Functional Role of Kallikrein 6 in Regulating Immune Cell Survival

Kallikrein 6 (KLK6) is a newly identified member of the kallikrein family of secreted serine proteases that prior studies indicate is elevated at sites of central nervous system (CNS) inflammation and which shows regulated expression with T cell activation. Notably, KLK6 is also elevated in the serum of multiple sclerosis (MS) patients however its potential roles in immune function are unknown. Herein we specifically examine whether KLK6 alters immune cell survival and the possible mechanism by which this may occur.Using murine whole splenocyte preparations and the human Jurkat T cell line we demonstrate that KLK6 robustly supports cell survival across a range of cell death paradigms. Recombinant KLK6 was shown to significantly reduce cell death under resting conditions and in response to camptothecin, dexamethasone, staurosporine and Fas-ligand. Moreover, KLK6-over expression in Jurkat T cells was shown to generate parallel pro-survival effects. In mixed splenocyte populations the vigorous immune cell survival promoting effects of KLK6 were shown to include both T and B lymphocytes, to occur with as little as 5 minutes of treatment, and to involve up regulation of the pro-survival protein B-cell lymphoma-extra large (Bcl-XL), and inhibition of the pro-apoptotic protein Bcl-2-interacting mediator of cell death (Bim). The ability of KLK6 to promote survival of splenic T cells was also shown to be absent in cell preparations derived from PAR1 deficient mice.KLK6 promotes lymphocyte survival by a mechanism that depends in part on activation of PAR1. These findings point to a novel molecular mechanism regulating lymphocyte survival that is likely to have relevance to a range of immunological responses that depend on apoptosis for immune clearance and maintenance of homeostasis

The ability of KLK6 to reduce murine T and B cell death depends in part on activation of PAR1.

<p>To determine the involvement of PAR1 in KLK6-mediated reductions in lymphocyte death, the ability of KLK6 (10 µg/ml) to reduce cell death under resting conditions, or in response to dexamethasone (0.1 uM), was compared between wild type and PAR1^−/− splenocytes. Comparisons were made between the total percentage of 7AAD⁺ dead cells, or between those additionally labeled to recognize CD3⁺ T cells, or B220⁺ B cells. (A) KLK6 (10 µg/ml) significantly reduced the overall percentage of 7AAD⁺ dead cells under resting conditions and in response to dexamethasone (0.1 µM). KLK6-mediated reductions in overall cell death in both paradigms (A) were seen to encompass both CD3⁺ T lymphocytes (B) and B220⁺ B lymphocytes (C). The absence of PAR1 (PAR1 KO) impaired the ability of KLK6 to significantly reduce the overall percentage of cell death and that associated with T cells and B cells under resting conditions. The absence of PAR1 also impaired the ability of KLK6 to reduce dexamethasone-induced CD3⁺ T cell death. Data shown are mean ± SEM of triplicate cultures examined in parallel; One Way ANOVA with SNK post hoc test for multiple comparisons, P = 0.002***, P≤0.003**, P<0.05*. Results shown are representative of that seen in at least 3 independent experiments.</p

KLK6 differentially regulates PARP cleavage and Bcl-2 family member protein levels.

<p>Western blots illustrate KLK6 (10 µg/ml)-induced changes in the amount of cleaved PARP (116 kDa intact form, 89-kDa fragment, A), or in levels of Bcl-2 family members (B), observed in murine splenocytes derived from WT mice and cultured for a 24 hr period under resting conditions, or in the presence of camptothecin (1.0 µM), or ConA (5 µg/ml). Relative to non-KLK6-treated control samples, KLK6-treatment promoted reductions in PARP cleavage in WT resting (98% reduction) and camptothecin (89% reduction) co-treated cultures (A). Significant PARP cleavage was not seen with ConA treatment at the 24 hr time point examined. B, The pro-survival protein Bcl-XL was elevated in cultures exposed to camptothecin or ConA when co-treated with KLK6. Relative to non-KLK6 treated sister wells, detection of Bcl-XL was elevated by 40-fold in the presence of KLK6+camptothecin and 80-fold in the presence of ConA+KLK6. KLK6-induced changes in Bcl-XL were not seen in resting cultures. KLK6 suppressed detection of the pro-apoptotic protein Bim by 83 and 84% in resting and Con A treated cultures respectively, and by 16% in cultures treated with camptothecin. Actin was used to control for loading in each case. All Western blots shown are representative of from 2–5 separate cell culture experiments.</p

The ability of KLK6 to decrease death of murine splenocytes relates to its ability to delay the apoptotic cascade.

<p>(A) Histogram and corresponding dot plots (B) demonstrate that KLK6 significantly reduced the percentage of dead cells (Annexin V-FITC⁺ and PI⁺) observed in cultures of splenocytes under resting conditions and after exposure to dexamethasone (0.1 µM), or staurosporine (1 µM). Reduced cell death was accompanied by a significant increase in the live population (unlabeled cells) under resting conditions and with exposure to staurosporine. KLK6 exposure also resulted in a corresponding increase in the percentage of early apoptotic cells (Annexin V-FITC⁺, PI^-) in the presence of apoptosis inducing agents. Data shown are the mean ± SEM of triplicate cultures examined in parallel; One Way ANOVA with SNK post hoc test for multiple comparisons, P<0.001***, P<0.003**, P<0.007*. All results shown are representative of that seen in at least 3 independent experiments.</p

Differential effects of KLK1 and KLK6 on survival and proliferation of murine splenocytes.

