regulation of IL-1β, IL-8 (Belknap et al, 2007) and IL-6 (Boontham et al, 2003) along with COX-2 and signaling protein NF κB (Waguespack, Kemppainen, et al, 2004) in the developmental period of the black walnut extract (BWE) model of laminitis. Bacterial ligands and other cytokines result expression of IL-8 in various cell types including endothelial cells (Boontham et al, 2003).…”

Equine laminitis: a tissue microdialysis study of lamellar bioenergetics

“…This enhanced genetic expression of IL-1β coincided with a severe neutropenia , which is characteristic of the early stages of the BWE induction model (Galey et al, 1991). Furthermore, the increased levels of IL-1β were suggestive of a primary role for inflammatory cytokines in laminitis, as potential inducers of the vascular and metabolic changes (Waguespack et al, 2004b), which were previously regarded as primary events (Hood, 1999;Pollitt, 1999).…”

“…Using lamellar tissues from BWE-treated and control horses the differential expression of MAIL, IL-1β and IL-6 between the two groups was assessed. Results showed a near 3-fold, a 30-fold and a 160-fold increase in lamellar mRNA expression of MAIL, IL-1β and IL-6, respectively, 2.5 to 3 hours (at the time of neutropenia) after BWE administration when compared to a sham treatment (Waguespack et al, 2004b). The findings not only agreed with the previously identified increase in IL-1β in the developmental phase of laminitis ), but provided further support to the idea of inflammatory activation and involvement in laminitis pathogenesis.…”

“…One of the first studies investigating the role of inflammatory changes in experimentally induced laminitis reported the identification of the equine gene encoding for "Molecule possessing Ankyrin-repeats Induced by LPS" (MAIL) (Waguespack et al, 2004b), a nuclear regulator of the expression of inflammatory cytokines, namely in LPS-induced inflammatory signalling and in the up-regulation of IL-6 (Kitamura et al, 2000). Using lamellar tissues from BWE-treated and control horses the differential expression of MAIL, IL-1β and IL-6 between the two groups was assessed.…”

“…After demonstration of the dramatic increase in cytokine expression, in particular the elevation of IL-1β Waguespack et al, 2004b), a very important cytokine released by activated leukocytes (Newton, 1990), white blood cell diapedesis through the lamellar microvasculature during the early stages of BWE-induced laminitis was investigated. Leukocytes were not observed perivascularly in the lamellae of control horses, indicating an absence of a marginal pool of white blood cells in the lamellar circulation; in the BWE treated horses, leukocyte emigration, although highly variable, was confirmed at two different time points (i.e.…”