Impaired Motility of Neonatal PMN Leukocytes: Relationship to Abnormalities of Cell Orientation and Assembly of Microtubules in Chemotactic Gradients

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Abstract
To allow a further understanding of the pathogenesis of impaired stimulated locomotion by polymorphonuclear leukocytes (PMNs) in human neonates, we studied cellular orientation by neonatal PMNs in response to well-defined chemotactic gradients (Zigmond orientation chambers) and characterized the cytoplasmic microtubule (MT) complex of neonatal PMNs during cell orientation and movement. PMN suspensions obtained from 52 neonates demonstrated a diminished capacity to undergo orientation at all time intervals after exposure to gradients of N-formyl-methionyl-leucyl phenylalanine (f-Met-Leu-Phe) or C5a. Among responding (orienting) neonatal PMNs observed, only 70% (f-Met-Leu-Phe) or 59% (C5a) oriented accurately (toward chemotactic gradients) as compared to values of 96% (f-Met-Leu-Phe) or 92% (C5a) for adult controls. Furthermore, neonatal PMNs failed to alter their direction of orientation/migration when chemotactic gradients were reversed. Similar abnormalities were observed when 10-fold gradients of f-Met-Leu-Phe were employed over a concentration range between 10−7 and 10−11 M. Employing tubulin immunofluorescence, the cytoplasmic MT complex of-neonatal PMNs was assessed prior to and after cell exposure to uniform concentrations or gradients of chemotactic factors (CFs). MT assembly by neonatal PMNs studied under these experimental conditions was significantly diminished. Neonatal cell suspensions demonstrated 26 ± 5 (f-Met-Leu-Phe) or 27 ± 6 (C5a) MT/cell as compared to respective values of 36 ± 6 or 35 ± 5 for adult suspensions (P <.001). MT lengths of neonatal PMNs increased from 6.7 ± 1 µm (PBS) to 7.5 ± 1 µm (f-Met-Leu-Phe) or 7.3 ± 1 µm (C5a) as © 1984 Alan R. Liss, Inc. Anderson et al compared to values of 6.5 ± 1 µm (PBS), 11.1 ± 1 µm (f-Met-Leu-Phe), and 10.9 ± 1 µm (C5a) for adult PMNs exposed to gradients or uniform concentrations of CFs (P <.01 for both f-Met-Leu-Phe and C5a). Thus, the polymerized tubulin mass product of chemotactically stimulated neonatal PMNs (202 µm) was significantly (P <.001) diminished as compared to adult PMNs (360 µm). As shown by a [3H]colchicine binding assay, impaired MT assembly could not be attributed to diminished cytoplasmic tubulin content of neonatal PMNs, which was comparable to adult PMNs.