A Topical Wound Disinfectant (Ethacridine Lactate) Differentially Affects the Production of Immunoregulatory Cytokines in

A ppropriate wound management is a major issue in hospital care involving almost all fields of medical care. Despite the prophylactic use of a variety of clinically efficient antibiotics in many surgical procedures, the concomitant use of local antiseptics still represents a common and useful therapeutic measure to control wound infection. Particularly for immunocompromised patients, topical antiseptics have to be safe, ie, they must not impair the physiological wound healing process. However, recent publications^1–4 reported various interactions of commonly used local antiseptics using cell culture systems or animal models, respectively. These results suggest that local antiseptics profoundly affect not only the viability but also key functions of skin cell types, such as keratinocytes or fibroblasts, or even effector activities of peripheral leukocytes. The latter are inevitably involved in wound healing by producing a variety of regulatory cytokines.⁵ Nevertheless, reports showing a direct influence of topical antimicrobials on cells of the immune system remain scarce. The aim of the present study was, therefore, to investigate the effects of ethacridine lactate on granulocytes, monocytes, and T-lymphocytes and to screen for immunopharmacological activities beyond its antibacterial properties. In order to mimic the conditions in vivo for the contact of ethacridine lactate with immune cells, the authors used human whole-blood cultures prepared from 6 healthy volunteer donors. With this system, the capacity of the drug to influence the co-stimulated synthesis of several important mediators known to orchestrate wound healing was investigated. Specifically, the authors assessed the production of interleukin-6 (IL-6), interleukin-10 (IL-10), interleukin-12 (IL-12), prostaglandin E2 (PGE2), interferon-gamma (IFNγ), interleukin-5 (IL-5), and elastase. Moreover, it was of interest to examine the effects of ethacridine lactate in situations resembling impaired wound healing, leading to chronic wounds. This was studied by adding hydrocortisone to the whole-blood cultures, followed by an incubation with ethacridine lactate. In this part of the experiments, elastase, TGF-beta 1, IL-6, and MCP-1 were determined. Methods Volunteers and blood collection. For preparation of the cell cultures, blood of 6 healthy volunteers of both genders was used. Donors with symptoms of acute infection or with known chronic inflammatory diseases were excluded from the study. The same applied for individuals receiving a vaccination, surgery, or medications within the last 14 days before donation. The blood was drawn into a syringe prefilled with sodium-heparin (50 IU/mL of blood, ratiopharm, Ulm, Germany) and distributed into the wells of microculture plates (96-well plates, Greiner, Germany). Test substances. Ethacridine lactate was supplied by Chinosolfabrik (Seelze, Germany) and diluted to the desired concentrations with Hanks Balanced Salt Solution (HBSS, Biochrom KG, Berlin, Germany). Hydrocortisone was purchased from Sigma-Aldrich (Deisenhofen, Germany). Whole-blood culture system (WBCS) and stimulation of mediator production. The whole-blood culture was performed according to the method described by Meyaard.⁶ Briefly, after seeding the diluted blood into 96-well plates, ethacridine lactate was added in 5 different concentrations: 10, 3, 1, 0.3, and 0.1 µg/mL (final concentration in culture). All cultures were done in triplicate. The stimuli to induce the production of the different mediators were added. The type of stimulus used allowed discrimination between the responding cell type in the whole blood. Thus, the secretion of elastase, a serine protease abundantly expressed in azurophilic granules of neutrophil granulocytes, was induced by treating the WBCS with zymosan (Sigma-Aldrich), opsonized (pretreated with human AB-plasma), and diluted to give a final concentration of 1 mg/mL. The monocyte population in the WBCS was activated to release TGF-beta 1, IL-6, IL-10, and IL-12 by the use of lipopolysaccharide (LPS, from Escherichia coli serotype O55:B5, Sigma-Aldrich, final concentration 10 ng/mL). The synthesis of T-cell derived mediators was activated by the addition of 2 monoclonal antibodies against the surface markers CD3 (R&D Systems, Germany, final concentration 200 ng/mL) and CD28 (purchased from the German Cancer Research Institute, DKFZ, Heidelberg, Germany; final concentration 300 ng/mL). The stimulation control cultures received only the co-stimuli; the negative controls were cultured with cell culture medium alone. After addition of all stimuli and the test