Article Figures & Data
Figures
- FIGURE 1.
Generation and characterization of FcγRIIA transgenic mice. A, Map of the 72-kb human genomic DNA fragment containing the 20-kb human FcγRIIA gene used to create the transgenic mice is shown. The relative locations of the eight exons and the sites for digestion by selected restriction endonucleases are shown (Sf = SfiI, P = PmeI, Sa = SalI, Bs = Bss H II, B = BamHI). B, Detection of the human FcγRIIA gene in transgenic mice using PCR primers specific for human FcγRIIA. The expected 280-bp product is seen in lanes 1–4, representing mouse F07, 11, 32, and 40, respectively. Lane 5 presents the result for a littermate mouse that is negative for the transgene. Lane 6 contains φX-HaeIII DNA size markers.
- FIGURE 2.
Immunohistochemical analysis and Scatchard analysis of human FcγRIIA expression in transgenic mice. a, Bone marrow of line 11 IIA tg mouse. b, Bone marrow of nontransgenic control. c, Spleen of line 11 IIA tg mouse. d, Spleen of nontransgenic control. Primary staining with IV.3 was followed by alkaline phosphatase-conjugated secondary Ab and developed with substrate to give the red color for positive cells. All are ×400 magnification. Only the line 11 IIA tg samples (a and c) stained for human FcγRIIA on megakaryocytes and macrophages in marrow and spleen. e, 2.4G2 staining (anti-murine FcγRII/III) of thioglycolate-induced peritoneal macrophages (×1000 magnification), and f, colocalization of human FcγRIIA on the same set of macrophages by 125I-IV.3 binding. The Scatchard analysis indicates ∼65,000 IV.3 binding sites on the surface of line 11 transgenic mouse macrophages, equivalent to the receptor level on human macrophages (55).
- FIGURE 3.
Platelet FcγRIIA expression and function in transgenic mouse line 11. A, Measurement of FcγRIIA platelet surface receptor density. Scatchard plots for human platelets and line 11 FcγRIIA transgenic hemizygous mouse platelets binding 125I-IV.3 are shown, as described (53). The number of sites/platelet is comparable (1800, human; 1550, FcγRIIA transgenic mouse). B, Platelet aggregation was stimulated by human FcγRIIA using anti-FcγRIIA Ab IV.3 (arrow 1) followed by GAM (arrow 2), and plotted as extent of platelet aggregation versus time. Aggregation occurred only when GAM was added after prior addition of anti-FcγRIIA Ab. The extent of FcγRIIA-mediated platelet aggregation (curve A) was comparable with that of the positive control, thrombin (curve B). Platelet aggregation in response to 4A5 (curve C), as well as spontaneous aggregation, was negligible.
- FIGURE 4.
Immune thrombocytopenia following i.v. injection of anti-platelet Ab 4A5. The mean platelet counts (+/−SEM) over time are shown. Thrombocytopenia is more severe in IIA tg than wild-type mice, and the difference is significant (p < 0.05 by ANOVA, n = 6 in each group).
- FIGURE 5.
Immune thrombocytopenia following i.p. injection of anti-platelet Ab 4A5. Shown is the time course of mean platelet counts (+/−SEM) for line 11 IIA tg mice and IIA tg × γ-chain KO mice following injection of anti-platelet Ab. Saline and Ab isotype controls are as indicated. Both line 11 IIA tg mice and IIA tg × γ-chain KO mice demonstrate statistically significantly lower nadir platelet counts (p < 0.05 by ANOVA) than controls.
- FIGURE 6.
Nadir platelet counts for the mouse genetic lines following i.p. injection of anti-platelet Ab 4A5. IIA tg 11 mice reach thrombocytopenic nadirs that are significantly different from the thrombocytopenia in wild-type animals, as indicated by the asterisk (n = 6 in all groups; shown is mean ± SEM; p < 0.05 by ANOVA). IIA tg line 32 also demonstrated thrombocytopenia more severe than the thrombocytopenia in wild-type animals, as indicated by the asterisk (p < 0.05). There was no statistically significant difference in the nadir platelet counts induced by 4A5 between FcγRIIA transgenic lines 11 and 32. The IIA tg × γ-chain KO genetically crossed mice demonstrated significantly lower platelet nadir counts than wild-type mice and γ-chain KO mice (p < 0.05). Saline or isotype controls are as indicated.
Tables
Oligonucleotide Sequence Human FcγRIIA transgene PCR3, sense 5′-CTGGTCAAGGTCACATTCTTC-3′ 4INM, antisense 5′-CAATTTTGCTGCTATGGGC-3′ T5S, sense 5′-GGGTAGTTCCTTACAATTTTCCTG-3′ 16 M, antisense 5′-CAATGGAAGCAGCCACAGGTT-3′ 4.0-7S, sense 5′-GAGAAGAGAATTAGAGAGGTGAGG-3′ 4.0-6M, antisense 5′-GACTTACATGATTGCTCT-3′ 195M, antisense 5′-ATTGCGACTGCTGTAGCAGCCATT-3′ Mouse FcR γ-chain, wild-type gamma1, sense 5′-ACCCTACTCTACTGTCGACTCAAG-3′ gamma2, antisense 5′-TCACGGCTGGCTATAGCTGCCTT-3′ Mouse FcR γ-chain, knock-out locus Neo, sense 5′-CTCGTGCTTTACGGTATCGCC-3′ gamma2, antisense 5′-TCACGGCTGGCTATAGCTGCCTT-3′ Mouse whey acidic protein gene WAP-S, sense 5′-ATCCATGTCTCCATGCCTTCTTCT-3′ WAP-A, antisense 5′-TGTTGACAGGAGTTTTGCGGGTCC-3′ Mouse β-actin mβactinS, sense 5′-ATGGGTCAGAAGGACTCCTA-3′ mβactinA, antisense 5′-CTCGGTCAGGATCTTCATGA-3′
In this issue
