Regulation of in vitro and in vivo immune functions by the cytosolic adaptor protein SKAP-HOM

Abstract

SKAP-HOM is a cytosolic adaptor protein representing a specific substrate for the Src family protein tyrosine kinase Fyn. Previously, several groups have provided experimental evidence that SKAP-HOM (most likely in cooperation with the cytosolic adaptor protein ADAP) is involved in regulating leukocyte adhesion. To further assess the physiological role of SKAP-HOM, we investigated the immune system of SKAP-HOM-deficient mice. Our data show that T-cell responses towards a variety of stimuli are unaffected in the absence of SKAP-HOM. Similarly, B-cell receptor (BCR)-mediated total tyrosine phosphorylation and phosphorylation of Erk, p38, and JNK, as well as immunoreceptor-mediated Ca(2+) responses, are normal in SKAP-HOM(-/-) animals. However, despite apparently normal membrane-proximal signaling events, BCR-mediated proliferation is strongly attenuated in the absence of SKAP-HOM(-/-). In addition, adhesion of activated B cells to fibronectin (a ligand for beta1 integrins) as well as to ICAM-1 (a ligand for beta2 integrins) is strongly reduced. In vivo, the loss of SKAP-HOM results in a less severe clinical course of experimental autoimmune encephalomyelitis following immunization of mice with the encephalitogenic peptide of MOG (myelin oligodendrocyte glycoprotein). This is accompanied by strongly reduced serum levels of MOG-specific antibodies and lower MOG-specific T-cell responses. In summary, our data suggest that SKAP-HOM is required for proper activation of the immune system, likely by regulating the cross-talk between immunoreceptors and integrins.

FIG. 1.

Scheme showing the retroviral insertion into the SKAP-HOM locus, the expression pattern of SKAP-HOM in lymphoid organs, and the loss of SKAP-HOM protein in SKAP-HOM-deficient mice. (A) The VICTR20 retroviral insertion site in the SKAP-HOM locus was determined by restriction site mapping and comparing the wild-type and retroviral-targeted alleles. PCR products generated by oligonucleotide primers (a to c; also used for genotyping) positioned as shown were sequenced to identify the precise site of retroviral integration. Only the first 4 out of 12 coding exons are shown. (B) Mice were genotyped by PCR detecting the wild-type SKAP-HOM allele and the recombinant allele containing the neomycin resistance cassette. (C) Postnuclear lysates of total splenocytes of knock-out, wild-type, and heterozygous animals were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by blotting onto nitrocellulose sheets. Immunoblots were probed with a polyclonal rabbit anti-SKAP-HOM antiserum. (D) Total splenocytes and thymocytes were prepared from wild-type animals. Splenic B cells and T cells were obtained by negative selection with microbeads. CD4⁺ T cells and CD8⁺ T cells were obtained by positive selection with microbeads. Postnuclear lysates were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the resulting blots were probed as for panel C. (E) Loss of SKAP-HOM does not affect expression of ADAP and SKAP55 in the T-cell compartment. Postnuclear lysates of purified splenic T- and B-cell populations were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by anti-SKAP-HOM, anti-ADAP, and anti-SKAP55 Western blotting. LTR, long terminal repeat; KO, knockout; WT, wild-type; HT, heterozygous.

FIG. 2.

Normal T- and B-lymphocyte differentiation in SKAP-HOM-deficient mice. (A) Thymic T-cell precursors, splenic T cells, and lymph node T cells were analyzed by flow cytometry after staining with CD4-FITC and CD8-phycoerythrin. (B) Bone marrow B-cell precursors, splenic B cells, and lymph node B cells were analyzed after staining with B220-FITC and IgM-phycoerythrin. The percentages of cells within each lymphocyte population are indicated. BM, bone marrow; LN, lymph node.

FIG. 3.

Normal T-cell and BMM proliferation and normal Fc- and complement receptor-mediated phagocytosis in SKAP-HOM-deficient mice. (A) Purified splenic T cells were stimulated with increasing concentrations of CD3ɛ MAbs or with a combination of PMA plus ionomycin. Data of quadruplicate cultures are shown as means ± SEM; n = 5. (B) BMMs isolated from wild-type or knockout mice were cultured for 2 days and then plated in replicates in the presence of 10 ng/ml M-CSF. Cells were harvested at the indicated times and counted. (C) BMMs were incubated with sheep red blood cells opsonized with either IgG or IgM and C5-deficient (def.) sera. BMMs were scored for the percent of phagocytically active cells and for the ability of active cells to phagocytize RBCs (D). Results are representative of three separate experiments.

FIG. 4.

