Stimulation of monocytes with identified soluble mediators

After the identification of differentially and increasingly expressed T‐cell‐derived soluble mediators, monocytes were stimulated with the identified factors and the MMP‐9 mRNA concentration was measured by qRT‐PCR in order to test whether MMP‐9 induction can be reproduced solely by stimulation with these mediators. Therefore, 2 x 10⁶ THP‐1 cells / well (starved overnight with 1% FCS) were stimulated for 0, 2, 4, 6, and 24h with concentrations of 5ng/ml IL‐16, 5ng/ml sICAM‐1, 5ng/ml IL‐8, 25ng/ml Serpin E‐1, 5ng/ml MIF, 5ng/ml IL‐13 individually or in combination and 25 ng/ml TNF as a control. The concentrations were determined by dose response experiments. Subsequently, RNA isolation, cDNA synthesis, and qRT‐PCR were performed as described before (see 2.4 and 2.5).

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2.9 Cellular function of activated monocytes

The activating capacity of different anticoagulants was analysed by accessing the major cell functions. Therefore, monocytes were stimulated with the supernatant of HMWH‐stimulated T‐

cells. Afterwards, proliferation, apoptosis, and phagocytosis of the stimulated cells were analysed.

2.9.1 Proliferation

To analyse stimulation‐dependent monocytic proliferation, cell counts and ATP measurements were performed using a Neubauer chamber and the Via‐Light Plus kit, respectively. This kit is based on the measurement of ATP that is present in all metabolically active cells. The bioluminescent method utilizes luciferase to catalyze the formation of light from cell derived ATP and was performed according to manufacturer’s instructions. 2 x 10⁶ monocytic cells / well were stimulated for up to 5 days with HMWH‐stimulated T‐cell‐derived supernatant. Afterwards, cell lysis reagent was added for 10 min. To generate luminescent signals, ATP monitoring reagent plus (AMR plus) was added and the light emission was measured using a luminometer.

2.9.2 Phagocytosis

Phagocytosis, which is an early and crucial event in triggering host defense, was measured using the CytoSelect Phagocytosis Assay. This is a high‐throughput method to measure phagocytosis by which phagocytes (i.e., stimulated monocytes) are incubated with pre‐labeled Zymosan particles for 1 h. Non‐phagocytosed Zymosan particles are blocked, cells are permeabilised and

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lysed, and finally, Zymosan substrate is added and the amount of phagocytosed Zymosan particles is measured in the respective extracts on a plate reader at 405nm.

2.9.3 Apoptosis

To assess the degree of apoptosis in stimulated cells, terminal deoxy nucleotidyl transferased dUTP nick end labeling (TUNEL) which is an established method for detecting DNA fragments, was used. The TACS TdT kit contains a highly purified form of the TdT enzyme for the enzymatic incorporation of biotinylated nucleotides. Labelling was achieved using Biotin and horseradish peroxidase‐coupled Streptavidine. Determination of TdT labeling was performed by determination of the metabolization of the colorimetric substrates diaminobenzidine (DAB) or TACS Blue Label. Therefore, 2 x 10⁶monocytic THP‐1 cells / wellwere stimulated with HMWH‐

stimulated T‐cell‐derived supernatant. Cells were harvested by centrifugation at 500g for 5 minutes, media was discarded and cells were re‐suspended and fixed in 3.7% buffered formaldehyde (1 ml / 1 × 10⁶ cells). Afterwards, the suspension was centrifuged at 500 g, the fixative was discarded, and the cell pellet was re‐suspended in 80% ethanol (1 ml / 1 × 10⁶ cells).

1 x 10⁵ cells were spotted on a clean glass microscope slide and dried for 20 minutes at 45 C.

Slides were immersed in 70% ethanol for 10 minutes then air dried at room temperature for 2h.

A rehydration step was done in a succession of 100%, 95%, and 70% ethanol. Then, the slide was immersed in 1 x PBS. The in situ labelling procedure was done by transferring the sample in 1 x PBS for 10 minutes at RT after rehydration in ethanol. Proteinase K solution was added and incubated for 15‐30 minutes at 37 °C, then the samples were washed with deionized water for 2 minutes. Afterwards, slides were immersed in quenching solution (5 min at RT), and washed with 1% PBS (1 min RT). Subsequently, slides were immersed in 1 x TdT labelling buffer for 5

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minutes. 50 µl of labelling reaction was added, mixed, and incubated at 37 °C for 1h.

