Abstract
Phagocytosis and secretion of interleukins and growth factors put the macrophage in the centre of the wound healing process. For the last four years over 400 human ulcers have been treated in elderly and paraplegic patients by local application of monocytes prepared from a blood unit, in a unique, closed, sterile system. The process of preparation includes a step of hypo-osmotic shock, which induces monocyte/macrophage activation. This is different from any other known method of activation. In the present study we evaluated the efficacy of the hypo-osmotic shock. We found enhanced levels of IL-1 (P = 0·004) and IL-6 (P = 0·001) in the incubation medium (100% autologous serum) of the activated cells, as compared with controls, prepared in the same system. The IL-1 reached a plateau after 6 and 12 h incubation at 37°C, in both experimental and control incubation medium. The level of IL-6 was further elevated after 12 and 24 h incubation in experimental and control incubation mediums (P = 0·001). The phagocytosis of fluorescent beads was markedly enhanced after hypo-osmotic shock (P = 0·005).
The osmotic shock induced macrophages were compared to those stimulated with LPS, and osmotic shock was proved to be at least as efficient method of stimulation as LPS.
Keywords: monocytes, macrophages, activation, hypo-osmotic shock
INTRODUCTION
Macrophages evolve from monocytes and constitute 1–6% of the nucleated peripheral blood cells. The monocytes egress the blood stream into tissues and differentiate into different kinds of macrophages, depending on the nature of the signals in the local microenvironment [1,2].
Phagocytic abilities and diverse secretory potential put the macrophages in the centre of the wound healing process [1,3–5]. This biologically complex sequence of events, involves cellular and molecular processes, such as cell migration, inflammation, angiogenesis, collagen synthesis and deposition, and reepithelialization [5,6]. The macrophages have key functions in almost every stage of the process. Upon initiation of the inflammation stage, the macrophage secret IL-1 that induces the rapid recruitment of inflammation cells from the circulation into the wound [7–9]. As phagocytes, the macrophages help in digestion of bacteria and debridement [10]. In the later stages of the healing process they secret IL-6, which is believed to influence endothelial cell proliferation, and the initiation of angiogenesis [11–13]. The macrophage serves as the coordinator of this proliferative process, by producing growth factors such as PDFG BB, TGF-α, TGF-β1, VEGF, FGF, EGF and IGF-1.
In summary the macrophage produces practically all the interleukins and growth factors that participate in the wound healing process [8–14].
The application of rabbit anti mouse macrophage serum to wounds of young mice delayed wound healing, reducing it to the rate found in old mice [15]. Moreover, wound repair was enhanced in old mice by local injection of monocytes/macrophages derived from young mice [16]. After developing a method for preparation of human activated monocytes/macrophages from a blood unit in a closed sterile system, these cells were successfully used for treatment of human decubital ulcers [17,18].
The process of macrophage preparation includes a step of hypo-osmotic shock, which induces monocyte/macrophage activation, that is different from any other known method. In most methods various substances are introduced to the cell culture (LPS, INF-γ, etc.) [19–22]. Another known method is using hyperhypo-osmotic shock to induce cytokine secretion by monocytes/macrophage [23–27]. In the present study we evaluated the efficacy of the hypo-osmotic shock as a trigger for monocytes/macrophage activation.
MATERIALS AND METHODS
Preparation of monocytes
Hypohypo-osmotic shock stimulation
Monocytes were prepared in a closed sterile system as previously described [17] (patent pending No. PCT/US95/08351.D.Danon) with some modifications. Shortly, a whole blood unit collected into triple blood bag system was separated into packed red blood cells, white blood cells (buffy coat) and plasma. The bags containing the plasma and buffy coat were connected, using a sterile connecting device (Terumo Sterile Tubing Welder SC-201 A, Tokyo, Japan), to the macrophage preparation system (Fig. 1). Transfer of 60 ml CaCl2 (80 mm) to the plasma bag induced a coagulation that was completed after about two hours of incubation at 37°C. The obtained autologous serum served as growth medium to the monocytes.
Fig. 1.
A closed sterile system for preparation of monocytes from a blood unit. (1) 500 ml bag with 200 ml distilled water; (2) 50 ml bag with 20 ml NaCl 9%; (3) 50 ml bag with 20 ml CaCl2 80 mm; (4) 150 ml bag with 110 ml NaCl 0·9%; (5) 500 ml bag with 250 ml air, for the hypo-osmotic shock treated cells; (5′) 500 ml bag with 250 ml air, for the control cells; ABC-triple blood bags collection set.
