Cellular functions of activat ed THP‐1

2. Materials & Methods

3.11 Cellular functions of activat ed THP‐1

To assess different cell functions of monocytic cells under conditions also inducing MMP‐9 expression, different cell functions of stimulated monocytes were analysed such as proliferation, phagocytosis, and apoptosis.

3.11.1 Proliferation

First, to assess the proliferation of monocytes stimulated with T‐cell supernatant, we performed ATP measurements and cell counts using Via Light Plus kit and the Neubauer chamber, respectively. Monocytes stimulated with either HMWH‐treated T‐cell supernatant (treated monocytes) or with RPMI (un‐treated monocytes) were incubated up to 5 days; at each day, proliferation analysis was performed. As shown in Fig. 3.20A, HMWH‐stimulated T‐cell supernatant was able to induce the proliferation of these treated monocytes in comparison to untreated monocytes at different time points indicating that HMWH‐treated T‐cell supernatant has an activating effect on monocytic cells and is able to modulate monocytic cell function by enhancing THP‐1 proliferation over time. As a confirmatory approach, the number of monocytes was counted using Neubauer chamber yielding equivalent results (Fig 3.20 B). These results indicate that HMWH‐stimulated T‐cell supernatant ‐ including the secreted factors identified previously (see 3.7) ‐ is able to significantly induce a sustained proliferation of monocytic cells.

OD 405 nm

Fig. 3.21: THP‐1 phagocytosis is enhanced by HMWH‐treated Jurkat supernatant. 2 x 10⁶THP‐1 cells / well were starved overnight and then treated for 24h with the supernatant of Jurkat cells (treated with HMWH for 24h).

Afterwards, monocytic cells were incubated with zymosan particles for 30 minutes and the amount of engulfed particles was determined. Mean ± SD, n = 3 (measured in duplicates). *** p ≤ 0.005.

3.11.3 Apoptosis

Moreover, the degree of apoptosis in monocytes under these experimental conditions was assessed. Therefore, THP‐1 cells/well stimulated with HMWH‐stimulated T‐cell‐derived supernatant. After multiple fixation and dehydration steps, labeling was achieved using Biotin and horseradish peroxidase‐coupled Streptavidin. TdT labeling was performed by TACS Blue Label. Cells were harvested by As represented in Fig. 3.22, the degree of apoptosis was also enhanced in THP‐1 cells by HMWH‐treated Jurkat supernatant (i.e. 80% of apoptotic cells with respect to 100% of total cells),(Fig. 3.22 B grey, D blue), in contrast to the control (RPMI‐treated monocytes; Fig. 3.22 A grey, C blue), in which apoptosis was less prominent (i.e. 5% of apoptotic cells with respect to 100% of total cells). This indicates that the enhancement of monocytic functions ‐ MMP‐9 production (see 3.4), cytokine secretion (see 3.7), proliferation,

and phagocytosis ‐ in response to T‐cell‐derived factors following HMWH‐treatment is accompanied by an increased cell death of activated monocytes.

Fig.3.22: Apoptosis is enhanced in THP‐1 by HMWH‐treated Jurkat starved overnight and then treated with the supernatant of Jurkat

supernatant.2 x 10⁶THP‐1 cells / well were cells (treated with HMWH for 24h). After

multiple fixation and dehydration steps, labelling was achieved using Biotin and horseradish peroxidase‐coupled Streptavidin. TdT labelling was performed by TACS Blue Label. In each case, one representative experiment of n = 3 is shown.

4. Discussion 4.1 Topic overview

Matrix metalloproteinases (MMPs) represent a major group of enzymes that participate in the degradation of extracellular matrix (ECM) components and basement membranes, normal tissue remodeling, wound healing, inflammatory cell migration, and the processing and activation or inactivation of soluble factors (121). MMP‐9 has become a subject of growing interest in human pathology, especially in pathophysiological process such as inflammation, arthritis, cardiovascular diseases, cancer, and neurological diseases (122). It has been reported that blood sampling with different anticoagulants alters the expression of MMPs and tissue inhibitors of metalloproteinases (TIMPs) differentially thus influencing the concentration and the diagnostic validity of MMP‐9 (123). In this study, we aimed to evaluate the influence of direct and indirect effects of different anticoagulants on the regulation of MMP‐9, since it has been shown that esp.

MMP‐9 is an important regulator of many pathogenic and non‐ pathogenic processes and that changes in its levels are also reflected in body fluids, esp. blood (124).

