Expression pattern of Emk in mouse tissues by Western blot analysis. A Western blot analysis of Emk and α-actin protein in selected lymphoid tissues and cells is shown. Enriched populations of T and B lymphocytes were isolated from the spleens of BLNK^−/− and ZAP70^−/− mice, respectively. The 80- and 75-kDa forms of Emk arise by alternative splicing.

Histological analysis of hematoxylin-eosin-stained organs from Emk^+/+ and Emk^−/− mice. (A and B) Kidneys from 8-month-old Emk^+/+ (A) and Emk^−/− (B) mice. Magnification, ×200. (C and D) Lungs from 8-month-old Emk^+/+ (C) and Emk^−/− (D) mice. Magnification, ×200. (E) High-power magnification of a kidney from an 8-month-old Emk^−/− mouse, showing cellular components of lymphocytic infiltrates. Magnification, ×1,000. Abbreviations: G, glomerulus; T, tubule; BV, blood vessel; LI, lymphocytic infiltrate; B, bronchiolus; PC, plasma cell.

Expansion of Ter119⁺ HSA⁺ lymphocytes in spleens of Emk^−/− mice. (A) Graphical representation of the numbers of B220⁺, CD4⁺, and CD8⁺ splenocytes obtained from Emk^+/+ and Emk^−/− mice at 7 to 12 months of age (from Table 2). (B and C) Splenocytes of Emk^+/+ (B) and Emk^−/− (C). Splenocytes isolated from mice at ∼7 months of age were double stained with FITC-conjugated anti-HSA and PE-conjugated anti-Ter119. The stained cells were analyzed by flow cytometry gated on lymphocytes, and the results are shown as dot plots. The percentage of gated cells in each quadrant is indicated.

Reduced B-cell proliferative capacity in Emk^−/− mice. B220⁺ splenocytes isolated from 8-week-old Emk^+/+ and Emk^−/− mice were induced to proliferate by incubation with 20 μg of anti-IgM antibody per ml for the indicated times (A), with different concentrations of anti-IgM antibody (5 to 80 μg/ml) for 48 h (B), with PMA and ionomycin (C), or with anti-CD40 antibody (D). Cell proliferation was measured by [³H]thymidine incorporation using pooled splenocytes from two animals per genotype, and each experiment was repeated a minimum of three times. Standard deviations for triplicate samples are shown as error bars along the y axis.

Emk^−/− mice develop autoimmune glomerulonephritis. (A and B) Glomeruli of representative Emk^+/+ (A) and Emk^−/− (B) mice analyzed by hematoxylin and eosin staining. Magnification, ×630 (oil). (C and D) Glomeruli of representative Emk^+/+ (C) and Emk^+/+ (D) mice incubated with anti-mouse IgG coupled to Cy3 and visualized by indirect immunofluorescence. Magnification, ×400. (E) The glomerulus seen in panel D was incubated with FITC conjugated to anti-mouse C3 (anti-C3) and was visualized by direct immunofluorescence. Magnification, ×400. (F) Anti-IgG-Cy3 immunofluorescence staining of a representative Emk^−/− kidney. Magnification, ×200. (G) Electron micrograph of an Emk^+/+ kidney capillary loop. Magnification, ×7,700. (H) Electron micrograph of Emk^−/− kidney capillary loop. Magnification, ×10,000. Abbreviations: RBC, red blood cell; BL, basal lamina; ID, Ig deposit; CL, capillary lumen; EC, epithelial cell.

The immune response of Emk^−/− mice after challenge with T-cell-dependent and -independent antigens. Emk^−/− (open diamonds) and Emk^+/+ (open squares) littermates (8 weeks old) were injected intraperitoneally with NP-Ficoll (A) or NP-KLH (B). Serum samples were analyzed for IgM, IgG1, IgG2a, and IgG3 by ELISA as described in Materials and Methods. The results are expressed as the dilution at which the optical density is 0.2 over background. (A) The log values of the titers were analyzed statistically. T-cell-independent IgM and IgG3 responses were ca. 4-fold lower (P < 0.001) and ca. 3.5-fold lower (P < 0.05), respectively, in Emk^−/− mice than in their Emk^+/+ littermates after 17 days. (B) The T cell-dependent IgG2a response in Emk^−/− mice was ca. 7.5-fold higher than that in their Emk^+/+ littermates (P < 0.005) after 21 days. Similarly, the IgG1 response in Emk^−/− mice (B) was ca. 9.5-fold higher than in their Emk^+/+ littermates (P < 0.005).

Analysis of CD44 expression levels, proliferative capacity, and cytokine production in T lymphocytes of Emk^+/+ and Emk^−/− mice. (A and B) CD44 (pgp-1) expression levels were analyzed by FACS on CD4⁺ T cells isolated from spleens (A) and lymph nodes (B) of Emk^+/+ and Emk^−/− mice. (C) Cell proliferation (CD4⁺ T cells) was measured by [³H]thymidine incorporation after stimulation with either anti-CD3 or anti-CD3 plus anti-CD28. Standard deviations for triplicate samples obtained from two Emk^+/+ and two Emk^−/− mice are shown as error bars along the y axis. (D and E) CD4⁺ splenic T cells pooled from two to three animals were driven to differentiate to Th1, Th2, or neutral phenotypes, and ELISAs for IFN-γ (D) or IL-4 (E) were performed as described in Materials and Methods. Each experiment was performed a minimum of three times, and standard deviations are shown as error bars along the y axis.

Targeted disruption of the mouse Emk gene. (A) Structure of the targeting construct. The genomic organization of the mouse Emk gene was disrupted by replacing part of exon 2 and all of exons 3 and 4 of the Emk gene with the neomycin phosphotransferase cDNA driven by the thymidine kinase promoter (pTK-neo) as a selectable marker. Restriction sites in the introns flanking the targeted exons are indicated. Exons 2 to 8 are represented by shaded boxes. Small arrows depict the location of PCR primers used for genotyping. The probe used for Southern blot analysis is indicated by a bar under the targeted locus. (B) PCR analysis of genomic DNA isolated from tail clippings of F₂ Emk mice. Emk^+/+ mice produced a 310-bp PCR fragment, Emk^−/− mice generated a 437-bp fragment, and Emk^+/− mice gave rise to both products. (C) Western blot analysis of Emk protein. Lysates were prepared from 293 cells (lane 1) or from MEFs derived from Emk^+/+ (lane 2), Emk^+/− (lane 3), or Emk^−/− (lane 4) animals. A 250-μg portion of total cellular protein was resolved by SDS-PAGE, and Western blotting was performed using affinity-purified Emk antibody. Endogenous Emk is expressed as two alternatively spliced mRNAs encoding protein products of ∼75 and 80 kDa. (D) Immune complex kinase assays. Homogenates of testis and brain were prepared from Emk^+/+, Emk^+/−, and Emk^−/− mice. Emk was immunoprecipitated using 2.5 mg of total tissue protein, and kinase assays were performed in vitro using GST-Cdc25C(200–256) as a substrate. Proteins were resolved by SDS-PAGE, and radiolabeled proteins were visualized by autoradiography. ³²P-labeled substrate was excised from the gel, and radioactivity was quantitated by Cerenkov counting.