As there is a large variation between the blood units in several parameters, we have designed the control sample to be prepared from the same unit as the experimental. A second culture bag was therefore added to the preparation system. The buffy coat was equally divided, by weight, into two culture bags, of which only one was submitted to hypo-osmotic shock (Fig. 1). Transfer of 100 ml distilled water into one bag caused hypo-osmotic shock. After 45 s 10 ml of 10 fold concentrated saline were added to re-establish isotonicity. In parallel 110 ml of saline were transferred into the control culture bag. The set was centrifuged at 600 × g for 10 min. The supernatant of both bags was transferred to the previously containing distilled water bag (‘sink bag’). Equal volumes of autologous serum (30 ml) were transferred into each bag. The bags, containing equal amount of white blood cells and serum, were incubated at 37°C for one hour, after which the supernatant of the culture bags was transferred to the ‘sink bag’. The bags were rinsed with fresh serum, which was also transferred into the ‘sink bag’. At this stage all the nonadherent cells were rinsed away, a layer of monocytes was adherent on the inner surface of the bottom plastic membrane of both culture bags. A new equal volume (30 ml) of serum was transferred into each bag.
At this point a mixed population of activated monocytes and macrophages is obtained in the culture bag. The term ‘activated monocytes’ will be used for the reasons of simplicity, although this population contains cells that are already differentiated macrophages, according to their morphology, function in the wounds and the fact that macrophages are heterogeneous in their rate of maturation [1,2].
LPS stimulation
The buffy coat was equally divided, by weight, into two culture bags and both bags were treated as the control described above. A layer of macrophages was adherent on the inner surface of the bottom plastic membrane of both culture bags and a new equal volume (30 ml) of serum was transferred into each bag. At that point 10 µg/ml of LPS from Salmonella enteritidis (Sigma, L6011) were added to one of the bags. The incubation time of the cells with LPS was according to the terms of the experiment.
Activation assessment
Monocyte activation was assessed by assays of interleukins levels in the supernatants from the monocyte cultures after given incubation times. Phagocytosis capacity of florescent beads, by monocytes collected from the culture bags, was evaluated by florescence activated cell sorter (FACS).
Interleukins
Samples of supernatants were collected and stored at −60°C for further interleukins titration by ELISA kits. In order to refer the interleukins production to the number of monocytes, they were marked with CD-14 florescent antibody (CD-14 is a common marker for both monocytes and macrophages, thus including all the relevant cell population).
Sample collection
The culture bags, containing the adherent cells, were incubated with 30 ml of autologous serum for 24 h. Aliquots of 1·5 ml supernatant were sampled from each bag after 6,12 and 24 h incubation. The level of interleukins in the serum before incubation served as base line. All samples were centrifuged at 12000 g for 5min, the supernatant transferred into a clean Eppendorf tubes and stored at −60°C.
CD-14 marking
After 24 h incubation the cells were harvested by rubbing the inner surfaces of the plastic bags against each other, the cell suspension was transferred into 50 ml sterile polyethylene tubes and centrifuged at 600 g for 10 min. The supernatant was discarded and the cells were resuspended in 1 ml of serum. Cells were counted in a Newbauer haemocytometer after suspension in trypan blue (1:1 in 0·4% trypan blue solution) for evaluation of cell count and percentage of vital cells. The cells were concentrated to 10 × 106/ml by centrifugation (600 ×g for 10 min) and addition of serum to the sediment, as required. Then, 50 µl suspension (containing 5 × 105 cells) were transferred into a new conic tube. Ten microlitre of CD-14 were added (CD-14 FITC mouse anti-human, monoclonal, Ancell, Italy) and incubated at 4°C, for 25min in the dark. At the end of the incubation the cells were washed twice with 1 ml cold PBS (600 ×g for 10 min) and resuspended in 1 ml cold PBS. The samples were measured for fluorescence by FACS, for the evaluation of macrophage percentage in the collected cell population.
Interleukins quantification
IL-1β and IL-6 were titrated using Edogen ELISA kits, Cambridge, MA, USA. Samples were measured in duplicates and the mean result of the interleukin concentration in supernatant was corrected to 106 monocytes. The level of the interleukins in pure serum of each blood unit was subtracted from each sample.
Phagocytosis
Phagocytic ability was assessed by measuring the percentage of the macrophage population that phagocytised fluorescent latex beads.
Preparation of fluorescent beads suspension
SIGMA Latex beads, Amine Modified, Fluorescent, Aqueous Suspension:2·5%Solids, Mean diameter 1·01µ (S.D. = 4·7%) were used as stock suspension. Working solution was prepared by adding 50 µl of stock solution to 10 ml PBS.