With the knowledge that (i) platelets and blood leukocytes (i.e., eosinophils, neutrophils, lymphocytes, and monocytes) contain high amounts of MMP‐9, (ii) MMP‐9 activation by plasminogen activator maintains its release into the blood following its activation via plasmin‐

related mechanisms, and (iii) MMP‐9 has a role in the activation and inactivation of some immunological function (i.e., leukocyte migration, modulating chemokine, and cytokine activity) (125, 126), this study was designed to identify the involved cell types and the influence of blood sampling with different anticoagulants on MMP‐9 expression. The focus of the analysis was set

on the functional assessment of the cells in the presence of the anticoagulants since it is still unclear which processes lead to the increased MMP‐9 production and which may lead to contradictory findings between different studies. Furthermore, the effect of anticoagulants on blood cell types, the molecular and cellular mechanism that lead to their activation/interaction, and the production of soluble mediators in the corresponding cells were studied. Thus, effects and mechanisms should be identified that might play role in future therapeutic targets for certain cardiovascular diseases (esp. stroke).

To assess the impact of the respective substances on the suitability of MMP‐9 as a biomarker, the essential impact of blood sampling was studied, so to measure the true expression of MMP‐

9 in blood which is still a poorly considered process, and which may lead to some technical pitfalls and misinterpretation, esp. for investigators studying the role of MMP‐9 in pathophysiology or clinicians measuring blood MMP‐9 levels as a biomarker (127). In addition to that, the assessment of ECM remodeling in tissue using MMP‐9 serum or plasma levels is still characterized by controversial results due to the use of different anticoagulants during sample collections (e.g., EDTA, HMWH, LMWH, and citrate) (128).

Since it has been reported that cytokine production by different cell types (esp. monocytes) is essential for the upregulation of MMP‐9 (129), the soluble mediators should be identified which are produced by monocytes, T‐cells, or B‐cells in response to the respective anticoagulants (e.g., high or low molecular weight heparin) and which can induce MMP‐9 expression and might further regulate cellular functions. .

To best of my knowledge, this is the first study elucidating the influence of different types of anticoagulants on MMP‐9 expression by major cell types of the blood and characterizing the molecular mechanisms regulating these effects.

4.2 Direct stimulation with anticoagulants has no influence on MMP‐9 expression of monocytes, T‐cells, and B‐cells

To elucidate which cell types within the blood may be responsible for the increased MMP‐9 mRNA and protein expression reported in the literature (29), (130), (131), especially in anticoagulant‐treated samples, the major cell types in the blood (monocytes, T‐cells, and B‐ cells) were analyzed. In these experiments, the cell lines THP‐1 (monocytes), Jurkat (T‐cells), and HT (B‐cells), respectively, were used. The cells were starved for 24h and then stimulated up to 24h with EDTA, HMWH, LMWH, or citrate. Subsequently, the MMP‐9 mRNA expression was determined. The principle finding was that direct stimulation with the anticoagulants used had no influence on MMP‐9 expression by the corresponding cell types suggesting that an indirect mechanism (e.g., an interaction of several cell types) might play a role and exert the influence on MMP‐9 expression. Although these initial findings indicate that direct stimulation of single cell types with anticoagulants has no clear impact on MMP‐9 production, the use of other anticoagulant concentrations or alternative time courses could be helpful in future studies to confirm the data in this context. Furthermore, we tried to use in this experiment the most suitable cell lines available for our study representing the major blood cell types. However, further studies may focus on these or other cell types in blood as well as alternative cell lines. For instance, other studies have shown that direct or indirect stimulation with anticoagulants

(esp. heparin) is sufficient to significantly induce and increase MMP‐9 expression levels in leukocytes and platelets and change their gelatinolytic activity (96). Moreover, since there is an association between MMP‐9 polymorphisms and certain diseases (132), it would be helpful to use cell lines carrying those mutations/polymorphisms for further studies.

Concerning heparin, the presence of HMWH in test tube does not increase the release of MMP‐

9 from monocyte directly which is also revealed in another published study (133). This suggests that an indirect mechanism might play a role during this process. Other anticoagulants (i.e., EDTA, citrate, LMWH) have no clear impact on MMP‐9 expression. This finding is difficult to interpret, but it could be ascribed to the zinc‐chelating properties of EDTA, the non‐induction effect of citrate corresponding the alteration in MMP‐9 activity (134), and the lower affinity of LMWH to bind to proteins, ECs, and macrophages (135). Further relevant data have shown that the ideal anticoagulant mediates a positive impact on the determination of MMP‐9 levels in blood, i.e., by stabilizing the protein concentration in the time interval between blood sampling and analysis (114).

4.3 Significant Induction of MMP‐9 expression by heparin in a co‐culture including monocytes, T‐cells, and B‐cells

To determine whether the MMP‐9 expression is influenced by an interaction of different cell types, a mixture of monocytes, T‐cells, and B‐cells was used. Following starvation of the respective cell lines overnight, they were stimulated with anticoagulants (EDTA, citrate, HMWH, LMWH) up to 24h and the expression of both MMP‐9 mRNA and protein was assessed. We observed a significant, approx. 7‐fold increase in MMP‐9 mRNA expression in the co‐culture of

monocytes, T‐cells, and B‐cells after addition of HMWH in contrast to the stimulation with other anti‐coagulants, i.e., citrate, EDTA, or LMWH which had no effect on MMP‐9 mRNA levels.

