Laura Mayo-Bond

Interaction of Fcγ receptor type IIIB with complement receptor type 3 in fibroblast transfectants: Evidence from lateral diffusion and resonance energy transfer studies

To explore potential inter-receptor interactions between Fc gamma RIIIB, a GPI-linked protein, and the leukocyte integrin CR3, we have prepared transfected 3T3 fibroblast cell lines expressing Fc gamma RIIIB, CR3, or both Fc gamma RIIIB and CR3. We test the hypothesis that Fc gamma RIIIB and CR3 are physically associated in membranes using fluorescence recovery after photobleaching (FRAP) and resonance energy transfer (r.e.t.) microscopy. Cells expressing Fc gamma RIIIB alone displayed a diffusion coefficient (D) of 3.4 x 10(-9) (+/- 2.9 x 10(-9) cm2/second and a mobile fraction (m.f.) of 0.73 (+/- 0.10). In contrast, Fc gamma RIIIB exhibited D = 2.5 x 10(-9) (+/- 1.4 x 10(-9) cm2/second (n.s.) and a m.f. of 0.48 (+/- 0.08) (p < 0.01) on cells expressing both Fc gamma RIIB and CR3, thus indicating that co-expression of CR3 constrains the lateral diffusion of Fc gamma RIIIB. To further test for a direct physical interaction between these gene products, (r.e.t.) microscopy was performed. Donor-labeled anti-CR3 and acceptor-labeled anti-Fc gamma RIIIB on cells expressing both receptors yielded a r.e.t. photon count rate of 8.9(+/- 6.4) kilocounts/second (kC/s), whereas CR3-to-CR3 measurements gave 1.6(+/- 0.6) kC/s (p < 0.01). Moreover, the addition of exogenous agents such as N-acetyl-D-glucosamine, but not indomethacin, diminished the magnitude of these interactions in transfectant membranes. These data support the notion that a subpopulation of Fc gamma RIIIB is physically associated with CR3 and that this association can be affected by exogeneous compounds.

Urokinase-deficient and urokinase receptor-deficient mice have impaired neutrophil antimicrobial activation in vitro.

Leukocytes express both urokinase‐type plasminogen activator (uPA) and the urokinase receptor (uPAR, CD87). We have shown that neutrophil recruitment to the lung during P. aeruginosa pneumonia is impaired in uPAR‐deficient (uPAR−/−) mice but is normal in uPA−/− mice. However, both uPA−/− mice and uPAR−/− mice have impaired lung clearance of P. aeruginosa compared with wild‐type (WT) mice. To determine the role of uPA and uPAR in antibacterial host defense, we compared neutrophil bacterial‐phagocytosis, respiratory burst, and degranulation among uPA−/−, uPAR−/−, and WT mice. Nutrophil phagocytosis was significantly diminished comparing uPA−/− and uPAR−/− mice with WT mice at all time points. The generation of superoxide by both uPA−/− and uPAR−/− neutrophils was about half of that seen in WT neutrophils. Degranulation of azurophilic granules was significantly diminished in uPA−/− neutrophils compared with either uPAR−/− or WT neutrophils. By contrast, agonist‐stimulated release of specific granules was not diminished in either uPA−/− or uPAR−/− mice compared with WT. We conclude that the uPA/uPAR system modulates several of the crucial steps in neutrophil activation that result in bacterial killing and effective innate host defense.

An in vivo animal model of gene therapy for leukocyte adhesion deficiency.

Leukocyte adhesion deficiency (LAD) is an inherited disorder of leukocyte function that is caused by defects in the CD18 gene and is associated with diminished cell surface expression of CD11/CD18 proteins. We have developed an in vivo model for gene therapy of LAD. Recombinant retroviruses were used to transduce a functional human CD18 gene into murine bone marrow cells which were transplanted into lethally irradiated syngeneic recipients. A reliable flow cytometric assay for human CD18 in transplant recipients was developed based on: (a) the availability of human specific CD18 monoclonal antibodies and (b) the observation that human CD18 can form chimeric heterodimers with murine CD11a on the cell surface. Human CD18 was detected on leukocytes in a substantial number of transplant recipients for at least 6 mo suggesting that the gene had been transduced into stem cells. Expression was demonstrated in several lineages of a variety of hematopoietic tissues, but was consistently highest and most frequent in granulocytes. Murine granulocytes demonstrated appropriate posttranscriptional regulation of human CD18 in response to activation of protein kinase C. No apparent untoward effects of human CD18 expression were noted in transplant recipients. These studies suggest a specific strategy for LAD gene therapy that may be effective and safe.