Preparation of cell suspension
The cells were harvested after 1 h incubation at 37°C, in 10 ml of autologous serum. The cell suspension was transferred into 15 ml sterile polyethylene tubes and centrifuged at 600 ×g for 10 min. The supernatant was discarded and the cells resuspended in 1 ml fresh serum. The cells were counted and concentrated to 1 × 106 cells/ml,as described above.
Phagocytosis
1 ml of cell suspension (1 × 106/ml) and 15 µl of fluorescent beads working suspension were mixed and incubated at 37°C for 3 h. Every 30 min the suspension was shaken. After incubation the cell suspension was washed twice in 1 ml PBS (600 ×g for 10 min) and CD-14 R-PE (CD-14 RPE mouse anti human, monoclonal, Ancell, Italy) marking was performed, as described above. The percentage of cells that phagocytized fluorescent beads was evaluated by FACS.
Statistical analysis
For evaluation of the effect of hypo-osmotic shock and time of incubation on interleukins production, repeated measured anova was used. Probes from treated and control cells were tested several times (after 6, 12, 24 h).
The results of interleukins concentrations were highly variable, due to high variability between the blood units. Therefore a log of concentration was used as the outcome variable. The interleukins results were presented individually for each blood unit, in order to emphasize the difference between the control and the shock treated cells.
All calculations were performed using SPSS 8·0 for Windows [SPSS Inc.]. For evaluation of phagocytosis Student's T-test calculations were performed using Excel for Windows.
RESULTS
In all the experiments described, 100% vitality of cells by trypan blue count was found in macrophages prepared by either hypo-osmotic shock or isotonic saline.
Effect of hypo-osmotic shock
Interleukins secretion
A wide range of interleukin levels was measured in both the osmotic-shock induced macrophages and their saline-treated controls. This phenomenon resulted from the unit-to-unit variation, and was overcome by the fact that each unit served as it's own internal control.
IL-1
The levels of IL-1 found in the supernatant of macrophages treated with hypo-osmotic shock were 3·5–40 folds higher than those measured in the supernatant of their saline-treated controls, in all the units tested (P = 0·004), as detailed in Table 1. The increase in IL-1 secretion was apparent already after the first 6 h of incubation and was maintained throughout the following 24 h, as depicted in Fig. 2.
Table 1.
Quantification of IL-1β levels in hypo-osmotic shock or LPS stimulated monocytes and their respective controls, measured in the supernatant of the individual blood samples, as function of the incubation time. The lowest effect of hypo-osmotic shock after 6 h resulted in a 3·5 fold increase (sample No. 8), and the highest-in a 40 fold increase (sample No. 4)
| IL-1β levels at 6 hours | IL-1β levels at 12 hours | IL-1β levels at 24 hours | ||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Blood sample no. | Control | Osmotic shock | Control | LPS | Control | Osmotic shock | Control | LPS | Control | Osmotic shock | Control | LPS |
| 1 | 1 | 30 | 208 | 639 | 1 | 25 | 365 | 746 | 1 | 20 | 381 | 719 |
| 2 | 1 | 72 | 27 | 282 | 1 | 100 | 89 | 328 | 1 | 123 | 70 | 339 |
| 3 | 11 | 163 | 89 | 712 | 6 | 190 | 127 | 755 | 4 | 204 | 147 | 809 |
| 4 | 11 | 405 | 54 | 393 | 8 | 400 | 117 | 392 | 7 | 393 | 144 | 388 |
| 5 | 11 | 39 | 17 | 676 | 58 | 400 | 18 | 872 | 55 | 697 | 18 | 978 |
| 6 | 17 | 58 | 108 | 656 | 26 | 39 | 125 | 780 | 30 | 53 | 266 | 924 |
| 7 | 21 | 58 | 221 | 1033 | 34 | 62 | 528 | 1034 | 39 | 72 | 717 | 1167 |
| 8 | 5 | 17·5 | 48 | 603 | 12 | 25 | 52 | 587 | 12 | 29 | 85 | 624 |
Note: hypo-osmotic shock and LPS treatment are performed on different blood units
Fig. 2.
Values of IL-1β concentrations in the supernatants of culture bags from 8 individual blood samples, in control (isotonically treated): (○), and hypo-osmotically treated monocytes (•) represented on the ordinate in a logarithmic scale. IL-1β concentration, expressed as pg/ml containing 106 monocytes, was measured after 6,12 and 24 h incubation. It can be seen that in both groups a plateau was reached after 6–12 h. Samples treated by hypo-osmotic shock reached a statistically significant higher level. (P = 0·004)
Macrophages treated with hypo-osmotic shock behaved in this set of experiments similarly to cells incubated with LPS (Table 1): Stimulation with this ‘conventional’ macrophage-inducer resulted in a 3–40 folds elevation in IL-1 levels, when compared to its matched controls, after 6 h incubation (P = 0·0002).