Impaired B-cell proliferation in SKAP-HOM-deficient mice. (A) Purified splenic B cells from wild-type or SKAP-HOM-deficient mice were stimulated with the indicated concentration of soluble anti-IgM F(ab′)₂ or soluble anti-IgM; n = 10; results are means ± SEM. The mean counts per minute of maximally IgM F(ab′)₂-stimulated wild-type B cells was 7,045 ± 893 cpm versus 407 ± 69 cpm of unstimulated cells. For SKAP-HOM-deficient B cells, the mean counts per minute was 6,417 ± 663 versus 628 ± 60, respectively. (B) Purified splenic B cells were stimulated with the indicated concentration of LPS (0.31 to 20 μg/ml). (C) Purified splenic B cells were activated with either anti-CD40+IL-4, LPS (2.5 μg/ml), or PMA plus ionomycin (Ion.). The stimulation index was calculated as the ratio of counts per minute (cpm) of stimulated to unstimulated proliferation. Nonstimulated B cells are shown as controls; n = 8; shown are means ± SEM. *, P < 0.05; **, P < 0.01.

FIG. 5.

Constitutive serum immunoglobulin levels and humoral immune responses in SKAP-HOM-deficient mice. (A) Constitutive serum immunoglobulin levels in nonimmunized wild-type and SKAP-HOM-deficient animals; n = 11; shown are means ± SEM. (B) Humoral immune response after immunization with the T-dependent antigen DNP-KLH. Twelve-week-old mice were injected at days 0 and day 21 intraperitoneally with DNP-KLH. Mice were bled before boostering (on day 21, corresponding to primary response) and after boostering (on day 28, corresponding to secondary response). Anti-DNP-specific levels of IgM, IgG1, IgG2a, and IgG3 were measured by ELISA. Four animals were investigated for each group. (C) Proliferative response of splenocytes after the secondary immunization to DNP-KLH. Lymphocytes isolated from the spleen at day 28 (after boostering) were stimulated with the indicated concentrations of DNP-KLH for 72 h. Results are given as the mean stimulation index of three independent experiments (means ± SEM). Ion., ionomycin.

FIG. 6.

Normal proximal BCR-mediated signaling in the absence of SKAP-HOM. (A) Freshly isolated, indo-1-loaded splenic B cells were stimulated with anti-IgM F(ab′)₂ (10 μg/ml) or, as a positive control, with ionomycin (100 nM). Intracellular calcium levels were measured by flow cytometry. (B) Purified splenic B cells were left unstimulated or treated with soluble anti-IgM F(ab′)₂ (10 μg/ml) for the indicated minutes. Cellular lysates were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and blots were probed with anti-phosphotyrosine (anti-pTyr) MAb 4G10. The expression levels of total Erk1 and Erk2 are shown as loading controls in the lower panel (additional data not shown). KO, knockout; WT, wild type.

FIG. 7.

Attenuated BCR-mediated adhesion but normal F-actin polymerization and conjugate formation of SKAP-HOM-deficient B cells. (A) Purified splenic B cells were stimulated with anti-IgM F(ab′)₂ and then allowed to adhere to tissue cell culture dishes coated with either fibronectin (A) or recombinant murine ICAM-1 (B). Adherent cell numbers were determined by microscopy. Results are expressed as means ± SEM of six independently performed experiments. (C) Purified B cells were treated with anti-IgM F(ab′)₂, permeabilized with saponin, and then stained with FITC-phalloidin. The cellular F-actin polymerization was assayed by flow cytometry after 1, 5, and 10 min. Results are expressed as percent increase of mean fluorescence intensity (MFI). The shown graph is representative of five independently performed experiments. (D) Purified B cells were loaded with OVA peptide overnight. T cells of OT-II TCR-transgenic mice were loaded with CFSE and coincubated with the peptide-pulsed B cells for 8 h. Conjugate formation was subsequently assessed by flow cytometry. Results are expressed as percentages of loaded B cells conjugated with T cells. The shown graph is representative of four independently performed experiments. unstim., unstimulated.

FIG. 8.

SKAP-HOM-deficient mice show reduced severity of EAE. (A) EAE was induced following immunization with MOG 35-55 (200 μg MOG in CFA). The severity of EAE is presented as mean clinical scores (means ± SEM; n = 9). The accompanying table in the box shows the incidence of EAE within the experimental groups at days 7, 10, and 15, respectively. The shown graph is representative of three independently performed experiments. The difference in the mean clinical score was significant at days 7, 8, 13, 15, and 17 (P < 0.05). (B) Serum levels of anti-MOG-specific antibodies in sera collected at day 14 after immunization of mice with MOG 35-55. Concentration of specific IgM and IgG were measured by ELISA. (C) Proliferative response to MOG 35-55 of lymphocytes isolated from draining lymph nodes at day 14 following immunization with MOG 35-55 (n = 5). Lymph node cells were cultured with or without MOG 35-55 for 72 h. The mean counts per minute of maximally MOG-stimulated wild-type lymph node cells was 4,431 ± 856 cpm versus 1,059 ± 211 cpm of unstimulated cells. For SKAP-HOM^−/− lymph node cells, the mean cpm was 1,969 ± 860 versus 610 ± 191, respectively. WT, wild type; KO, knockout.