Short- and long-term effects of IL-1 and TNF antagonists on periodontal wound healing

The present study tested the effects of local injection of IL-1 and TNF soluble receptors on a periodontal wound-healing model in nonhuman primates. In this model, periodontal lesions were developed for 16 wk, followed by open flap surgery. Starting at the time of surgery, groups of animals received localized injections of both soluble cytokine receptors or else PBS three times per week for 3, 14, or 35 days. Periodontal wound healing was analyzed for each group at the end of the treatment regimen. Fourteen days after surgery, a significant decrease was observed between the animals treated with soluble receptors and the untreated group with respect to recruitment of inflammatory cells in deep gingival connective tissue. Concurrent apoptosis of inflammatory cells in those tissues increased significantly in treated animals compared with untreated animals. All other outcome parameters of periodontal wound healing were likewise significantly improved in treated animals compared with untreated animals. In marked contrast, however, 35 days after surgery, there was a significant increase in the number of inflammatory cells that had infiltrated into deep gingival connective tissue in treated compared with untreated animals. Outcome parameters of periodontal wound healing worsened in treated animals when compared with untreated. These results indicate that proinflammatory cytokines may play different functional roles in early vs late phases of periodontal wound healing. Short-term blockade of IL-1 and TNF may facilitate periodontal wound healing, whereas prolonged blockade may have adverse effects.     

Expression of stromal-derived factor-1 is decreased by IL-1 and TNF and in dermal wound healing

Stromal-derived factor-1 (SDF-1) is a CXC chemokine that is believed to be constitutively expressed by stromal cells of numerous tissues. In this report, we demonstrate that dermal fibroblasts and vessels of noninflamed tissues express SDF-1. Unexpectedly, we found that expression of SDF-1 is regulated by inflammation. Expression of SDF-1 by primary cultures of human gingival fibroblasts is potently inhibited by activated macrophages via secretion of IL-1alpha and TNF-alpha. Levels of SDF-1 mRNA also decrease in acutely inflamed mouse dermal wounds. We propose that SDF-1 functions as a homeostatic regulator of tissue remodeling, whose expression stabilizes existing dermal architecture.     

Induction of macrophage TNF alpha, IL-1, IL-6, and PGE2 production by DTH-initiating factors

The elicitation of delayed-type hypersensitivity (DTH) reactions in mice is due to the sequential action of two different, antigen-specific, Thy-1+ cells. We have previously cloned the early-acting DTH-initiating cell from nude mice that were immunized and boosted by contact sensitization with oxazolone (OX). This cell clone, WP-3.27, releases an antigen-specific factor (OX-F) that sensitizes mast cells such that specific antigen challenge will induce serotonin release which mediates the early phase of DTH. In normal mice contact sensitized with picryl chloride (PCl), a similar polyclonal factor (PCl-F) has a similar activity and is also known to bind to macrophages. Thus, we measured macrophage production of TNF alpha, IL-1, IL-6, and PGE2 in response to the hapten affinity-purified DTH-initiating factors OX-F and PCl-F. Both factors induced significant release of each cytokine and PGE2. The production of TNF alpha, IL-1, and IL-6 was measured by bioassays. Northern blot analysis showed rapid accumulation of cytokine mRNA (2-4 hr), while maximal production of PGE2 occurred at approximately 8 hr. These macrophage activating properties of OX-F and PCl-F were not due to contamination with LPS as determined by the low levels of LPS present in OX-F and PCl-F and by the failure of polymyxin B to inhibit factor-induced PGE2 and TNF alpha production. Also, macrophage activation was shown not to be due to the action of several lymphokines known to be produced by WP3.27. Separation of OX-F and PCl-F by preparative isoelectric focusing showed a similar pattern: there were two major peaks of PGE2-inducing activity observed for both factors (for PCl-F at pI of 2-3 and 5.0, and for OX-F at pI of 3.5-4 and 5.0), but not for a sham factor produced by WEHI-3 cells. The ability of DTH-initiating factors to rapidly induce macrophage cytokine release and PGE2 synthesis 4-6 hr later may suggest a role for these mediators during the respective early vascular and late cellular phases of inflammation in DTH.     

IL-1 plays a critical role in oral, but not dermal, wound healing.