Subsequently, 1 x TdT stop buffer was added for 5 minutes, and samples were washed 2 x in PBS for 2 minutes. Finally, samples were immersed in TACS‐Blue label solution for 2‐7 minutes, washed several times in deionized water for 2 minutes each, and processed for counterstaining using nuclear fast red according to the manufacture’s protocol.

2.10 Statistical analysis

Statistical analysis was performed using unpaired t‐test with Welch’s correction and Mann–

Whitney U‐test for significance. P values <0.05 (∗), and <0.01 (**), were considered significant.

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3 Results

3.1 Direct stimulation of different cell types with anticoagulants

To elucidate which cell types within the blood may be responsible for the increased MMP‐9 mRNA and protein expression following blood sampling reported in the literature, especially in heparin‐treated samples, the major blood cell types(monocytes, T‐cells, B‐cells) were analyzed.

Therefore, the cell lines THP‐1 (monocytes), Jurkat (T‐cells), and HT (B‐cells), respectively, have been used. In these experiments starved cells were stimulated up to 24h with EDTA, heparin (i.e.

HMWH), or citrate. Unexpectedly, neither the stimulation with EDTA or citrate nor HMWH had a significant stimulatory effect on the MMP‐9 mRNA expression in monocytes (Fig.3.1), T‐ cells (Fig.3.2), or B‐cells cells (Fig.3.3) at different time points.

These results indicate that direct stimulation of monocytes, T‐cells, or B‐cells with the respective anticoagulants does not induce MMP‐9 expression significantly. This suggests that an indirect mechanism might play an important role for the regulation of MMP‐9 expression in response to the reported anticoagulants.

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3.2 Significant Induction of MMP‐9 expression by HMWH in a co‐culture including THP‐1, Jurkat, and HT 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 (i.e., THP‐1, Jurkat, and HT cells) was used.

Following starvation of the cells overnight, the cell mixture was stimulated with the respective anticoagulants and incubated for 0h, 2h, 4h, 6h, and 24h. Finally, the intracellular MMP‐9 mRNA expression as well as the amount of secreted MMP‐9 protein in the supernatant was determined.

The analysis of MMP‐9 mRNA expression in the co‐culture of THP‐1, Jurkat, and HT cells revealed that MMP‐9 expression increased significantly after addition of HMWH (Fig. 3.4 A). In contrast, stimulation with other anticoagulants such as EDTA (Fig. 3.4 B) or citrate (Fig. 3.4 C) had no MMP‐9‐inducing effect in this co‐culture model. Equivalently the stimulation of a mixture of THP‐1, Jurkat, and HT with HMWH‐treated Jurkat supernatant (Fig.3.5 A), but not HMWH‐treated HT supernatant (Fig. 3.5 B), increased the amount of MMP‐9 levels significantly over time, whereas the stimulation of a mixture of THP‐1, Jurkat, and HT with EDTA‐treated Jurkat‐supernatant/‐HT‐supernatant (Fig. 3.5 C, D) or citrate‐treated Jurkat‐supernatant/‐HT‐

supernatant (Fig. 3.5 E, F) did not result in any induction effect on MMP‐9 levels.

These results indicate that MMP‐9 expression in (at least) one of the cell types included in this experiment depends on an interaction with another involved cell type in response to HMWH, e.g., via direct cell‐to‐cell interaction or the stimulation with a soluble mediator. Therefore,

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3.4 Significant induction of MMP‐9 expression in THP‐1 cells in response to culture supernatant derived from HMWH‐treated Jurkat cells

With respect to the known ability of monocytes/macrophages to produce large amounts of MMP‐9 during tissue invasion (120), it was reasonable to speculate that during the interaction of 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.

Therefore, starved monocytes were stimulated in the next step with the supernatant of HMWH‐treated Jurkat cells for 0, 2, 4, 6, and 24h (Fig. 3.9). As a control, THP‐1 cells were also stimulated with the supernatant of HMWH‐treated HT cells (Fig. 3.9) as well as EDTA‐ or citrate‐

treated Jurkat and HT cells (data not shown). Comparable to the results obtained in the experiments in which double co‐cultures were performed, no effect on MMP‐9 mRNA levels could be observed using supernatants from HMWH‐treated HT cells (Fig. 3.9) or EDTA‐ or citrate‐treated Jurkat and HT cells (data not shown). The analysis of MMP‐9 expression in monocytic cells in response to stimulation with medium derived from HMWH‐stimulated Jurkat cells however, resulted in a significant induction of MMP‐9 after 6 and 24 h (Fig. 3.9).

