Cladribine Exposure Results in a Sustained Modulation of the Cytokine Response in Human Peripheral Blood Mononuclear Cells

<div><p>Background and Objectives</p><p>Cladribine is a cytotoxic drug which ameliorates the clinical course of relapsing-remitting multiple sclerosis. In addition to cytotoxicity, the mode of action may include immunomodulatory mechanisms. This <i>in vitro</i> study was designed to investigate cladribine’s effects on cell function after the removal of cladribine to distinguish cytotoxic versus immunomodulatory effects.</p><p>Methods</p><p>Cells were incubated in the absence or presence of cladribine (1×10^-8 M to 1×10^-5 M) for 72 h. Cladribine was removed from the cell culture and surviving peripheral blood mononuclear cells were cultured up to 58 days to determine the immunomodulatory effects of cladribine on cell function (e.g., proliferation and cytokine release).</p><p>Results</p><p>In the long-term, brief cladribine exposure did not impair the proliferation of surviving peripheral blood mononuclear cells. However, it induced an anti-inflammatory shift in the cytokine milieu with significantly enhanced release of IL-4 (Days 9 and 44, p<0.01; Day 58, p<0.05) and IL-5 (Day 9, p<0.01), resulting in an increased IL-4/INF-gamma ratio (Days 9 and 44, p<0.01; Day 58, p<0.05). Additionally, a trend towards an increased IL-10 production was observed. No changes were found in the production of IFN-gamma, TNF-alpha, IL-6, IL-8, IL-17A, IL-23 or NGF-beta.</p><p>Conclusions</p><p><i>In vitro</i> cladribine exposure induces a sustained anti-inflammatory shift in the cytokine profile of surviving peripheral blood mononuclear cells. This immunomodulatory action might contribute to cladribine’s beneficial effects in the treatment of multiple sclerosis.</p></div

<i>In vitro</i> effects of cladribine on cell survival of PBMCs and proliferation of PBMCs, CD4⁺ cells and CD8⁺ cells Human immune cells were stimulated with anti-CD3/anti-CD28 antibodies (a, n = 4; c, n = 6) or PHA (b, n = 4; d, n = 3) in the absence or presence of cladribine (1×10⁻⁸ M to 1×10⁻⁵ M) for 72 h.

<p>Determination of cell survival in PBMCs (a, b) was performed by standard trypan blue exclusion method. Proliferation was determined separately in PBMC, CD4⁺ cells and CD8⁺ cells (c, d) by the incorporation of tritiated thymidine. Stimulation indices were calculated as the ratios of the counts per minute of stimulated samples to unstimulated and untreated samples. Data are depicted as mean + standard deviation (SD).</p

<i>In vitro</i> effects of initial cladribine treatment on cytokine secretion of PBMCs restimulated at Days 9, 16, 23, 30, 44 and 58 after transfer into cladribine-free medium.

<p>PBMCs from healthy blood donors (n = 5) were incubated in the absence or presence of cladribine. (1×10⁻⁸ M to 5×10⁻⁷ M) for 72 h. Then cells were washed three times and transferred into cladribine-free medium. Cells were restimulated with anti-CD3/anti-CD28 antibodies for 48 h at days 9, 16, 23, 30, 44 and 58; supernatants were collected and cytokine concentrations were determined. Data are depicted as box plot diagrams. Whiskers represent maximum and minimum values. The IL-4/IFN-γ ratio was defined as the ratio of IL-4 (pg/ml) to IFN-γ (pg/ml). *: p<0.05; **: p<0.01.</p

<i>In vitro</i> effects of initial cladribine treatment on cytokine secretion of PBMCs restimulated at Days 9, 16, 23 and 30 after transfer into cladribine-free medium.

<p><b>PBMCs from healthy blood donors (n = 4) were incubated in the absence or presence of cladribine</b>. (1×10⁻⁸ M to 5×10⁻⁷ M) for 72 h. Then cells were washed three times and transferred into cladribine-free medium. Cells were restimulated with PHA for 48 h at days 9, 16, 23 and 30; supernatants were collected and cytokine were determined. Data are depicted as box plot diagrams. Whiskers represent maximum and minimum values. The IL-4/IFN-γ ratio was defined as the ratio of IL-4 (pg/ml) to IFN-γ (pg/ml). *: p<0.05; **: p<0.01.</p

<i>In vitro</i> effects of initial cladribine treatment on the proliferation of PBMCs stimulated at Days 9, 16, 23, 30, 44 and 58 after transfer into cladribine-free medium PBMCs were initially incubated in the absence or presence of cladribine (1×10⁻⁸ M to 5×10⁻⁷ M) for 72 h.

<p>Cells were washed three times and then transferred into cladribine-free medium. Cells were restimulated with anti-CD3/anti-CD28 antibodies (a, n = 5) or PHA (b, n = 4) for 48h at Days 9, 16, 23, 30, 44 and 58; proliferation was determined by the incorporation of tritiated thymidine. Stimulation indices were defined as the ratios of the counts per minute of stimulated samples to unstimulated and untreated samples. Data are depicted as mean + standard deviation (SD).</p