IL-6
Hypo-osmotic shock had a similar, but even a more pronounced effect, on the secretion of IL-6, with levels 2·3–90 folds higher than secreted by the saline-treated controls (P = 0·001) as depicted in Table 2 and Fig. 3.
Table 2.
Quantification of IL-6 levels in hypo-osmotic shock or LPS stimulated monocytes and their controls, measured in the supernatant of individual blood samples, as a function of incubation time. A great variation was detected between different blood samples in response to the activation by both the hypo-osmotic shock and the isotonic treatment. The highest level measured was 20 000 pg/ml containing106cells, due to the fact that no further dilution of the supernatant was performed after 1:100. The lowest effect of hypo-osmotic shock results in 2·3 fold increase (sample No. 5) and the highest effect was 90 fold increase (sample No. 4) after 6 h.
| IL-1β levels at 6 hours | IL-1β levels at 12 hours | IL-1β levels at 24 hours | ||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Blood sample no. | Control | Osmotic shock | Control | LPS | Control | Osmotic shock | Control | LPS | Control | Osmotic shock | Control | LPS |
| 1 | 48 | 294 | 269 | 251 | 40 | 300 | 293 | 423 | 35 | 348 | 390 | 400 |
| 2 | 1 | 688 | 34 | 120 | 15 | 1250 | 43 | 185 | 33 | 1523 | 209 | 194 |
| 3 | 84 | 2428 | 116 | 457 | 96 | 20000 | 311 | 492 | 114 | 20000 | 439 | 488 |
| 4 | 223 | 20000 | 16 | 562 | 340 | 20000 | 54 | 809 | 383 | 20000 | 71 | 789 |
| 5 | 58 | 135 | 240 | 580 | 247 | 1678 | 624 | 776 | 935 | 20000 | 753 | 748 |
| 6 | 3·3 | 30·7 | 360 | 652 | 28·8 | 500 | 613 | 652 | 5·9 | 500 | 669 | 583 |
| 7 | 8·9 | 62·1 | 84 | 327 | 14·4 | 96·8 | 199 | 393 | 26·8 | 289 | 221 | 332 |
| 8 | 24·4 | 173 | 0 | 135 | 53 | 474 | 0 | 256 | 134 | 883 | 40 | 282 |
Note: hypo-osmotic shock and LPS treatment are performed on different blood units
Fig. 3.
Values of IL-6 concentration in the supernatants in culture bags of 8 individual blood samples, in control (isotonically treated): (○), and hypo-osmotically treated monocytes (•) represented on the ordinate in a logarithmic scale. The concentration of IL-6, expressed in pg/ml containing 106 monocytes, was measured after 6,12 and 24 h of incubation. The hypo-osmotically treated cells reached very high levels of IL-6. The supernatants were not diluted beyond 1:100, which results in values of 20000 pg/ml containing 106 monocytes.
LPS stimulated the macrophages to secret IL-6 to levels that were 0–135 folds higher than their saline-treated controls, after 6 h of incubation (P = 0·006). However, at 12 h a plateau was reached and after 24 h, the difference between the stimulated cells and the controls was not statistically significant anymore (Table 2).
Time dependent interleukin secretion
While the levels of IL-1, secreted by both osmotic-shock treated macrophages and their matched controls, reached a plateau after 6 h of incubation (Fig. 2), increasing levels of IL-6 were continuously measured, after 12 and 24 h, in both systems (P = 0·001; Fig. 3). Similarly, stimulation with LPS resulted in increasing levels of IL-1, which continued to rise with the incubation time (P = 0·02). The levels of IL-6, however, reached a plateau after 12 h of incubation in the LPS-induced cells, while IL-6 secreted by their controls continued to rise up to 24 h of incubation (P = 0·002). This caused the elimination of the difference between the LPS stimulated-macrophages and their controls, at 24 h of incubation (Tables 1 and 2).
Phagocytosis
In the osmotic-shock stimulated macrophages, 0 h of incubation refers to cells harvested from the culture bag immediately after adherence. In the system containing LPS, the cells were incubated with the inducer during the adherence stage. As shown in Fig. 4, there was a statistically significant difference, in the percent of phagocytosis -performing cells, between the osmotic-shock stimulated macrophages and their matching controls. These differences were observed when measured after 0h (P = 0·005), 6 h (P = 0·014) and 24 h of incubation (P = 0·008).
Fig. 4.
