Identification of T‐cell derived soluble mediators which activate MMP‐9 expression in

Based on the knowledge that (T‐cell‐derived) cytokines induce the expression of MMP‐9 by monocytes (82), and that the activated monocyte itself can synthesize and release further cytokines, the focus of the next analyses was set on the characterization of the mediators which might orchestrate this MMP‐9 expression. To better understand the interacting mechanism between the cells, their soluble mediators, and the effect on MMP‐9 expression, individual and co‐culture experiments were performed to identify the mediators that were able to increase the MMP‐9 production in monocytes under the applied experimental conditions. HMWH‐ treated THP‐1 culture revealed the expression of Rantes, IL‐ra, and MIF, whereas HMWH‐ treated HT cells expressed TNF, sICAM‐1, MIF, IL‐13 and Serpin E1. Interestingly, although B‐ cells did not mediate any influence on MMP‐9 expression in response to HMWH, they are shown here to produce TNF, a potent cytokine used as a positive control for MMP‐9 induction in this and other studies (78, 146). This might be due to an insufficient amount of TNF produced by B‐cells or the different time points analyzed (2h TNF stimulation in the positive control approaches vs. 24h in co‐culture experiments).

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HMWH‐treated Jurkat cells were characterized by the expression of IL‐16, IL‐13, Serpin E1, MIF, and sICAM‐1 (Fig. 4.1). To further search other precise T‐cell specific soluble mediator(s), triple and double co‐culture experiments were performed, by which HMWH‐treated THP‐1, Jurkat, and HT or HMWH‐treated THP‐1 and Jurkat cells showed the expression of Rantes, IL‐1ra, MIF, sICAM‐1, Serpin E1, IL‐8, IL‐13, and IL‐16. General importance of cytokines for the MMP‐9 production is also reflected by experiments which showed that the addition of IL‐1β or TNF to human macrophage cultures increased MMP‐9 production, whereas MMP‐9 production was down‐modulated when human macrophage cultures were treated with IFN‐γ or IL‐4 (147). In addition, monocyte‐derived mediators, esp. IL‐8, were expressed in the presence of T‐cells, which might contribute to its effect in an autocrine manner. It was also shown in response to a number of stimuli that IL‐8 was able to bind to heparin (148) and that IL‐15 induced IL‐8 production in human monocytes (149). IL‐16 and sICAM‐1, which are a modulators of T‐cell activation, might also contribute to this effect by which relevant studies showed that IL‐16 stimulates the expression and production of pro‐inflammatory cytokines by human monocytes (150), a finding which supports the assumption of a cytokine‐induced activation of an autocrine feedback loop in monocytes. In agreement with our results, a previous study showed that sICAM‐1 stimulates MMP‐9 expression (151) and is implicated in transient cellular interactions, which regulate leukocyte homing, activation, and effector functions (152). The analysis of the precise T‐cell‐specific soluble mediator(s) revealed that within the T‐cell‐derived cytokine panel produced in response to HMWH (IL‐16, IL‐13, Serpin E1, MIF, and sICAM‐1), IL‐16 is the only completely T‐cell‐specific factor (since the other factors were also produced by B‐ or monocytic cells in the absence of T‐cells). Therefore, we concluded that TNF, sICAM‐1, MIF, IL‐13 and

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Serpin E1 (i.e., HT derived mediators) or Rantes, IL‐ra, and MIF (i.e., THP‐1 derived mediators) might not play a role for the inducing effect on MMP‐9 expression and that IL‐16 might be the major driver of monocytic MMP‐9 expression. However, although at this point of the study a regulation of MMP‐9 by T‐cell‐derivedIL‐16 was presumed, the possibility of further influencing effects was not be ruled out (e.g., the presence of supporting factors either constitutively or inducibly expressed by monocytes, T‐cells, or B‐cells that might not play a role in the absence of T‐cell‐derived factors, but may acquire some importance as mediators enhancing the effect of monocyte stimulation).

Fig. 4.1: Secretion of soluble mediators by T‐cells in response to HMWH.