Consistent with these observations, the amount of secreted MMP‐9 protein increased significantly over time in the supernatant of HMWH‐stimulated co‐cultured cells. Therefore, the conclusion was drawn that an interaction of at least two cell types is responsible for the increased MMP‐9 expression in blood samples with respect to HMWH stimulation, and the following experiments were designed and performed to further investigate this heparin‐driven interaction (see below). Other anticoagulants, however, appear to execute a lesser impact on MMP‐9 production/secretion in the cell mixture. This differential effect might be ascribed to the high negative charge density of HMWH since heparin is a highly sulfated glycosaminoglycan known to bind to the hemopexin domain (C‐terminus) of MMPs and often found associated with MMPs on the cell surface (117). In contrast, direct or indirect stimulation of cells (i.e., monocytes, T‐cells, and B‐cells) with EDTA or citrate did not show any effect on MMP‐9 at different time points (0 up to 24h), when compared with the significant induction on MMP‐9 levels was shown by an indirect stimulation (i.e., via T‐cells) of monocytes with HMWH up to 24h.

Further experiments using other cell lines and different time points ‐ which may represent the effect of HMWH on the cells in greater detail, e.g., between 6 and 72h or more ‐ would be beneficial to better access the course of MMP‐9 induction.

Another relevant observation obtained from the literature is that after blood sampling, even lower amounts of heparin increased the amount and activity of circulating MMP‐9 (133). This could further raise questions regarding the usage of HMWH in clinical and diagnostic research, esp. during the analytical phase (after blood separation), by which the concentration of heparin

might play a significant effect on MMP‐9 expression. This should be further analyzed in dose response experiments. Similar to our findings, some studies showed that blood sampling with heparin alters the physiological processes in the blood, e.g., by influencing T‐cell proliferation (136), anti‐thrombin III activity, protein interactions, and the gelatinolytic activity of the MMP‐9 (13). In consequence, HMWH should be cautiously used in blood collection procedures due to a possible fraud in the MMP‐9 levels effectively present in the respective patient/blood donor, also questioning the suitability of MMP‐9 as a biomarker. Moreover, systemic treatment with HMWH in the clinic may also be evaluated critically with respect to the present results, since a general induction of MMP‐9 may lead to severe side‐effects such as an increased tendency towards bleeding or an aggravation of stroke (137). However, further studies revealing these effects might further help to understand the impact of heparin on MMP‐9 expression.

4.4 Significant induction of MMP‐9 expression by heparin in the co‐culture of monocytes and T‐cells

To investigate which of these 3 cell types interact and exert an effect on MMP‐9 levels in blood samples, it was reasonable to analyze MMP‐9 expression in co‐cultures of 2 cell types. Therefore, mixture experiments were performed including monocytes/T‐cells, monocytes/B‐ cells, and T‐cells/B‐cells. In addition to HMWH, these cell mixtures were also treated with citrate and EDTA as a control. Cells were starved overnight and then stimulated with the respective anticoagulants up to 24h. Finally, MMP‐9 mRNA expression and the amount of secreted MMP‐9 proteins were determined. Interestingly, a significant, approx. 7‐fold increase in both MMP‐9 mRNA and protein expression by HMWH was observed in a mixture of monocytes and T‐cells, a result comparable with the data obtained from the triple co‐culture. In

contrast, mixtures of monocytes and B‐cells or T‐cells and B‐cells (as well as stimulation with anticoagulants other than heparin) had no significant effect on MMP‐9 amounts. This indicates that a specific interaction between monocytes and T‐cells is responsible for the significant induction of MMP‐9 expression and that MMP‐9 expression in one of these cell types depends on the HMWH‐dependent stimulation by the other cell type, either mediated directly, i.e., by cell‐to‐cell‐interactions, or indirectly with a soluble mediator.

Since some published studies have shown that activated T‐cells are able to activate monocytes and to induce their MMP‐9 secretion (138), it seemed likely to speculated that T‐cells might represent the HMWH‐governed cell type which stimulates MMP‐9 expression of monocytes. This effect might also be further influenced by auto‐regulatory events (139, 140), since it has been shown that different stimuli activate monocytes, enhance their pro‐coagulant activity, and induce significant increases in MMP‐9 and TIMP‐1 secretion following incubation with anti‐CD3 antibody‐stimulated T‐cells (139).

