Rap1a null mice have altered myeloid cell functions suggesting distinct roles for the closely related Rap1a and 1b proteins

Abstract

The Ras-related GTPases Rap1a and 1b have been implicated in multiple biological events including cell adhesion, free radical production, and cancer. To gain a better understanding of Rap1 function in mammalian physiology, we deleted the Rap1a gene. Although loss of Rap1a expression did not initially affect mouse size or viability, upon backcross into C57BL/6J mice some Rap1a-/- embryos died in utero. T cell, B cell, or myeloid cell development was not disrupted in Rap1a-/- mice. However, macrophages from Rap1a null mice exhibited increased haptotaxis on fibronectin and vitronectin matrices that correlated with decreased adhesion. Chemotaxis of lymphoid and myeloid cells in response to CXCL12 or CCL21 was significantly reduced. In contrast, an increase in FcR-mediated phagocytosis was observed. Because Rap1a was previously copurified with the human neutrophil NADPH oxidase, we addressed whether GTPase loss affected superoxide production. Neutrophils from Rap1a-/- mice had reduced fMLP-stimulated superoxide production as well as a weaker initial response to phorbol ester. These results suggest that, despite 95% amino acid sequence identity, similar intracellular distribution, and broad tissue distribution, Rap1a and 1b are not functionally redundant but rather differentially regulate certain cellular events.

FIGURE 1. Rap1a deletion strategy and detection

A. Knockout strategy showing replacement of exon 4 with neo resistance gene. Positions of genotyping primers are shown as well restriction enzymes used in gene manipulation or analysis. Bgl, BglII; N, NdeI; Pst, PstI; P, PvuII; S, SpeI; Stu, StuI. Numbered regions indicate location of exons, solid black bars show short and long arms used in homologous recombination. B. PCR analysis of Rap1a null mice. Using a common primer 5’ of the targeted sequence (in exon 3) and 3’ primers complementary to either exon 4 or the neo^R gene, PCR reactions detected Rap1a ^+/+ (upper band only) −/− (lower band only) or +/− (both bands) mice. Con, control plasmid template; F1, chimeric founder mouse. C. Western blot of Rap expression in mouse neutrophils. Cells were blotted for Rap1a, total Rap1 or Rap2 expression using specific antibodies. Glyceraldehyde 3 phopshate dehydrogenase (GAPDH) was used as an internal loading control. Data are shown for 3 separate Rap1a ^+/+ and ^−/− mice.

FIGURE 2. Analysis of T cell development and function

A. Flow cytometric analysis of CD4 and CD8 expression on thymocytes from wild-type and Rap1a-deficient mice. B. Flow cytometric analysis of CD4 and CD8 expression on wild-type and Rap1a-deficient spleen (top) and peripheral blood (bottom). C. Splenocytes from wild-type and Rap1a-deficient mice were stimulated with 2.5 µg/ml anti-CD3 in microwells. Cultures were pulsed with [³H]thymidine for the last 18 hours of a 72 hour incubation. D. CD4+ cells were purified from wild-type or Rap1a-deficient spleen and stimulated with 4 µg/ml plate bound anti-CD3, 0.5 µg/ml anti-CD28 or 10 ng/ml IL-4 + 10 µg/ml anti-IFN-γ, for Th2 cultures or 2 ng/ml IL-12 + 10 µg/ml anti-IL-4 for Th1 cultures. After 5 days in culture, cells were re-stimulated with anti-CD3 alone and supernatants were recovered after 24 hours for cytokine testing by ELISA. Data represent mean ±SEM from 6 +/+ and 5 −/− mice.

FIGURE 3. Analysis of B cell development and function

A. Flow cytometric analysis of B220 and CD43 expression in bone marrow from wild-type and Rap1a-deficient mice. B. Flow cytometric analysis of B220 and CD3 expression on spleen and peripheral blood cells from wild-type and Rap1a-deficient mice. C. Splenocytes from wild-type and Rap1a-deficient mice were stimulated with 2.5 µg/ml anti-IgM in the presence or absence of 5 ng/ml IL-4 in microwells. Cultures were pulsed with tritiated thymidine for the last 18 hours of a 72 hour incubation. D. Serum was isolated from wild-type and Rap1a-deficient mice. Concentrations of individual immunoglobulin isotypes were tested by ELISA.

FIGURE 4

Analysis of macrophage and granulocyte development. Flow cytometric analysis of CD11b and Gr-1 expression on bone marrow and spleen cells from wild-type and Rap1a-deficient mice.

FIGURE 5. Loss of Rap1a affects macrophage haptotaxis, adhesion and phagocytosis

A. Adhesion of wild-type and Rap1a^−/− macrophages to the (α₄β₁ integrin binding) fibronectin H296 peptide or vitronectin-coated cell culture wells was determined 90 min post plating following fixing and staining of adherent cells. B. Haptotaxis of wild-type and Rap1a^−/− macrophages through fibronectin H296 peptide-coated Transwell filters was measured at indicated time points. C. Phagocytosis of IgG-coated sheep red blood cells by bone marrow-derived macrophages was measured at the indicated time points after exposure. D. Phagocytosis of C3bi-opsonized sheep RBCs by macrophages was measured 30 min after exposure. All data are mean +/− SD for cells derived from 3 mice/genotype and are representative of at least 4 independent experiments (2 experiments using a total of 4 mice were pooled for 5D).

FIGURE 6. Loss of Rap1a influences chemotaxis of leukocytes

The ability of B220+ splenic B cells (upper), CD4+ splenic T cells (middle), or Sca1+/lin- bone marrow hematopoeitic stem cells (lower) from wild-type and Rap1a^−/− mice to migrate towards CCL21 or CXCL12 chemokines was determined using a Transwell assay. Data represent mean +/− SEM for cells derived from 6 mice (4 for bone marrow) *p<0.05 with a two-tailed students t-test.

FIGURE 7. Loss of Rap1a diminishes the oxidative burst of bone marrow-derived neutrophils

A. The ability of 10 µM fMLP to stimulate superoxide production in neutrophils derived from wild-type or Rap1a^−/− mice was determined using HRP catalyzed activation of isoluminol. Data in upper panel show time courses of superoxide production from cells derived from a typical control and least responsive −/− mouse cells. The maximum response (second panel) and total superoxide produced (third panel) were reduced while the time to reach maximum response was delayed (lower panel) upon averaging data (mean +/− SE) from 12 mice/genotype (data obtained from 4 independent experiments). *p<0.02 (t-test) for comparisons between +/+ and −/− mouse-derived cells. B. Stimulation with 200 ng/ml PMA promoted superoxide production in cells derived from wild-type or Rap1a^−/− mice measured by isoluminol chemiluminescence. Upper panel shows a typical time course. Pooled data in second panel shows a weaker overall maximal response in Rap1a^−/− neutrophils. The third panel shows a weaker initial (first 3 min) response to PMA. The lower panel shows a similar amount of superoxide was produced over 30 min in response to phorbol ester stimulation. Mean +/− SE, n=12 per genotype, *p<0.01 to phorbol ester stimulation.