substance at different concentrations, the whole-blood cultures were incubated at 5% CO₂ at 37^oC for different time periods. Elastase was routinely measured after 6 hours, the monokines after 24 hours, and the lymphokines after 48 hours of culture. Thereafter, the culture supernatants were harvested after centrifugation of the microwell plates (10 min, 600 g) and stored frozen at -20^oC until determination of all cytokines with specific ELISA systems. Measurement of cytokines. For the determination of the different mediators, antibody pairs from R&D Systems, Germany, were used. All ELISA tests were strictly performed according to the manufacturer’s instructions. Treatment of WBCS with hydrocortisone. In order to simulate the conditions of a suppressed immune system as can be expected as a reason for delayed wound healing, hydrocortisone was added to some of the whole-blood cultures. These cultures were supplemented with hydrocortisone at a final concentration of 250 ng/mL 2 hours before adding ethacridine lactate together with the co-stimuli opsonized zymosan (elastase), LPS (IL-6, TGF-beta 1), or anti-CD3/anti-CD28 (MCP-1). Control cultures included stimulus-treated whole-blood cultures with and without hydrocortisone as well as negative controls treated with cell culture medium only. At the end of incubation, the immunosuppressed cultures were processed as previously described. Determination of cyclo-oxygenase 1 (COX-1) activity. Human platelets in whole blood served as target cells for the determination of the activity of ethacridine lactate on COX-1. Whole blood from the 6 volunteers treated with heparin (50 IU/mL; ratiopharm) was pre-incubated for 1 hour in Monovette tubes (Sarstedt, Germany) with the different concentrations of the test substance as noted previously. Thereafter, blood coagulation was initiated by the addition of 0.75 mg of protamine sulfate (Sigma-Aldrich) per 50 IE heparin, together with a clotting trigger (sterile glass beads). One hour later, supernatants were harvested by centrifugation (15 min, 900 g) and stored frozen at -20^oC until determination of PGE2 with a specific ELISA (Cayman, SpiBio, Massy, France). Data processing and statistics. The donor-to-donor variability of the individual cytokine levels usually is large. Therefore, the cytokine concentrations measured by ELISA were normalized by calculating a stimulation index (SI) as described by Essa et al.⁷ Accordingly, the amount of cytokines produced by the sole addition of the different stimuli (LPS, opsonized zymosan, or anti-CD3/anti-CD28) was defined to have the SI = 1.0. Furthermore, 95% confidence intervals for the means of each dose level were calculated and are shown in the graphs as error bars. Results Effects of ethacridine lactate on monokine synthesis (IL-6, IL-10, IL-12). Generally, ethacridine lactate was shown to induce remarkable changes in the synthesis of cytokines from different leukocyte subpopulations. However, different reactivities of the cell types with regard to stimulatory or inhibitory actions of the drug were observed as well as marked differences concerning the responding cytokine type. The data revealed that a pre-incubation of granulocytes with ethacridine lactate followed by a co-stimulation with opsonized zymosan did not result in relevant changes of elastase release by neutrophils (data not shown). However, the release of prostaglandin E2 by platelets seemed to be inhibited slightly (19–44%), indicating the functional activity of COX-1 at the highest concentration of ethacridine lactate (data not shown) and with considerable inter-individual variations. In contrast, clear effects of ethacridine lactate were seen on the synthesis of monocyte-derived cytokines as illustrated by Figures 1 to 3. Obviously, the highest concentration of ethacridine lactate (10 µg/mL) uniformly inhibited the synthesis of the pluripotent immunoactivating cytokine IL-6 by LPS-stimulated whole-blood leukocytes substantially (Figure 1). However, upon further dilution of the test substance, the synthesis of IL-6 gradually returned to background levels. The immunoregulatory cytokine IL-10 was also markedly suppressed (Figure 2). However, at 1 µg/mL, this inhibition turned to a stimulating effect of at least 47% above the level of the stimulated controls. The most remarkable effects of the drug were observed on IL-12 release (Figure 3). The dose-response relationships are clearly in contrast to those seen with IL-10, as a strong stimulation of the IL-12 synthesis could be measured. The maximal effect on IL-12 synthesis was induced with 3 µg/mL of the drug, and even at higher dilutions, an increased production could be observed, now in a donor-independent manner. Because IL-12 is a potent