Decreased resistance to bacterial infection and granulocyte defects in IAP-deficient mice

Science

Volumn 274, Issue 5288, 1996, Pages 795-798

  Lindberg, Frederik P ^a   Bullard, Daniel C ^b   Caver, Tony E ^c   Gresham, Hattie D ^d   Beaudet, Arthur L ^b   Brown, Eric J ^a  

^a WASHINGTON UNIVERSITY SCHOOL OF MEDICINE   (United States)

^b BAYLOR COLLEGE OF MEDICINE   (United States)

^c University of Missouri   (United States)

^d VA Research Service   (United States)

Author keywords

[No Author keywords available]

Indexed keywords

FC RECEPTOR; INTEGRIN;

ARTICLE; BACTERIAL INFECTION; BACTERIAL PERITONITIS; GRANULOCYTE; HOST RESISTANCE; INFECTION RESISTANCE; LEUKOCYTE ACTIVATION; LEUKOCYTE MIGRATION; LIGAND BINDING; PHAGOCYTOSIS; PRIORITY JOURNAL; RESPIRATORY BURST;

AMINO ACID SEQUENCE; ANIMALS; ANTIGENS, CD; ANTIGENS, CD47; CARRIER PROTEINS; CELL MOVEMENT; ESCHERICHIA COLI INFECTIONS; FEMALE; GENE TARGETING; HETEROZYGOTE; IMMUNITY, NATURAL; INTEGRIN BETA3; MALE; MICE; MICE, INBRED C57BL; MICE, KNOCKOUT; MOLECULAR SEQUENCE DATA; NEUTROPHIL ACTIVATION; NEUTROPHILS; PEPTIDE FRAGMENTS; PERITONITIS; PHAGOCYTOSIS; PHENOTYPE; PLATELET MEMBRANE GLYCOPROTEINS; RESPIRATORY BURST;

BACTERIA (MICROORGANISMS); ESCHERICHIA COLI;

EID: 0029909981     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.274.5288.795     Document Type: Article

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27. f gene from pPol2neobpA (gift from P. Soriano) into exon 2, which encodes the signal peptide cleavage site and entire IAP IgV domain. This construct was dectroporated into AB2.1 embryonic stem cells [P. Soriano, C. Montgomery, R. Geske, A. Bradley, Cell 64, 693 (1991); M. R. Kuehn, A. Bradley, E. J. Robertson, M. J. Evans, Nature 326, 295 (1987); A. P. McMahon and A. Bradley, Cell 62, 1073 (1990)]. Digestion with Sac I and hybridization with the probe indicated was used to identify homologous recombinants. Multiple clones were isolated that correctly targeted the IAP gene, and injection of one of these resulted in germline transmission.
28. f gene from pPol2neobpA (gift from P. Soriano) into exon 2, which encodes the signal peptide cleavage site and entire IAP IgV domain. This construct was dectroporated into AB2.1 embryonic stem cells [P. Soriano, C. Montgomery, R. Geske, A. Bradley, Cell 64, 693 (1991); M. R. Kuehn, A. Bradley, E. J. Robertson, M. J. Evans, Nature 326, 295 (1987); A. P. McMahon and A. Bradley, Cell 62, 1073 (1990)]. Digestion with Sac I and hybridization with the probe indicated was used to identify homologous recombinants. Multiple clones were isolated that correctly targeted the IAP gene, and injection of one of these resulted in germline transmission.
29. f gene from pPol2neobpA (gift from P. Soriano) into exon 2, which encodes the signal peptide cleavage site and entire IAP IgV domain. This construct was dectroporated into AB2.1 embryonic stem cells [P. Soriano, C. Montgomery, R. Geske, A. Bradley, Cell 64, 693 (1991); M. R. Kuehn, A. Bradley, E. J. Robertson, M. J. Evans, Nature 326, 295 (1987); A. P. McMahon and A. Bradley, Cell 62, 1073 (1990)]. Digestion with Sac I and hybridization with the probe indicated was used to identify homologous recombinants. Multiple clones were isolated that correctly targeted the IAP gene, and injection of one of these resulted in germline transmission.
30. Mice were tail bled, and washed erythrocytes stained first with mAb miap301 to mouse IAP, then with fluorescein isothiocyanate-labeled secondary antibody and analyzed by flow cytometry. As a negative control, an isotype-matched mAb to keyhole limpet hemocyanine (KLH) was used. Single-cell suspensions of mouse bone marrow (from femurs and tibiae) were similarly analyzed, except that staining was done in the presence of excess human IgG (1 mg/ml) to block Fc receptor interactions.
31. 2-terminus [raised to KLH-conjugated pyro-QLLFSNVNSIEFTSC (single letter amino acid code: pyro-Q, pyro-Gln; L, Leu; F, Phe; S, Ser; N, Asn; V, Val; I, Ile; E, Glu; C, Cys)] followed by washing, incubation with peroxidase-labeled goat antibody to rabbit IgG, and detection by enhanced chemoluminesence (Amersham, Arlington Heights, IL).
32. 4 bacteria in 100 μl of pyrogen-free saline and observed as described (16). Mice back-crossed for eight generations yielded similar results (13).
33. 4, and after 24 hours total cell counts, differential counts, and colony-forming units of E. coli were assessed and PMN counts were calculated.
34. Sheep red blood cells (RBCs) opsonized with murine IgG2b antibody to sheep RBCs were prepared and incubated with murine bone marrow PMNs after a brief centrifugalion as described (22). After lysis of extracellular targets, RBCs phagocytosed per 100 PMNs (phagocytic index) were determined (22).
35. 6 murine bone marrow PMNs were added per well and hydrogen peroxide production assayed after 60 min with a scopoletin-based fluorescence assay (8).
36. 6 murine bone marrow PMNs were incubated 15 min at room temperature in 100 μl of Hanks' balanced salt solution with catalase, 0.1 mM fMLP, and either normal rabbit IgG (30 μg/ml), rabbit IgG to human IAP (30 μg/ml), or rabbit antiserum to human placental Arg-Gly-Asp binding integrins (30) at a dilution of 1:200. Then 45 μl of beads were added and the mixture incubated for 3C min at 37°C. Cells were washed twice, and PMN-bound fluorescent beads were counted under an ultraviolet microscope and expressed as beads bound per 100 cells (attachment index).
37. 2+ (VBS), and 10 μl of C5-deficient mouse serum (absorbed with sheep erythrocytes) for 1 hour at 37°C followed by washing and resuspension in 400 μl of VBS (23). Then 200,000 PMNs in 50 μl of VBS, 50 μl of 100 μM fMLP in VBS, and 15 μl of EC3bi were pelleted briefly and resuspended, followed by incubation at 37°C for 30 min. The number of EC3bi rosetted per 100 PMNs was determined microscopically. No EC3bi are ingested under these conditions (23).
38. We thank M. Williams, I. Lorenzo, and L. A. Hurley for technical assistance and P. Soriano for reagents. Supported by NIH grants GM38330 (E.J.B.), AI32177 (A.L.B.), GM15483 (D.C.B.), the Howard Hughes Medical Institute (F.P.L. and A.L.B.), the American Arthritis Foundation (E.J.B. and F.P.L.), the Research Service of the Department of Veterans Affairs (H.D.G.), and the Monsanto-Washington University Agreement.