NEUTROPHIL ELASTASE MEASUREMENT IN BLOOD OF HORSES WITH NATURALLY OCCURRING LAMINITIS

Introduction: The enzymatic theory of equine laminitis is supported by enhanced Matrix MetalloProteinase-9 (MMP-9) activity in laminar tissues that could be attributed to neutrophil infiltration and activation during the development phase of the disease.1,2 We hypothesized the role of neutrophil elastase (NE) in the conversion of pro-MMP into active form and evaluated the possibility of this theory by comparing plasmatic equine NE between horses suffering from laminitis and healthy horses.
Materials and Methods: NE was extracted from equine neutrophils (isolated from whole blood by gradient centrifugation), and purified by a double chromatographic step. Purified NE was used for the immunization of a guinea pig and a rabbit. At the end of immunization, animals were exsanguinated and the IgG purified by affinity chromatography. The anti-NE antibodies were used to develop and validate an ELISA technique for measuring NE in plasma from EDTA-blood samples. Mean normal blood value was established by measuring NE level in plasma from 37 healthy horses. Blood NE level was determined in the plasma of 29 horses suffering from acute laminitis and referred by the veterinarian to our equine clinic. The NE concentration was measured from blood collected either at the admission or within the first 4 days of hospitalization. Values from healthy and laminitis horses were transformed (natural logarithm) for the normality test and then compared by using an unpaired t-test. Significance was set at p<0.05.
Results: Purified equine NE was characterized by enzymatic assay and its molecular weight was 29 KDa as determined by polyacrylamide gel electrophoresis. The immunoreactivity of the antibodies isolated from guinea pig and rabbit antisera was controlled by positive radial immunodiffusion. The ELISA was sensitive (lowest limit of detection: 0.56 ng/ml) and specific for measuring NE in plasma from EDTA-blood samples. The mean plasmatic NE value ± SD in healthy horses was 32.53±28 ng/ml. In horses suffering from laminitis the mean plasmatic value was 202±187.22 ng/ml, significantly different from the normal value (p<0.0001).
Discussion: Increased mean level of NE in blood of laminitis horses traduces the systemic nature of laminitis with neutrophils recruitment, activation and degranulation. These results are in agreement with a previous study which demonstrated increased MPO concentrations in blood, laminar tissues and skin of induced laminitis horses during the development phase of the disease.3 Similarly elevation of MMP-9 level in the laminar tissues is accompanied by an increased blood concentration in horses developing laminitis.1,2 As for MPO, increased NE concentrations in blood of laminitis horses could have led to or derived from an infiltration of NE in laminar tissues. NE is a serine protease mainly involved in host defense and tissue remodeling4 but various serine proteases comprising the elastases are implicated in the activation of pro-MMP into active MMP.5 Thus, by its direct ability to degrade extracellular matrix protein and by activating MMP, NE could play an important role in the enzymatic pathway of laminitis by participating to the degradation of basement membrane and dermo-epidermic separation in the laminar tissues. This study contributes to better understanding of the exact pathogenesis of laminitis and may open the field for new treatment options.

References:
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