In the next experimental steps it was assessed whether the heparin‐driven activation monocytes by T‐cells is mediated independently of direct cell‐to‐cell contacts but via the production of monocyte‐activating soluble mediators resulting in a subsequently increased monocytic MMP‐9 production.

4.5 Significant induction of MMP‐9 expression in monocytes in response to culture supernatant derived from heparin‐treated T‐ cells or human heparin plasma‐treated T‐cells

To validate the speculation that during the interaction between monocytes and T‐cells, the T‐

cells are responsible for the secretion of a soluble monocyte‐stimulating factor to which the

monocytes react with increased MMP‐9 expression, starved monocytes were stimulated up to 24h with the supernatant of HMWH‐treated T‐cells (and B‐cells as well as EDTA‐ or citrate‐

treated T‐/B‐cells as a control). Indeed, the analysis of MMP‐9 expression in response to stimulation with medium derived from HMWH‐stimulated T‐cells revealed that a significant increase in MMP‐9 expression was induced in monocytic cells after 6 and 24 h. In contrast, no effect on MMP‐9 mRNA levels could be observed using supernatants from HMWH‐treated B‐

cells, nor EDTA or citrate‐treated T‐ and B‐cells. In an alternative approach and to test whether the amount/the concentration of HMWH in blood tubes is sufficient to induce cytokine production by T‐cells and MMP‐9 production of monocytes, T‐cells were stimulated with heparin‐plasma derived from human donors before the supernatant was transferred to monocytes. Even under this modified condition equivalent results were obtained. This present finding supports the suggestion that the monocytes are the main producers of MMP‐9, and that the supernatant of heparin‐stimulated T‐cells contains soluble mediators which are able to significantly induce MMP‐9 expression in monocytes. These findings are in good agreement with a previous study showing that monocytes are the main producers of MMP‐9 and a predominant source of MMP‐9 in the blood (29). Furthermore, blood monocytes and tissue macrophages secrete MMP‐9 in large quantities (141) and the regulation of MMP‐9 expression is influenced by an interaction of these cells and other cell types as well as by cellular interaction with ECM components (142). Future in vivo studies, investigating the role of anticoagulants (esp. HMWH) for the secretion of monocyte‐activating cytokines by T‐cells (i.e., the indirect effect of HMWH on MMP‐9 induction) and the modulatory effects exerted by different mediators and receptors on these cells, could reveal the mechanism(s) of interaction

between T‐cells and monocytes (and potentially further cell types) in the presence of HMWH.

Moreover, they may provide new and attractive targets to pharmacologically manipulate the expression of MMP‐9 observed in various diseases since the crosstalk between heparin, T‐cells, and monocytes (including its functional properties and its potential modulation of diverse cellular processes) is still not well defined (143).

4.6 HMWH versus LMWH

To investigate the stimulatory effect of other types of heparin on MMP‐9 expression, the effect of two different variants of LMWH (i.e., Clexane and Fragmin) were also analyzed. In a clinical study it was indicated before that LMWH possess both anticoagulant and non‐anticoagulant functions and that the anticoagulant activity of LMWHs down‐regulates MMP‐9 (144). Thus, it was reasonable to study the effect of LMWH on MMP‐9 induction effect in monocytes. Regarding this context, THP‐1 cells were stimulated in an initial control experiment directly with Clexane or Fragmin. In a second approach, the effect of LMWH on the MMP‐9 mRNA was characterized in cell mixtures, esp. a THP‐1/Jurkat mixture, since our previous data showed that T‐ and monocytic cells play an important role in the induction of MMP‐9. Stimulating THP‐1 cells directly with HMWH or LMWH (i.e., Clexane or Fragmin) showed no effect on MMP‐9 induction, which was in good agreement to the results of the direct HMWH stimulation of THP‐ 1 cells performed in the initial phase of the study. These results indicate that direct stimulating of the monocytes with LMWH ‐ like HMWH ‐ has no significant effect on MMP‐9 expression. In agreement with our findings, it was shown in a recent study that LMWH (combined with doxorubicin) down‐regulates MMP‐9 expression in Hepatocellular cancer HepG2 cells (145). However, in the double cell type mixture experiments, surprisingly, neither Clexane nor

Fragmin showed a stimulatory effect on MMP‐9 levels, in contrast to the significant effect of HMWH after 24h. These results suggest that induction of MMP‐9 expression also depends on the type of heparin used. Studies on Clexane or Fragmin stimulated monocytes and T‐cell have not been investigated yet.

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

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

Im Dokument Cellular and molecular characterization of the effects of different anticoagulants on the regulation of matrix metalloproteinase 9 (Seite 85-0)

Cellular functions of activat ed THP‐1

2. Materials &amp; Methods

3.11 Cellular functions of activat ed THP‐1

2. Materials & Methods