Phagocytosis by monocytes stimulated with either hypo-osmotic shock or LPS. Monocytes treated by hypo-osmotic shock (
) showed higher phagocytic activity than their control (□), saline-treated monocytes, at all incubation time points. LPS induced cells (▪) showed increase phagocytosis only after 24 h of incubation. Values represent the means ± standard deviations for cells prepared from 8 blood units.
In LPS stimulated cells a statistically significant difference in phagocytosis could be seen only after 24 h of incubation, when compared to their matching controls (P = 0·003).
DISCUSSION
In the present study we demonstrated that a simple hypo-osmotic shock, in a closed, sterile system, induces activation of monocytes. These activated and differentiated cells are successfully applicated in wound healing of human ulcers in elderly and paraplegic patients [17,18].
Among the different methods known to activate monocytes the most widely used is the addition of endotoxins and interferon γ [19–22]. It has also been shown that hyperosmotic stress stimulates cytokine production in vitro [23–27].
Activation for a specific target was reported, in which monocytes were incubated in presence of minced peripheral nervous tissue [28]. In contradistinction, monocytes were incubated in the presence of inhibitors, in order to modulate the wound healing process [29]. The use of hypo-osmotic shock and a closed, sterile system have the advantage of suitability for clinical use.
A certain degree of activation seemed to take place by the very adhesion of the cells to the plastic membrane (Tables 1 and 2), as previously noted by others [30]. However, the hypo-osmotic shock enhanced the differentiation and activation of the macrophages very markedly, as can be measured by both interleukin secretion and phagocytosis. Upon induction extremely high levels of IL-1 and IL-6 were detected (3·5–40 folds and 2–90 folds, respectively).
Unlike most published papers, which report average numbers resulting from several blood samples, each unit in the current study served as its' own control. The results were reported separately for each blood sample, thus emphasizing the great variation between the various blood donors.
Whether read as average or extremes, the levels of IL-1 and IL-6 in the supernatant of the culture medium increased with time. But while IL-1 levels reached a plateau after 6–12 h, the levels of IL-6 continued to increase in the following 12–24 h of incubation. This finding may contribute to the understanding of the wound healing process, as IL-1 is related to the inflammation stage [7–9] while IL-6 plays an important role in later stages of wound healing, such as angiogenesis and fibroblast proliferation [11–13].
The hypo-osmotic shock seems to be at least as efficient as other previously published methods, such as induction with LPS. Studies of monocytes incubated with this inducer, for 24 h at 37°C, showed an increased secretion of IL-1 into the medium, from 500 pg/ml to 1700 pg/ml (about 3 fold) [31]. Stimulation of monocytes with other agents such as phytohemaglutinin (PHA) and phorbolmeristateacetate (PMA), resulted in increase in IL-1 from 0 to 125 pg/ml, under similar experimental conditions [32], while the levels of IL-6 measured after PHA and PMA stimulation increased from 750 to 1500 pg/ml (two fold). The authors did not use, however, a matching control cell culture, without PMA stimulation, for the determination of a base line.
When the effects of the hypo-osmotic shock method was compared to monocyte stimulation with LPS (10 µg/ml), within our system, similar results were obtained, regarding IL-1 secretion after 6, 12 and 24 h of incubation. The levels of IL-6 were also markedly elevated after 6 and 12 h of incubation (P < 0·03), but no significant difference was depicted after 24 h between the supernatants of the LPS stimulated cells and their controls (P = 0·3).
We could not find any previously published data regarding the response to stimulation with LPS in less than 24 h of incubation. In our system, however, a significant effect on secretion of both IL-1 and IL-6 was detected already after 6 h of incubation. While the highest measured levels of IL-1 after 24 h incubation were 1200 pg/ml, the highest IL-6 levels reached 850 pg/ml.
The activation by hypo-osmotic shock enhances the phagocytic capacity of cells, collected immediately after adherence (P = 0·005), while LPS stimulated cells showed higher phagocytosis ability only after 24 h of incubation (Fig. 4).
Hypo-osmotic shock activated monocytes can be collected after one hour of incubation and can be applied to the wound. Once in the wound the activated cells will assume their function, according to the surrounding microenvironment [2]. For this reason the preferred procedure is to use the macrophage suspension for treatment after one hour incubation. Longer incubation time can still be effective. It seams that differentiated monocytes can adapt to a new microenvironment. When differentiated mice peritoneal monocytes were applied to experimental wounds, they enhanced the rate of wound healing [16].
In view of these results, we conclude that the hypo-osmotic shock is an effective, safe, easy and inexpensive method, for activation of monocytes. This rapid, sterile source of activated monocytes, suitable for human use in chronic nonhealing wounds, may open options for additional clinical applications in the future.
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