To delete ILK in vivo in T cells, we bred ILKflox/flox
mice to transgenic mice expressing Cre recombinase under the direction of the Lck proximal promoter. The resulting Cre+
mice provided a well-validated system for dissecting the role of ILK in leukocyte biology through Cre-mediated deletion in developing T cells (7
). Screening of tail DNA for inheritance of Cre recombinase and floxed ILK alleles was performed by PCR as previously described (2
). Representative data from the genotyping of mice are shown in Fig. . Thymic T cells from Lck-Cre+
mice had a marked reduction in ILK protein levels, as seen in Fig. .
FIG. 1. T cell-specific deletion of ILK in mice. (a) ILKflox/flox mice were bred to transgenic mice expressing Cre recombinase under the direction of the Lck proximal promoter, and the progeny were genotyped by PCR for the inheritance of Cre recombinase and ILK (more ...)
The genotypes of more than 900 offspring followed the predicted Mendelian distribution. Mice showed no gross developmental abnormalities and were fertile. As Cre driven by the Lck proximal promoter is activated early in T-cell development, we next analyzed thymuses from mice with ILK deletion versus those from littermate controls. Thymic cellularity was comparable in 3- to 4-week-old mice; however, there was a threefold reduction of thymic T cells in ILK-deficient mice by 6 to 8 weeks of age (Fig. ). Flow-cytometric analysis of thymocytes from 6- to 8-week-old mice revealed a diminution in both the percentages (~1.5-fold) and absolute numbers (~14-fold) of the double-positive (CD4+ CD8+ DP) thymic T-cell population, accompanied by a relative increase in the percentage of double-negative (CD4− CD8− DN) and single-positive (CD4+ or CD8+ SP) cells (Fig. , top). In contrast, thymocytes from 3- to 4-week-old mice were phenotypically indistinguishable from wild-type controls (data not shown). T-cell receptor (TCR) profiles of thymocytes of the 6- to 8-week-old mice also revealed a concordant decrease in the TCRmed populations (Fig. , middle), which normally represent DP cells, and a relative increase in TCRhi single-positive (SP) cells. Staining with annexin V and propidium iodide (PI) demonstrated an approximate three- to fourfold increase in the annexin V+/PI+ dead cells in the ILK-deficient thymus (Fig. ). To rule out the possibility of an early developmental defect caused by ILK deficiency, we analyzed the DN-thymocyte precursor population in greater detail (Fig. ). An increase in the percentage of cells falling in the Lin− DN gate was observed in the 6- to 8-week ILK-deficient thymus due to the depletion of the DP compartment. However, the proportion and absolute number of cells in the DN1 through DN4 stages of development, as identified by CD25 and CD44 costaining, was comparable between wild-type and ILK-deficient mice. Taken together, these results suggest that ILK deletion does not cause a thymocyte developmental defect but rather confers an increased susceptibility to cell death in the thymus that primarily affects DP T cells. Of note, no changes were noted in other thymic constituents such as TCRγδ T cells (data not shown).
FIG. 2. Diminished thymic cellularity in Lck-Cre+/ILKflox/flox mice. (a) Thymuses from mice were dissected and strained into single-cell suspensions. Cumulative data of total cell counts from 46 mice are graphed, stratified by age. *, P < (more ...)
We conducted studies on peripheral T cells in parallel with our initial characterization of thymic T cells. While thymic T cells demonstrated >99% ILK deletion as assessed by quantitative PCR (Fig. ), peripheral T cells from spleen and lymph nodes demonstrated an approximately >20-fold enrichment of ILK-competent cells, suggesting these cells have a competitive advantage in trafficking, survival, or proliferation in peripheral lymphatic organs. To further characterize the effects of ILK deficiency on the peripheral T cells, we then crossed both our Lck-Cre+
mice and Lck-Cre+
controls with Rosa-YFP reporter mice, which allowed us to identify YFP+
, Cre-expressing/ILK-deleted T cells using flow cytometry (24
). Concordant with the quantitative PCR studies, we observed a significant enrichment for YFP−
T cells that have “escaped” deletion and are ILK competent in the lymph nodes of YFP indicator+
mice compared to those of YFP indicator+
controls (approximately one-third of T cells in the ILK-deficient reporter mice [Fig. and ]). We then examined whether ILK deficiency had effects on specific peripheral T-cell subsets. Flow-cytometric studies demonstrated normal numbers and proportions of CD4+
peripheral T cells in the ILK-deficient mice compared to the littermate controls (Fig. ). Furthermore, the CD4:CD8 ratios were unchanged in the ILK-deficient YFP+
populations. Additionally, the distribution of the CD45RB epitope in TCR+
cells (which inversely correlates with activation in murine T cells) was also comparable in the ILK-deficient mice compared to the littermate controls in both the lymph nodes (Fig. ) and thymocytes (data not shown). We thus observed a global enrichment for “ILK-sufficient cells” in the triple-transgenic animals, rather than subset-specific effects. Discrepancies in the number of resident T cells and the relative presence of ILK did not translate into consistent differences in thymic, splenic, or abdominal lymph node architecture between the ILK knockout (KO) mice and littermate controls.
FIG.3. Peripheral enrichment for ILK-competent cells. (a) Thymuses and peripheral lymph organs were harvested from Lck-Cre+/ILKflox/flox and control mice, and the T cells were purified by positive selection. Real time PCR was used to determine the relative (more ...)