These results support the suggestion that the monocytes are the main producers of MMP‐9 and that the supernatant of HMWH‐stimulated T‐cells is able to significantly induce MMP‐9 expression in monocytes.

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MMP-9 mRNA expression ¹⁵ Jurkat cells

Fig.3.9: Induction of MMP‐9 expression in THP‐1 cells by incubation with supernatant from HMWH‐stimulated Jurkat cells. 2 x 10⁶THP‐1 cells / well were starved overnight and subsequently stimulated with the supernatant of Jurkat or HT cells (treated with HMWH for 24h) for the indicated time points. MMP‐9 mRNA expression was determined using qRT‐PCR (housekeeping gene: GAPDH); mean ± SEM, n = 3 (measured in duplicates). * p ≤ 0.05;

*** p ≤ 0.005.

3.5 Significant induction of MMP‐9 expression in THP‐1 cells by incubation with supernatant from Jurkat cells stimulated with human plasma derived from HMWH‐containing monovettes

The basic idea of this experiment was to see whether it is also possible to increase the MMP‐9 production in monocytes under clinical circumstances, i.e., by addition of supernatant from T‐

cells which were stimulated with human plasma from heparin‐containing monovettes. Therefore, following starvation overnight, monocytes were incubated up to 24h with the supernatant from T‐cells or B‐cells which have been stimulated for 24h with heparin‐plasma derived from normal donors.

The results demonstrated in Fig. 3.10 indicate that even stimulation with the supernatant of Jurkat cells which were treated with human heparin‐plasma is sufficient to induce MMP‐9 expression in THP‐1 cells. In general, this induction started at 2h, reached a significant level after 4h and showed an increase up to 2h. As expected, this significant increase was not seen in

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a control experiment in which monocytes were incubated with the supernatant of human heparin plasma‐stimulated B‐cells (Fig. 3.10).

MMP-9 mRNA expression 10 Jurkat cells 8

HT cells

(fold induction) 6

4 2

0

0 2 4 6 24 (h)

Fig. 3.10: Induction of MMP‐9 expression in THP‐1 cells by incubation with supernatant from Jurkat cells stimulated with human heparin‐plasma. 2 x 10⁶ THP‐1 cells / well were starved overnight. Afterwards, THP‐1 cells were stimulated with the supernatant of heparin plasma‐treated Jurkat or HT cells at the indicated time points. MMP‐9 mRNA expression was determined using qRT‐PCR (housekeeping gene: GAPDH); mean ± SEM, n = 3 (measured in duplicates). * p ≤ 0.05; ** p ≤ 0.01.

3.6 High molecular weight heparin versus Low molecular weight heparin

To further investigate the stimulatory effect of other types of heparin on MMP‐9 expression, the effect of two different types of LMWH (i.e., Clexane and Fragmin) was analyzed. Regarding this context, THP‐1 cells were stimulated in a control experiment directly with Clexane or Fragmin.

Moreover, the effect of LMWH on the MMP‐9 mRNA experiment in cultured cell mixtures, esp.

THP‐1 and Jurkat cells, was assessed since our previous data showed that the interaction of T‐cells with monocytic cells plays an important role in the induction of MMP‐9 levels. As demonstrated in Fig. 3.11, stimulating THP‐1 cells with HMWH or LMWH (i.e., Clexane

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Fig. 3.12: LMWH (Clexane or Fragmin) has no significant MMP‐9–inducing effect in a mixture of THP‐1 and Jurkat cells. 1 x 10⁶THP‐1 and Jurkat cells (i.e., 2 × 10⁶ cells / well in total) were starved overnight. Afterwards, the mixture was stimulated with HMWH or Clexane (A) or with or without Fragmin (B) for 24h. Then, MMP‐9 mRNA expression was determined using qRT‐PCR (housekeeping gene: GAPDH); mean ± SD, n = 3 (measured in duplicates). * p ≤ 0.05.