inducer of the synthesis of IFNγ by T-helper 1 lymphocytes, it was interesting to compare this high IL-12 response with that of the Th1- and Th2-derived cytokines, ie, IFNγ and IL-5, respectively. Effects of ethacridine lactate on lymphokine synthesis (IFNγ, IL-5). Indeed, IFNγ production by whole-blood leukocytes from different volunteers was also increased. The leukocytes of 2 of the 6 donors produced extraordinarily high amounts of IFNγ upon co-stimulation in the presence of ethacridine lactate (Figure 4). The lymphocytes from the other 4 donors were also activated, although to a lesser extent, resulting in SI values between 1.30 and 2.2. However, the amount of IFNγ released did not seem to correlate directly with the generally higher IL-12 response. In contrast to IFNγ and IL-12, the synthesis of IL-5 was profoundly inhibited by the test substance, an effect that was detected in the cultures of 5 of the 6 volunteers (Figure 5). With 10 µg/mL of ethacridine lactate, the inhibition of the IL-5 synthesis ranged between 60% and 80% and was still detected at 3 µg/mL in the cultures of 2 of the volunteers. The opposite effect was observed with the lymphocytes of 1 of the donors, which were stimulated substantially in their IL-5 secretion, despite an increased release of IL-12 and IFNγ in the presence of ethacridine lactate in his cultures. Effects of ethacridine lactate in hydrocortisone suppressed cultures. The release of elastase by hydrocortisone pretreated granulocytes in whole blood was not changed significantly after treatment with ethacridine lactate (data not shown). The effects observed with hydrocortisone pretreatment on IL-6 synthesis were almost comparable to the dose-response curves presented in Figure 1, however, with slightly higher inter-individual variations (data not shown). Generally, the wound disinfectant was not able to counteract the inhibitory actions of hydrocortisone on IL-6 synthesis. The most important results obtained with hydrocortisone-treated cultures were those found for TGF-beta 1 and MCP-1, demonstrating again opposing activities of this topical antiseptic. Figure 6 shows the results for TGF-beta 1. Hydrocortisone exerted only a weak inhibitory effect on TGF-beta 1 release by LPS-stimulated monocytes, although this inhibition seemed to be enhanced by ethacridine lactate. The inhibition at the highest concentration of the drug ranged between 20% and 45% (Figure 6) and still was clearly detectable with 3 µg/mL of the test substance. Even at the highest dilution (0.1 µg/mL) of ethacridine lactate the SI values remained slightly below the hydrocortisone reference value without the test substance, an effect consistently found in the cultures of all donors. Although ethacridine lactate inhibited the production by whole-blood cultures of MCP-1 at high concentrations, the drug was able to stimulate the release of MCP-1 at lower doses by the cells in 3 of the 6 volunteers (Figure 7). Only the culture of 1 donor exhibited a clear activation of MCP-1 synthesis with 10 µg/mL of ethacridine lactate. Lymphocytes from the other donors remained inhibited at all concentrations of ethacridine lactate. Discussion The current study demonstrates for the first time that the topical disinfectant ethacridine lactate, routinely used for treating wound infections since 1923, exhibits potent immunomodulatory effects regarding the release of cytokines in human whole-blood cultures. This is concluded from data showing a profound inhibition of the release of IL-5 and IL-10, whereas the synthesis of IL-12 or IFNγ was stimulated strongly and dose-dependently. Moreover, the patterns of IL-10 release and the response of the leukocytes concerning MCP-1 indicate that this wound disinfectant displays a strong immunomodulatory profile rather than just inhibiting or stimulating leukocytes. For example, MCP-1 is essential for controlling the migration behavior of monocytes and lymphocytes during the different phases of wound healing as noted by Engelhardt et al.⁸ Altogether these data support a new concept of an enhancing effect of ethacridine lactate on antibacterial defense reactions, mainly accomplished by a preferential activation of Th1 lymphocytes. The doses used in the authors’ cultures comply with concentration levels that can be expected in the tissue surrounding the wound rather than in the wound itself. The substance effects will, therefore, primarily apply to cells freshly recruited to the wound area from the surrounding blood capillaries, which is an ongoing process. Investigations concerning effects of topical antiseptics on different cell types involved in wound healing were recently the subject of a few articles.