We next explored potential mechanisms related to altered cell trafficking and/or survival that might explain the decreases in thymic cellularity and overall enrichment for ILK competent cells in the spleen and lymph nodes. As previous studies suggested a role for ILK in chemokine-triggered effector functions in leukocytic cell lines (3
), we performed chemotaxis assays of T cells in transwell chambers. T cells from the Lck-Cre+
littermate mice served as biological controls to rule out theoretical confounders related to Cre expression (13
thymocytes from 3- to 4-week-old mice exhibited classic “bell-shaped” results in chemotaxis to the lymphocyte chemokines CXCL12 (stromal cell derived factor [SDF]-1α) and CCL19 (macrophage inflammatory protein [MIP]-3β) (Fig. and ). In contrast, chemotaxis of the Lck-Cre+
T cells, which were indistinguishable from the wild type by flow cytometry analysis, was significantly diminished to both agonists across the broad range of concentrations tested. This defect was T cell-specific, as we saw no difference in chemotaxis of purified lymph node B cells from the same animals (Fig. ). It was also not due to modulation of the CXCL12 receptor, CXCR4
, on the KO T cells (Fig. ). We then repeated the chemotaxis studies using flow-sorted YFP+
lymph node T cells from the triple-transgenic animals, which confirmed the findings observed with the thymocytes (Fig. ). Importantly, YFP−
T cells from Lck-Cre+
mice, which “escaped” deletion and are ILK competent, demonstrated normal chemotaxis. We saw no effect of ILK deletion on levels of β1-integrins, nor reproducible changes to recombinant vascular cell adhesion molecule (VCAM)-1 in static adhesion assays (not shown).
FIG. 4. Abnormal chemotaxis of ILK-deficient T cells. Chemotaxis assays were performed on thymocytes from 3- to 4-week-old mice in transwell chambers to the lymphocyte chemokines (a) CCL19 (n = 4, *P < 0.01) and (b) CXCL12 (n = (more ...)
A growing body of evidence implicates ILK in cell survival pathways (1
) that in T cells might account for altered thymic development or result in abnormal stress responses. As noted above, T cells from 3- to 4-week-old ILK-deficient mice were indistinguishable from wild-type T cells as assessed by flow cytometry, including no significant differences in the baseline levels of apoptosis as assessed by PI or annexin V staining (data not shown). We next examined whether stressing these T cells ex vivo might unmask differences between ILK-deficient and ILK-competent cells in susceptibility to apoptosis using three complementary methodologies. We found a significant increase in apoptosis following 12 h of serum deprivation in the isolated ILK KO thymocytes using a cell death ELISA for cytoplasmic histone-associated DNA fragmentation (Fig. ). These findings were confirmed by a striking increase in the cleavage product of caspase-3, as assessed by Western blot analysis (Fig. ), and a concordant increase in propidium iodide staining in flow cytometry studies (Fig. ). Interestingly, in contrast to demonstrated differences in cell survival of Lck-Cre+
T cells under stress, we observed no significant differences of in vitro T-cell proliferation upon stimulation with anti-CD3 (Fig. ) or phorbol-12-myristate-13-acetate-ionomycin (data not shown). Proliferation studies were also performed using YFP+
lymph node T cells from the triple-transgenic animals, and yielded similar results (data not shown).
FIG. 5. Diminished survival of ILK KO thymocytes ex vivo. Thymocytes from 3- to 4-week-old mice were isolated and ex vivo cell survival was assessed after 12 h of serum deprivation by (a) cell death detection ELISA (n = 4); (b) Western blot analysis probing (more ...)
FIG. 6. ILK deletion has no effect on T-cell proliferation. In vitro proliferation assays of lymphocytes were performed using whole lymph nodes or LN T cells purified (91 to 96%) by negative selection column from Lck-Cre+/ILKWT/WT (WT) and Lck-Cre+ (more ...)
We next assessed the effects of ILK deletion on intracellular signaling pathways that participate in leukocyte trafficking and survival. In vitro, ILK can serve as a membrane-proximal upstream modulator of Akt, which has been implicated in both pathways (23
). The chemokine CXCL12 induced robust Akt activation as assessed by phosphorylation on the activating site, Ser 473 (Fig. ), as previously described (29
). We observed a reproducible decrease in Ser 473 phosphorylation in ILK-deficient T cells compared to ILKWT/WT
cells after 5 min of CXCL12 stimulation. We also performed in vitro assays of Akt activity and found a concordant decrease in Akt kinase function following chemokine stimulation (Fig. ).
FIG. 7. Effect of ILK deletion on T-cell signaling pathways. Freshly isolated thymic T cells from Lck-Cre+/ILKWT/WT (WT) and Lck-Cre+/ILKflox/flox mice (KO) were incubated with or without CXCL12/CCL19 for the indicated time periods at 37°C. (more ...)
We finally tested whether ILK deletion modulated the activation of small GTPases such as Rac1 which are important for membrane ruffling and lamellipodia formation as well as chemokine-triggered cell movement (4
). In an in vitro kinase assay, we
observed significant activation of Rac by the chemokines CXCL12 and CCL19 in T cells isolated from Lck-Cre+
mice (Fig. ). However, T cells from Lck-Cre+
mice showed comparable levels of basal Rac activity though markedly inhibited chemokine-triggered activation. In contrast to the observed abnormalities in Akt and Rac signaling in T cells, kinetic studies showed that chemokine-triggered ERK1/2 phosphorylation was unchanged, thus ruling out global cell signaling changes secondary to ILK deficiency (Fig. ).