Immune System Dysfunction and Autoimmune Disease in Mice Lacking Emk (Par-1) Protein Kinase

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 ofBLNK ^−/− and ZAP70 ^−/−mice, respectively. The 80- and 75-kDa forms of Emk arise by alternative splicing.

Analysis of CD44 expression levels, proliferative capacity, and cytokine production in T lymphocytes ofEmk ^+/+ 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 twoEmk ^−/− mice are shown as error bars along they 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.

Reduced B-cell proliferative capacity inEmk ^−/− mice. B220⁺ splenocytes isolated from 8-week-old Emk ^+/+ andEmk ^−/− 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.

The immune response of Emk ^−/−mice after challenge with T-cell-dependent and -independent antigens.Emk ^−/− (open diamonds) andEmk ^+/+ (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 theirEmk ^+/+ littermates (P < 0.005) after 21 days. Similarly, the IgG1 response inEmk ^−/− mice (B) was ca. 9.5-fold higher than in their Emk ^+/+ littermates (P < 0.005) .

Expansion of Ter119⁺ HSA⁺lymphocytes in spleens of Emk ^−/− mice. (A) Graphical representation of the numbers of B220⁺, CD4⁺, and CD8⁺ splenocytes obtained fromEmk ^+/+ and Emk ^−/− mice at 7 to 12 months of age (from Table 2). (B and C) Splenocytes ofEmk ^+/+ (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.

Histological analysis of hematoxylin-eosin-stained organs from Emk ^+/+ andEmk ^−/− mice. (A and B) Kidneys from 8-month-old Emk ^+/+ (A) andEmk ^−/− (B) mice. Magnification, ×200. (C and D) Lungs from 8-month-old Emk ^+/+ (C) andEmk ^−/− (D) mice. Magnification, ×200. (E) High-power magnification of a kidney from an 8-month-oldEmk ^−/− 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.