3.7 Identification of T‐cell derived soluble mediators which activate MMP‐9 expression in THP‐1 cells

The basic idea of this experiment was to identify the (combination of) T‐cell‐derived mediator(s) which is able to increase the MMP‐9 production in monocytes. Therefore, following starvation overnight, individual cell lines as well as double and triple cell line mixtures were cultivated and directly stimulated with HMWH. Cell culture supernatants were then profiled for the expression of multiple cytokines and chemokines. As demonstrated in Fig. 3.13A, HMWH‐stimulated THP‐1 released three types of mediators, macrophage migration inhibitory factor (MIF), IL‐1‐ra, and Rantes. Jurkat released IL‐13, IL‐16, MIF, sICAM1, and Serpin E1 (Fig. 3.13B), whereas HT cells released IL‐13, MIF, TNF‐α, SICAM‐1, and Serpin E1 (Fig. 3.13C). Additionally, in a mixture of HMWH‐stimulated TH P‐1 and Jurkat cells, IL‐1‐ra, IL‐8, IL‐16, IL‐13, Serpin E1, MIF, SICAM‐1, and Rantes could be detected (Fig. 3.13D). In a triple cell mixture consisting of THP‐1, Jurkat, and HT cells, IL‐1‐ra, IL‐8, IL‐13, IL‐16, IL1‐ra, SICAM‐1, MIF, Rantes, and Serpin E1 were identified (Fig.

3.13E). This suggests that the T‐cell‐specific soluble mediators (e.g., IL‐16, sICAM‐1, or SerpinE‐1) individually or in a combination are able to stimulate and induce MMP‐9 secretion from monocytes. A direct MMP‐9‐inducinginfluence of the monocyte‐derived factors (either individually or in combination) IL‐1‐ra, MIF, or Rantes was not supposed, since monocyte do not produce increased MMP‐9 levels under this condition. Furthermore, an influence of B‐

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cell‐derived factors (MIF –although in higher concentration ‐, IL‐13, sICAM‐1, Serpin E1, TNFα), again either alone or in that combination, could not be assumed due to the same reasons, on MMP‐9 expression.Thus, a potential influence was supposed for IL‐16, since it was produced from T‐cells. In addition, monocyte‐derived mediators, esp. IL‐8, which are expressed in the presence of T‐cells, may contribute to this effect in an autocrine manner.

Fig. 3.13: Identification of cytokines and chemokines expressed by THP‐1, Jurkat, and HT cells in response to HMWH. Starved cells were incubated as single cell lines (A‐C) or double (D) and triple (E) co‐culture approaches and stimulated with HMWH for 24h. Cytokine/chemokine expression was analyzed using theProteome Profiler XL array kit. One representative experiment of n = 3 is shown.

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3.8 LMWH vs. HMWH: identified soluble mediators

To further to investigate whether LMWH has the same effect as HMWH on stimulating T‐cells leading to the release of soluble mediators and the subsequent induction of MMP‐9 by monocytes, the secretion of these mediators in response to stimulation with LMWH (here:

Clexane) was analyzed in THP‐1 and Jurkat cells. As demonstrated in Fig. 3.14, no secreted cytokines/chemokines could be detected in individual THP‐1 cultures as well as double co‐

cultures of THP‐1 and Jurkat cells following stimulation with LMWH for 24h. This supports the suggestion, that only HMWH is able to induce the production of the identified soluble mediators from Jurkat cells and subsequent MMP‐9 production in monocytes.

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3.10 Identification of Serpin E1 and/or MIF as alternative supporting factor for MMP‐9 induction

Since the presence of further factors ‐ either constitutively expressed by monocytes or T‐cells or expressed by B‐cells (which may not play a role alone, i.e., in the absence of T‐cell‐derived factors) ‐ may be of importance, the role of the mediators which at least slightly (although not significantly) enhanced the MMP‐9 expression by monocytes was analyzed to elucidate whether they may act as alternative supporting factor for MMP‐9 induction (comparable with the role of IL‐8). In this context, we stimulated THP‐1 cells using a combination consisting of IL‐ 16, MIF, and Serpin E1 (Fig. 3.17B) or IL‐16, IL‐13 MIF, and Serpin E1 (Fig. 3.17C) resulting in a slight significant induction on MMP‐9 expression (3‐4‐fold) even in the absence of sICAM‐1 and IL‐8, but only when Serpin E1 and MIF were present. The presence of IL‐13, however, has no further enhancing effect on MMP‐9 (see also Fig. 3.15). With respect to the results of the experiment in which THP‐1 were stimulated with IL‐16, IL‐8, and sICAM‐1 (significant 7‐8‐fold induction, this supports our suggestion that IL‐16, sICAM‐1, and IL‐8 play an essential role on MMP‐9 expression and that Serpin E1 and/or MIF might play a role as an alternative supporting factor for MMP‐9 induction.

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3.11 Cellular functions of activated 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.

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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,

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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

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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.

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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

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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,

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,

Im Dokument Cellular and molecular characterization of the effects of different anticoagulants on the regulation of matrix metalloproteinase 9 (Seite 56-0)