“In vitro” evaluation of granulocyte functions in healthy dogs and in dogs with neutrophilic leukocytosis

“IN VITRO” EVALUATION OF GRANULOCYTE FUNCTIONS IN HEALTHY DOGS AND IN DOGS WITH NEUTROPHILIC LEUKOCYTOSIS Paltrinieri S., Comazzi S., Sartorelli P., Mussinelli F. Istituto di Patologia Generale Veterinaria – Milano - Italy Extensive evaluation of the different steps of the phagocytosis in neutrophils (PMNs) are not available in healthy dogs nor in dogs with inflammatory diseases. In this work we designed a panel of tests to investigate all the steps of phagocytosis in the same animal. Among the techniques reported in literature those performable with small amount of blood were choosen, to test dogs with spontaneous diseases, on which the isolation of large numbers of cells is often difficult. 6 control dogs (group 1) and 6 with neutrophilic leukocytosis (group 2), due to different diseases (post surgical infections, pyotorax, uroperitoneum, enteritis) were tested. PMNs were isolated from 3 ml of EDTA-preserved blood using discontinous gradient sedimentation technique with Percoll ( = 1.092/1.071). Chemotaxis was evaluated in a modified Boyden chamber against recombinant human interleukin 8 (rhIL-8), and with both the leading front and the cell count methods. Opsonized fluoresceinated yeasts were used to evaluate the uptake ability, expressing the results as percentage of phagocytic PMNs and as phagocytic index (PI = nr of ingested yeasts/100 PMNs). Adherence was measured in microtiter plates, by spectrophotometric evaluation of the release of acid phosphatase after washing of non adherent cells; before washing, superoxide production was evaluated measuring the cytocrhome C reduction: plastic contact and phorbol myristate acetate (PMA) were used as non-specific and specific activators of adherence and respiratory burst. Percoll isolation yeld a pure PMNs population, but the recovery rate of group 2 dogs was significantly lower (P<0.05) than in controls. The rhIL-8 is a good chemoattractant of canine PMNs and the leading front method should be used. Group 2 dogs had an higher response to rhIL-8 (P<0.01) than the controls (P<0.05). Higher values of the percentage of phagocytic PMNs (P<0.01) and of the PI (P<0.01) was detected in group 2 dogs. Basal adherence was very variable among healthy dogs; plastic contact increased the adherence (P<0.05), while PMA did not. These findings were detected also in group 2 dogs, without differences with the controls. Basal superoxide production was always very low. PMA increased the respiratory burst of the controls (P<0.05) and not those of group 2 dogs, maybe due to the individual variations. The selected tests allowed a simultaneous evaluation of the steps of phagocytosis in healthy dogs, although the individual variations in adherence and respiratory burst may suggest some modification of the method. In dogs with neutrophilia uptake ability and chemotaxis are increased,possibly due to pre-activation; the individual variations among these dogs may be due to the aetiology of the disease.