^9–12 They demonstrated that fibroblasts, neutrophils, and lymphocytes were negatively affected by some but not all types of these pharmaceuticals. To the authors’ knowledge, none of these was tested in whole blood. The key question arising from the present results obtained in a whole-blood culture system is whether the different cytokines affected in their synthesis by ethacridine lactate will contribute to its antibacterial activities. Tissue disruption and repair, whether initiated by trauma, microbes, or foreign materials, proceeds via an overlapping and complex pattern of events regulated mainly by the immune system.¹³ Thus, the different phases of wound healing, including coagulation, inflammation, epithelization, formation of granulation tissue, matrix deposition, and the remodeling phase, are mediated largely by molecular signals, primarily cytokines and growth factors. Therefore, it is reasonable to assume that the cytokine profile measured with whole-blood cultures reflects the local situation in open wounds much better than cultures of isolated leukocytes, which lack many of the cellular and noncellular elements present in vivo. Whole-blood culture models resemble the conditions in vivo very closely, because they allow a whole variety of interactions of the drug with the cellular and soluble components of the blood. Wounds in vivo also display particular features that cannot be mimicked in vitro. Thus, the activities of ethacridine lactate observed in the authors’ experiments may be modulated in some way in different types of wounds. Nevertheless, the present results clearly demonstrate that ethacridine lactate, in general, is able to modulate immunoregulatory activities of the human immune system under in vivo–like conditions. The activities the authors observed clearly fit into the concept of enhancing the local, leukocyte-mediated defense against microbial infection. Moreover, the authors could show that this happens at concentrations that can be expected to be prevalent in the tissues adjacent to the wounds. The modulation of TGF-beta 1 production, which in these experiments was found to be further suppressed by ethacridine lactate in hydrocortisone-treated whole-blood cultures, is a good example of the complex interplay of mediators involved in wound healing. Latent TGF-beta 1, released in large quantities following injury, for example, by degranulating platelets, elicits a rapid chemotactic response of peripheral blood leukocytes like neutrophils and monocytes. Neutrophils arriving shortly after injury are followed by actively recruited monocytes, reported to peak 48 hours post injury.¹⁴ These “wound monocytes” under the local influence of a plethora of mediators, among them TGF-beta 1, the platelet-derived-growth factors (PDGFs), PGE2, or chemokines like MCP-1, are essential to drive wound repair.¹⁵ Assuming that this local activation of monocytes promotes the inflammatory response in the early phase of wound healing, this is indispensable in situations of contaminated wounds. A therapeutic enhancement of antibacterial defense mechanisms at this time could help to clear the wound infection thereby accelerating the resolution of inflammation. TGF-beta 1, on the other hand, is known to suppress the production of defense-enhancing cytokines, especially in monocytes. An inhibition of macrophage functions by an excess of TGF-beta 1 production may, therefore, be considered harmful, particularly in infected wounds. It has been demonstrated that even a single dose of TGF-beta 1 given at the time of bacterial challenge may be detrimental to the host.¹⁶ In contrast, locally applied TGF-beta 1 profoundly accelerates wound healing by down-regulating inflammation and stimulating keratinocyte proliferation and fibroblast activation.¹⁴ An excess of TGF-beta 1 production in this later phase of wound healing often results in hyperfibrosis and local immunosuppression, which reflects at best the dual role of TGF-beta.¹⁷ Together with the modulation of TGF-beta 1 production, the slight inhibition of the COX-1 activity by ethacridine lactate suggests a moderate anti-inflammatory effect of this disinfectant. The switch from an inhibitory effect on IL-10 by ethacridine lactate at high concentrations to a stimulation in the cultures of all 6 donors further supports the view of an immunomodulatory characteristic of this drug. In humans, neutrophils, monocytes/macrophages, B-lymphocytes, and mast cells can produce IL-10.^18,19 This cytokine is currently under intense investigation as a potential candidate to antagonize inflammatory and antigen-specific immune responses in several clinical trials.