Selective Migration of CD18+ Neutrophils into the Tissue Results in Reversal of the Phenotype in Canine Leukocyte Adhesion Deficiency.

Leukocyte adhesion deficiency-1 (LAD-1) is a genetic immunodeficiency disease characterized by recurrent, life threatening bacterial infections. The phenotype of LAD-1 stems from the inability of leukocytes from affected children to adhere and migrate to sites of infection as a result of mutations in the leukocyte integrin CD18. Canine leukocyte adhesion deficiency (CLAD) represents the homologue of the severe phenotype of LAD-1 in humans. In previous studies we have demonstrated that less than 500 CD18+ neutrophils/μl following non-myeloablative stem cell transplantation from matched littermate donors resulted in reversal of the CLAD disease phenotype. In this report, we describe two CLAD dogs (D128 and D144) with donor microchimerism and improvement from a severe to a moderate clinical phenotype of CLAD following matched littermate hematopoietic stem cell transplant. Prior to transplant both dogs displayed the hallmarks of CLAD phenotype with fever, severe infections, leukocytosis and were following the natural history of CLAD, which invariably leads to death by 6 months of age. Both dogs are maintained on only prophylactic antibiotics and are alive more than two years post-transplant. The calculated level of CD18+ donor neutrophils was approximately 100 CD18+ neutrophils/μl in dog D128 and 50 CD18+ neutrophils/μl in dog D144, respectively. To determine how this persistently low level of CD18+ neutrophils/μl measured in the peripheral blood resulted in the moderation of the severe disease phenotype of CLAD, we assessed the level of CD18+ leukocytes in mucosal tissues. Mucosal tissues have a constant bacterial presence that is kept under control in part by a constant influx of neutrophils from surrounding periodontal tissues. The emigration of neutrophils from the circulation is a critical step during immune surveillance and inflammatory reactions, and is governed by a coordinated interaction involving a spectrum of adhesion and signal molecules. The oral rinse assay enables a non-invasive, in vivo measurement of neutrophil migration into tissues. Both dogs displayed evidence of selective extravasation of CD18+ donor cells into extravascular site as shown by FACS analysis with eleven-fold (D128) and five-fold (D144) more donor-derived CD18+ leukocytes present in saliva compared to the peripheral blood. The percentage of donor chimerism was confirmed using DNA microsatellite markers that distinguished donor from host. These results indicate that a level of less than 100 CD18+ neutrophils/μl in peripheral blood improves the CLAD phenotype from a severe to a moderate clinical phenotype. Moreover, measurement of neutrophils in peripheral blood alone fails to take into consideration of the total number of CD18+ donor derived cells in the recipient CLAD animals post-transplant. These results suggest that measurement of donor-derived CD18+ neutrophils in the tissue in this disease provides a more accurate assessment of the clinical effect than the measurement of CD18+ leukocytes in the peripheral blood and they support the treatment of CLAD by gene therapy, where a low number of gene corrected cells is anticipated with current gene therapy vectors and transduction conditions.