Intradermal Neutrophil Mobilization Induced by Complement Fragments and Endotoxin is More Effective in Neonatal Than in Adult Cattle

D. Zwahl en) for the intensity of extravascular cellular accumulation, using set scales for the granulocytes: PMN and eosinophils. Th e granulocytic accumulation was assessed separately in the upper dermi s (epithelium to adnexal structures) and in the lower dermi s (downwards from the deepest adnexa l structures). Th ese areas were scored as followed: 0 = less than six extravascular granulocytes present in the section ; I = six to 40 granulocytes present ; 2 = more than 40 granul ocytes present with moderate number of focal collections and/o r relati vely few scattered; 3 = more than 40 granulocytes present with extensive foci and relati ve few granulocytes scatte red or relati ve few foci and moderate numbers scattered; and 4 = extensive foci and marked numbers of scattered granulocytes. Th e scores for the two areas assessed were then add ed to provide a total score for the section (possible range 0 to 8). The relati ve percentage of PMN and eosinophils in the inflammato ry infiltrate was evaluated by counting 200 cells, 100 in the upper dermis and 100 in the lower derm is (% PMN x total score div ided by 100 = corrected PMN score). Stat istical anal ysis of data was accomplished by using a computer software program based on International Math emati cal and Statistical Library (Houston, TX) routines. This program first tested the distribution of the values and the homogeneity of the variances . As this showed that para metri cal methods were possib le, the program perfo rmed a one-way analysis of variance, followed by a multiple compari son to the controls using the Dunnet test. Stati stical significance was established at the P < 0.05 level. Few extravascular granulocytes were found 4 hours after injection of PFS (Table I) in both adults (Fig. I) and neonates. In contras t, all three chemotactic agonists induced a marked and significant inflammatory reaction in the skin of animals of both age groups (Table I). Th e reaction did not differ qua litat ively among the chemotaxins used and consisted of dense periv ascular granulocytic infiltrates mainly around larger venul es within the deeper dermis (Fig. 2) and of granulocyte accumulation around capillaries within the whole dermi s. Th e infiltrate consisted solely of granulocytes; there were no mononucl ear cells present. Th e total intradermal granulocytic score was comparable in adults and neonates after und iluted and 1/5 diluted ZAP and after LPS (Ta ble I). Th e total PMN score after C5a was higher in neonates than in adults (P < 0.05; Table I). To take into account that the inflammatory infiltrate in Host defenses in neonates are immunologically immature and obviously contribute to the high mortality in neonates. 1.9 One of the more important of the many described abnormalities in the host defens e system of the newborn seems to involve neutrophil functio n. " Recentl y in vitro studies with bovine neonatal polymorphonucl ear leukocytes (PMN) demonstrated major functional differences between neonatal PMN of calves and other species. Differences included enhanced chemotax is,' ? which ind icated hyperm ot ility of these cells. Th e early phase of the acute inflammatory tissue reaction is characterized by PMN emigration and plasma exudation from blood vessels. Both effects are elicited by chemo tactic agents, i.e., peptides or lipid s that cause directional migration of leukocytes in vitro" and in vivo . ' Because in vitro assays may not precisely reflect in vivo conditions,' we investigat ed the effect of different chemo taxins on intrader mal PMN mobilization in five colstrum-fed newborn calves less than 48 hours old and in five adult cows. Th e newborn calves consistently had increased neutroph il numbers (6.02 ± 2.09 x 106/ml of blood), and very low eosinophil numbers (0.08 ± 0. 10 x 106/ ml of blood), a typical finding in newborn calves. In contrast, adult catt le had 2.24 ± 0.49 x 106 neutroph ils/ ml, and 0.82 ± 0.40 x 106eosinophils/ ml of blood. Zymosan activated plasma (ZAP) was genera ted by activating bovine plasma with Saccharomyces yeast zymosan at a concentration of2 mg/ml (zymosan A, Sigma , Deisenhofen FRG). Escherichia coli 0 558 :5 endo tox in (Sigma) was d iluted in pyrogen-free saline (PFS) to a concentra tion of 1013 M per 100 ILl (assumed MW 12,000). Partially pur ified C5a was prepared as described in detail elsewhere . 10 Th e protein content of the active fraction was 2.7 mg/rnl . One hundred ILl of chemoa ttractants and saline control were injected intradermally at the base of the tail using tuberculin syringes with 26-gauge needles. Two sites per anima l were injected with undiluted ZAP, with C5a diluted I : 5 in PFS, and with liposa ccharide (LPS); one site each was injected with ZAP dilu ted I : 5 in PFS, and with PFS, respectively. Four hours after injection , 6 mm circular skin biopsies were taken under local anesthesia. After fixation with 4% form aldehyde, the biopsies were routinely processed for histologic examination and the slides stained with hematoxylin and eosin and with Lendrums chro motro p-3 R. To enumerate the granul ocytes within the skin we used a method described previously. " Briefly, the samples were coded and scored blind ly (by two observe rs; D. R. Roth and R.