²⁰ Hence, its inhibition by ethacridine lactate may partially be responsible for the observed strong increase in IL-12 production in the cultures of all donors. It is noteworthy to mention that under conditions of stress, the hypothalamic-pituitary-adrenal axis is also strongly activated by corticotropin-releasing hormone produced by the central nervous system in response to peripheral inflammation associated with the concomitant release of cytokines, such as IL-6 and, later, IL-10. These events mark the commonly observed “shift” to a Th2 response in individuals in situations of chronic stress.²¹ Considering the obvious inhibition of IL-6 and IL-10 by ethacridine lactate at high concentrations, this could imply an additional mechanism possibly counteracting a stress-induced suppression of local immune responses, thereby promoting antibacterial immunity. Conversely, the stimulation of IL-10 observed at lower concentrations of ethacridine lactate can also be regarded as useful during angiogenesis and formation of granulation tissue. Increased IL-10 levels at this stage of wound healing could help to antagonize the functions of IFNγ and IL-12, as both cytokines represent potent endogenous inhibitors of both processes. Lower levels of ethacridine lactate will be present deeper in the tissues by means of diffusion, therefore spatially separating its inhibitory from its stimulatory effects on the production of IL-10. As with all other types of defense reactions, the tissues adjacent to the local inflammation of the wound have to be protected from the aggressive actions of the immune system needed to cleanse the tissue of microbes. This can be expected to be well accomplished by an increased presence of IL-10. Similarly, the strong stimulation of IL-12 by ethacridine lactate suggests a profound change on the antibacterial defense, because IL-12 is ultimately involved in the induction and activation of T-lymphocytes of the Th1 subtype.^22,23 In this context, IL-12 is a highly potent stimulant to increase the functional activities of Th1 lymphocytes as well as natural killer cells. Both cell populations secrete IFNγ but with different kinetics. This effect of IL-12 is strongly associated with a cellular process called “macrophage activation,” a typical hallmark of cell-mediated immune responses. The activation of monocytes/macrophages is the consequence of a positive feedback loop, mainly based on the IL-12-triggered increase of IFNg production. Insofar, an enhanced production of IL-12 should increase bactericidal activities of wound macrophages and, thus, help to clear contaminated wounds. However, the role IFNg plays during wound healing is less evident. The interplay between IFNγ and TGF-beta 1 was reported to be very complex as can clearly be shown by the pathogenic role TGF-beta 1 plays in the pathology of fibrosis in humans. Several recent clinical trials as well as animal models have demonstrated a strong antifibrotic action of IFNγ mediated in part by its inhibitory effect on the expression of the mRNA for TGF-beta 1, as described by Ziesche and Block.²⁴ Nonetheless, there is no doubt in the potentiating activities of IFNg concerning antimicrobial defense reactions. IL-6 instead is known to profoundly activate epidermal keratinocyte proliferation necessary for closing wounds.^25,26 This is also demonstrated by the fact that gene knockout mice for IL-6 showed a delay in cutaneous wound healing.²⁷ On the other hand, the inherent pro-inflammatory potential of IL-6 is reflected by reports showing an overexpression of IL-6 in psoriatric skin lesions.²⁸ Therefore, the inhibition of its production by ethacridine lactate could also result in a limitation of wound inflammation. Conclusion The changes in the cytokine profile induced by ethacridine lactate in the present study favor the concept of a second mechanism of action beyond its disinfecting properties, ie, a preferential shift toward a Th1-type immune reaction. Among the major functions of Th1 lymphocytes during immune responses is the promotion of macrophage activation to kill and digest phagocytosed bacteria.²⁹ Regarding this, the present results reveal some newly emerging activities of ethacridine lactate, namely the capacity to enhance antibacterial defense reactions in microbially contaminated wounds. In many cases, impaired wound healing is the result of an underlying disease (autoimmunity or cancer), a rigorous therapeutic regimen (immunosuppression), or even stress. A topical antiseptic that would not only avoid additional compromise of the host’s defense reactions but also would positively support the immune system in its efforts to control wound infection will be of substantial clinical importance. Future research will have to be performed in order to complete clinicians’ knowledge on the immunopharmacological profile of this interesting wound disinfectant.