NONOPSONIC PHAGOCYTOSIS OF PSEUDOMONAS AERUGINOSA: INSIGHTS FROM AN INFANT WITH LEUKOCYTE ADHESION DEFICIENCY

Children with leukocyte adhesion deficiency type I are at risk for overwhelming infection because their neutrophils lack surface beta 2 integrins (CD18/CD11) that normally interact with endothelial cell adhesion molecules and mediate migration to sites of bacterial invasion. In vitro studies of phagocytic cells from an infant with leukocyte adhesion deficiency type I demonstrated that complement receptor 3 (CD18/CD11b) mediates nonopsonic phagocytosis of some Pseudomonas aeruginosa strains and might play a control role in the control of Pseudomonas infections at sites where there are low levels of opsonins. In patients with leukocyte adhesion deficiency (LAD) type I neutrophils lack the surface beta 2 integrins that normally bind to endothelial intercellular adhesion molecules (ICAM) and mediate migration to sites of bacterial invasion. 1, 2 The beta 2 integrins consist of two membrane-attached protein chains, an alpha chain (CD11) and a smaller beta chain (CD18). The beta chain is encoded by a gene on chromosome 21q22 that contains a region that is unusually susceptible to mutation. 3, 4 Mutations in the CD18 gene result in failure of expression or abnormal expression of the CD18/CD11 surface receptors (Table 1) so that interactions with their ligands cannot occur, resulting in the leukocyte adhesion deficiency phenotype.Table 1: The beta 2 integrins and their ligandsChildren with LAD suffer from recurrent and severe infections including omphalitis, pneumonia, perianal abscess, gingivitis, otitis media, gastroenteritis, and meningitis with a variety of different organisms including staphylococci, streptococci, Proteus spp., Escherichia coli, and Pseudomonas. 5 Two clinical patterns are recognized: type I or severe LAD, in which death usually occurs in infancy; and type II, with partial expression of beta 2 integrins, in which milder disease occurs and survival into adulthood is possible. 2 Although the primary defect in LAD is failure of migration of neutrophils to sites of infection, the defective surface receptors also have other vital roles in defense against bacterial infection. One of the beta 2 integrins, complement receptor 3 (CR3; CD18/CD11b), is the major receptor for phagocytosis of complement-opsonized particles 6 and is involved in nonopsonic phagocytosis of bacteria. 7, 8 We studied phagocytosis by neutrophils and macrophages harvested from an infant with severe LAD1. The studies were performed to gain a clearer understanding of the role of CR3 in defense against bacterial infection. Case report. The affected infant was transferred to Children’s and Women’s Health Centre of British Columbia at 17 days of age with a 12-day history of weight loss, poor feeding, diarrhea, severe omphalitis and perianal and ischial abscesses. Peripheral white blood cell count was 55 × 10 9 /l and remained persistently elevated. A needle aspirate of the necrotic buttock lesion contained no pus cells, but P. aeruginosa, Enterococcus faecalis and Enterobacter cloacae were isolated. A clinical diagnosis of LAD1 was made and confirmed by flow cytometry demonstrating expression of CD18 on <4% of neutrophils before and after stimulation with phorbol myristate acetate. The hypermutable region of the CD18 gene was cloned and sequenced, demonstrating the insertion of an adenosine before the guanosine at position 615 causing a frame-shift mutation. The infant underwent cord blood transplantation at 5 months of age, resulting in correction of the leukocyte defect and resolution of all lesions after engraftment. Methods. Informed consent and ethical approval for the study were obtained. Heparinized venous blood was obtained from the child with LAD, from adult controls and from umbilical cords of healthy newborn infants. Neutrophils and macrophages were separated from the blood by Ficoll-Paque (Sigma) density gradient centrifugation. Phagocytosis assay. Phagocytosis was assessed visually as previously described. 