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4.7.1 Stimulation with individual soluble mediators has no effect on MMP‐9 expression by monocytes

To validate the impact of the identified soluble mediators on MMP‐9 expression and with respect to the knowledge that cytokines regulate MMP‐9 expression in different cell types (153), monocytes were stimulated in an initial approach using these factors individually. In these initial experiments, starved monocytes were stimulated first using IL‐16, since it was the most prominent cytokine induced in response to heparin in double co‐culture experiments of monocytes and T‐cells. Unexpectedly, only a slight numerical tendency towards 2‐fold increased MMP‐9 levels, but no significant induction was observed under this condition, indicating that other soluble mediators might play an essential role on MMP‐9 expression. Additionally, an individual stimulation of THP‐1 cells with IL‐8, which was then used since it was secreted exclusively in monocyte‐ and T‐cell‐containing co‐cultures but not individual cultures, showed no effect on MMP‐9 expression levels. This indicates that neither IL‐16 nor IL‐8 alone have any elevating effect on MMP‐levels in monocytes. Comparing our initial findings in the culture experiments with other studies, no data were found in the literature concerning direct stimulation of monocytes with IL‐16 yet. However, a recent study showed that IL‐16 was able to significantly induce MMP‐9 levels via enhancing the binding activity of transcription factors NF‐

κB, AP‐1, and Sp‐1 in VSMCs, and the p38‐MAPK phosphorylation was significantly stimulated in IL‐16‐treated VSMCs (154). Moreover, p38‐MAPK inhibitor SB203580 prevented the activation of Sp‐1 binding in IL‐16‐treated VSMCs and inhibited IL‐16‐induced MMP‐9 expression as well as migration and invasion of these cells. These results ‐ in combination with the data concerning the missing effect of individual IL‐16 stimulation obtained in the present study ‐ showed that IL‐

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16 is nevertheless a promising potential player in MMP‐9 induction (possibly also representing a new target in the treatment of vascular diseases such as atherosclerosis), but that additional molecular mechanism appear to interact to induce or inhibit MMP‐9 levels in monocytes.

Studying the stimulatory effect of IL‐16 on different cell types can be helpful regarding this context.

In addition to IL‐16 and IL‐8, the other identified T‐cell‐derived soluble mediators were analyzed regarding a potential impact on MMP‐9 expression. Therefore, starved monocytes were also stimulated using MIF, IL‐13, Serpin E1, and sICAM‐1, respectively. Regarding this context, there were no considerable effects of the respective soluble mediators on MMP‐9 induction, although mild increased MMP‐9 values were detected in the presence of sICAM‐1 (3‐fold, statistically significant), MIF, and Serpin E1 (2‐fold each, not significant). With respect to MIF, the observed absence of any significant effect is in contradiction to some reports which have shown that this factor is able to induce MMP‐9 expression directly, but this may be ascribed to the use of different cell types in that study such as macrophages and VSMC (155). Studies revealing the role of MIF on MMP‐9 expression in monocytes not yet established.

Furthermore, a study using a mouse model showed that in IL‐13^−/−mice, MMP‐9 expression was reduced in monocytes (156). In consequence, these findings are not in agreement with the findings presented here, since individual stimulation of starved monocytes using IL‐13 did not show any significant effect on MMP‐9 levels. However a precise assessment of this effect remains difficult, since it is not clear whether the lack of IL‐13 has a direct or an indirect effect on MMP‐9 in this model (e.g., via a down regulation of IL‐13‐dependent regulatory proteins).

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To my knowledge, studies analyzing the role of Serpin E1 and sICAM‐1 on MMP‐9 expression in monocytes have not been performed yet. In contrast to the mediators mentioned above, a slight, but statistically significant inducing effect of MMP‐9 was accomplished with sICAM‐1. In absolute values, however, this increase was only 3‐fold and did not reach the values observed in the triple and double co‐culture experiments (8‐fold), indicating that additional factors are involved in MMP‐9 regulation. In agreement with these results, a previous study reported that sICAM‐1 may contribute to MMP‐9 expression in MC3T3‐E1 cells (157). In summary, individual T‐cell‐derived soluble mediators appear to be unable to significantly induce MMP‐9 levels suggesting that there is an interaction between several soluble mediators playing a role in this mechanism.