<p>To differentiate between possible effects of KLK6 on cell survival versus proliferation, murine splenocytes were labeled with CFSE and cultured in defined media in the presence of 1 or 10 µg/ml of KLK1, KLK6, or vehicle alone (Control), for periods of 24 or 72 hr. At harvest dead cells were labeled using PI and samples were examined by flow cytometry. A, Stimulation of cultures with either 1 or 10 µg/ml of KLK6, but not KLK1, significantly reduced the number of dead (PI⁺) cells observed at either the 24 or 72 hr time points (72 hr shown, B). The intensity of CFSE labeling in PI^- cells was determined using the proliferation platform of the Flow Jo Program and labeling peaks observed after 24 hr are shown in (C). KLK1 (10 µg/ml), but not KLK6 promoted a significant increase in the percent of cells divided at the 24 hr time point (D); Data are expressed as mean ± SEM, One Way ANOVA with SNK post hoc test; P<0.001**, P<0.02*. (SSC, side scatter). Parallel observations have been made in a least three separate cell culture experiments.</p

KLK6 promotes survival of Jurkat T cells in part by slowing the apoptotic cascade.

<p>To address the effect of KLK6 on apoptosis, Jurkat T cells were grown under resting conditions (A), or in the presence of apoptosis inducing agents (B to D), then labeled with Annexin V-PE and 7AAD prior to flow cytometry. Under resting conditions (A), and in the presence of camptothecin (B, 1.0 µM), ConA (C, 5 µg/ml), or ConA plus camptothecin (D), KLK6 (10 µg/ml) promoted an increase in the percentage of live cells (unlabeled) and a decrease in the percentage of dead cells (Annexin V⁺ and 7AAD⁺) at both acute (4 hr), subacute (24 hr), and in some cases the more chronic time point examined (48 hr). Interestingly, in the presence of apoptosis inducing agents (B to D), KLK6 also promoted a corresponding increase in the percentage of Jurkat T cells positive for Annexin V, but negative for 7AAD, and therefore classified as in the early stages of apoptosis. These results suggest KLK6 may delay but not block the apoptotic cascade. Data shown are for triplicate cultures examined in parallel and are expressed as mean ± SEM, One Way ANOVA with SNK post hoc test for multiple comparisons; P<0.001***, P≤0.005**, P≤0.02*. All results shown are representative of those seen across at least three experiments using independent cell culture preparations.</p

As little as a 5 minute pulse of KLK6 is sufficient to promote splenocyte survival.

<p>To gauge the minimal time of exposure to KLK6 necessary to observe its pro-survival effects, splenocytes were pulsed with KLK6 (10 µg/ml) for periods of 5, 30 or 60 min, or in the presence of KLK6 for the full 24 or 48 hr period of culture examined, prior to labeling with PI for analysis of dead cells by flow cytometry (A and B). A 5 min pulse with KLK6 promoted significant survival of explanted resting splenocytes over a 24 hr culture period that was similar in magnitude to that seen with longer pulses, i.e. 30 or 60 min and 24 hr (A). A 5 min pulse with KLK6 was also sufficient to promote survival when cells were exposed to dexamethasone (0.1 µM) for 24 hr (C). After longer periods of culture however (B, 48 hr), only more the more prolonged period of KLK6 stimulation (48 hr) exerted significant pro-survival effects (B). Data shown are the mean ± SEM of triplicate cell culture samples run in parallel and examined by One Way ANOVA with the SNK post hoc test for multiple comparisons; P<0.001**, P≤0.005*. Results shown are representative of at least 3 separate experiments using independent cell culture preparations.</p

KLK6 over expression in Jurkat T cells reduces cell death.

<p>Jurkat T cells were stably transduced with a vector in which the human KLK6 gene is constitutively expressed under the control of a CMV promoter, or with an empty vector (Control), and levels of live (Annexin V-PE^- and 7AAD⁻), early apoptotic (Annexin V-PE⁺ and 7AAD⁻), or dead (Annexin V-PE⁺ and 7AAD⁺) cells determined by flow cytometry under resting conditions, or after 24 hr periods of exposure to 0.1 or 0.25 µM staurosporine. (A) Histogram and corresponding dot plots (B), demonstrate KLK6 over expression produces effects largely parallel to those afforded by treatment of cultures with recombinant KLK6. There was a decrease in the percentage of dead cells and an increase in the number of live cells in Jurkat T cells over expressing KLK6, relative to those expressing an empty vector. KLK6 over expression also reduced cell death in the presence of 0.1 or 0.25 µM stuarosporine relative to that seen in cells stably transduced with empty vector. Reductions in cell death were likely to reflect in part a delay in apoptosis, since after 24 hr exposure to 0.25 µM Staurosporine, KLK6 over expression not only reduced the number of dead cells and increased the number of live cells, but cells in the early stages of apoptosis (AnnV-PE⁺, 7AAD⁻) were also significantly elevated. Data are expressed as mean ± SEM of triplicate cultures examined in parallel; One Way ANOVA with SNK post hoc test for multiple comparisons, P<0.001***, P = 0.002**, P = 0.003*. All data shown is representative of that seen in at least 3 independent cell culture experiments.</p