9 Neutrophils and monocyte-derived macrophages were coincubated with washed strains of P. aeruginosa (strain P1, a nonmucoid revertant derived from a mucoid isolate from a patient with cystic fibrosis and the LAD patient’s own nonmucoid isolate, strain 808) at a phagocyte:bacteria ratio of 1:100 for 60 min or with control particles. Control particles included complement-coated sheep erythrocytes (PMN Microbiologicals) to assess CR3 function, and IgG-coated sheep erythrocytes to assess phagocytosis via Fc receptors. Separate tubes were incubated at 4°C and 37°C for 1 h to distinguish phagocyte surface binding of bacteria from phagocytosis. The suspensions containing P. aeruginosa were treated with lysozyme (5 mg/ml) to disrupt extracellular bacteria and then washed and precipitated onto glycerol-coated glass slides by cytocentrifugation (Cytospin 2; Shandon). The slides were dried overnight, fixed with methanol, stained with Giemsa (BDH Laboratory Supplies) and ingested bacteria enumerated by light microscopy. Results. A defect in phagocytosis in neutrophils and macrophages from the infant with LAD1 was demonstrated by the inability of these cells to ingest IgM complement-coated erythrocytes (CR3-mediated), a cystic fibrosis isolate of P. aeruginosa (P1) or the infant’s isolate of E. cloacae (Fig. 1). Phagocytosis of IgG-coated erythrocytes was present but reduced relative to controls. Phagocytosis of the patient’s isolate of P. aeruginosa (strain 808) was normal (Fig. 1). Adult phagocytes (from two healthy volunteers) and cord blood phagocytes (from three healthy neonates) were competent for the phagocytosis of all particles (Fig. 1). In addition phagocytes obtained from the infant with LAD were fully competent for phagocytosis of both strains of P. aeruginosa after cord blood transplantation.Fig. 1: Neutrophil phagocytosis of various particles at 37°C. Data shown for neutrophils from a patient with LAD1, two adult controls and three cord blood samples, expressed as mean ± se with background (4°C control) subtracted. ∗ and ∗∗, significant difference from adult and cord blood neutrophil controls at P ≤ 0.02 and P < 0.00001, respectively, by Student’s t test. Data following cord blood transplantation in the infant are also presented for the two strains of P. aeruginosa.Discussion. These data provide the first direct evidence that CR3 is an important receptor for nonopsonic phagocytosis of some strains of P. aeruginosa. However, normal ingestion of the patient’s own P. aeruginosa isolate suggests that her propensity for infection was caused by the primary defect in neutrophil recruitment and not by defective phagocytosis. These observations are significant in the context of sites of infection where there are small concentrations of opsonins, such as the lung in cystic fibrosis, where CR3 mediates migration of phagocytes through the endothelium, 10 and is also the apparent receptor for phagocytosis of P. aeruginosa. Normal human tracheobronchial secretions have small concentrations of complement and IgM, but IgG and IgA are generally present. 11, 12 However, the pulmonary alveolar macrophage has greatly reduced levels of receptors for complement and the Fc portion of IgG. 13 It is reasonable to assume that pulmonary alveolar macrophages rely on nonopsonic phagocytosis more than do other tissue macrophages; the latter benefit from the assistance of a full array of serum opsonins. Therefore in that we have shown that CR3 is an important receptor for nonopsonic phagocytosis of P. aeruginosa; nonopsonic phagocytosis of bacteria mediated by CR3 is probably a critical element in bacterial clearance from the lung after pulmonary challenge. Phagocyte surface molecules may play a particular role in determining the outcome of infections in children with cystic fibrosis and previously healthy individuals exposed to respiratory challenge with P. aeruginosa and other bacteria (e.g. those receiving mechanical ventilation). In contrast the fact that CR3 was not required for phagocytosis of strain 808, the clinical isolate of P. aeruginosa from the patient described here, suggests that some strains express surface structures that can be recognized by other pattern recognition molecules such as CR1, mannose receptor, CD14, L-selectin, mannose receptor, scavenger receptor or CDw17. 6 It is likely that such variation in receptor use during phagocytosis is the manifestation of a bacterial mechanism for evasion of host immunity. Indeed some previous studies have demonstrated that exopolysaccharides produced by some strains of P. aeruginosa reduce binding of the organism to phagocytes in vitro. 14 Furthermore the events triggered in phagocytic cells after ingestion through different classes of receptors may result in diverse pathogen-directed inflammatory responses and explain some of the observed differences in outcome in patients infected with P. aeruginosa. Acknowledgments. Work was supported with a grant from the Medical Research Council of Canada to DPS. AJP is funded by a fellowship from the Pediatric Infectious Disease Society through an educational grant from Pfizer, Inc. We are grateful for technical assistance from Ken Crookall and Susan Ursuliak and to the staff of BC Children’s Hospital who cared for the infant described in this report. We are grateful to the family of the infant for understanding and cooperation.

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