4.7.2 A combination of T‐cell‐derived IL‐16, sICAM‐1 and monocyte‐derived IL‐8 is able to induce MMP‐9 expression in monocytes

To investigate whether a combination of the identified mediators may contribute to the induction of MMP‐9, the effect of IL‐16 and IL‐13, IL‐16 and sICAM‐1, as well as IL‐16 and IL‐8 was monitored in the next experiments. Interestingly, we showed that IL‐16 and IL‐8 as well as IL‐16 and sICAM‐1 significantly induced the MMP‐9 expression approx. 3‐fold, whereas the combination with IL‐13 has no further stimulatory effect. This indicates that IL‐16, sICAM‐1, and/or IL‐8 in combination play an important regulatory role in monocytic MMP‐9 expression.

Moreover, monocytes were stimulated with cytokine cocktails including 3 or more soluble mediators: IL‐16, MIF, and Serpin E1; IL‐16, IL‐13, MIF, and Serpin E1, as well as IL‐16, sICAM‐1, and IL‐8. Regarding this context, a significant increase was observed in all cases, with a 4‐fold induction using IL‐16, MIF, and Serpin E1 or IL‐16, MIF, Serpin E1, and IL‐13. I In the case of IL‐

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16, sICAM‐1, and IL‐8, a 7‐fold MMP‐9 increase was monitored, thus reaching an induction level comparable with the co‐culture experiments. The latter case indicates that especially IL‐16, sICAM‐1, and IL‐8 are able to collectively induce MMP‐9 and that a combinatory mechanism plays a role in inducing MMP‐9 levels. The limited but comparable effects of the other combinations including Serpin E1 and MIF suggest that these factors (either alone or in combination) exert a mild intensifying effect on IL‐16 and might be able to substitute ‐ at least in part ‐ the effects of sICAM‐1 and/or IL‐8. IL‐13, however, appears not to have an important or (or even supporting) influence. Interestingly, only a combination of these 2 factors with monocyte‐derived IL‐8 (or an alternative supporting factor such as Serpin E1) proved to be effective to further intensify MMP‐9 expression. These findings reflect a crucial crosstalk between both monocytes and T‐cells, representing a mechanism in which para‐ (IL‐16, sICAM‐1) and autocrine (IL‐8) mediators are able to induce MMP‐9 expression in response to HMWH.

Our data emphasize the importance of the T‐cell‐derived soluble mediators IL‐16 and sICAM‐1 that are able to stimulate monocytes to raise MMP‐9 levels in the presence of HMWH.

Studies on the effect of cytokine combinations on MMP‐9 expression (esp. by monocytes) have not been performed in detail yet. Therefore, future studies regarding the combinatory effect of T‐cell‐derived soluble mediators appear to be promising. However, further studies concerning the effect of other soluble mediators on the induction of MMP‐9 expression in monocytes or other blood cells, such as neutrophils, platelets, or other macrophage types (M1, M2, or foam cells) appear to be necessary, since these cell types are known to be a prominent source of MMP‐9 (158) and might provide the basis for future studies. Future work using animal models of cardiovascular (esp. stroke) (29) or inflammatory diseases (e.g., rheumatoid arthritis) (38)

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should provide further insights to the in vivo relevance of this mechanism. Moreover, further clinical studies regarding chemokine‐driven monocyte subset recruitment in to brain, heart, or liver with potential implications on MMP‐9 expression in the respective tissues may represent a promising approach for therapeutic intervention.

However, since monocyte‐derived IL‐8 ‐ although not effective alone – has been shown here to be a crucial element by which the monocytes amplify the signals received from T‐cells, the impact of the T‐cell‐derived mediators on monocytic IL‐8 production were assessed in the next step.