Wound healing is a well-orchestrated complex process leading to the repair of injured tissues. After injury, proinflammatory cytokines act as important modulators of the inflammatory process. IL-1 expression has been regarded as necessary for healing; however, its effects have also been implicated in delayed wound repair. Currently, there is no consensus or direct evidence that IL-1 activity plays a central role in the healing process. The present investigation was undertaken to define the role of IL-1R signaling in the healing outcome of an excisional wound in the palate or scalp of mice that had targeted deletions of the IL-1R type 1 (IL-1R1(-/-)) compared with matched wild-type mice. Histomorphometric analysis was undertaken to assess the degree of healing and the recruitment of polymorphonuclear and mononuclear phagocytes. After 14 days, wild-type mice exhibited complete closure of intraoral wounds, while IL-1R1(-/-) animals had only partial closure (50%). In the IL-1R1(-/-) mice, healing tissues exhibited a persistent inflammatory cell infiltrate, which did not occur in wild-type animals. Treatment with antibiotics significantly diminished the persistent inflammatory infiltrate and improved healing in the experimental animals. In contrast to oral wounds, the rate of healing and recruitment of polymorphonuclear cells in scalp wounds was similar in IL-1R1(-/-) and wild-type mice. The present data underscore the importance of IL-1 in wound healing in a challenging environment and identify its principal role in facilitating the healing process by protecting an open wound from bacterial insult. In a less challenging environment, the production of new connective tissue and its coverage by migrating epithelium are minimally affected by the absence of IL-1 activity.     

Cytokine production and killer activity of NK/T-NK cells derived with IL-2, IL-15, or the combination of IL-12 and IL-18

NK cell populations were derived from murine splenocytes stimulated by IL-2, IL-15, or the combination of IL-12 and IL-18. Whereas NK cells derived with the latter cytokines consisted of an homogeneous population of NK cells (DX5+CD3-), those derived with IL-2 or IL-15 belonged to two different populations, namely NK cells (DX5+CD3-) and T-NK cells (DX5+CD3+). Among NK cells, only those derived with IL-12/IL-18 produced detectable levels of cytokines, namely IFN-gamma, IL-10, and IL-13 (with the exception of IL-13 production by NK cells derived with IL-2). As for T-NK cells, IL-2-stimulated cells produced a wide range of cytokines, including IL-4, IL-5, IL-9, IL-10, and IL-13, but no IFN-gamma, whereas IL-15-derived T-NK cells failed to produce any cytokine. Switch-culture experiments indicated that T-NK cells derived in IL-2 and further stimulated with IL-12/IL-18 produced IFN-gamma and higher IL-13 levels. Next, we observed that NK/T-NK cell populations exerted distinct effects on Ig production by autologous splenocytes according to the cytokines with which they were derived. Thus, addition of NK cells derived in IL-12/IL-18 inhibited Ig production and induced strong cytotoxicity against splenocytes, whereas addition of NK or T-NK cells grown in IL-2 or IL-15 did not. Experiments performed in IFN-gammaR knockout mice demonstrated that IFN-gamma was not involved in the killer activity of IL-12/IL-18-derived NK cells. The hypothesis that their cytotoxic activity was related to the induction of target apoptosis was confirmed on murine A20 lymphoma cells. Experiments performed in MRL/lpr mice indicated that IL-12/IL-18-derived NK cells displayed their distinct killer activity through a Fas-independent pathway. Finally, perforin was much more expressed in IL-12/IL-18-derived NK cells as compared with IL-2- or IL-15-derived NK cells, an observation that might explain their unique cytotoxicity.     

Regulation of angiogenesis: wound healing as a model

Normal tissue function requires adequate supply of oxygen through blood vessels. Understanding how blood vessels form is a challenging objective because angiogenesis is vital to many physiological and pathological processes. Unraveling mechanisms of angiogenesis would offer therapeutic options to ameliorate disorders that are currently leading causes of mortality and morbidity, including cardiovascular diseases, cancer, chronic inflammatory disorders, diabetic retinopathy, excessive tissue defects, and chronic non-healing wounds. Restoring blood flow to the site of injured tissue is a prerequisite for mounting a successful repair response, and wound angiogenesis represents a paradigmatic model to study molecular mechanisms involved in the formation and remodeling of vascular structures. In particular, repair of skin defects offers an ideal model to analyze angiogenesis due to its easy accessibility to control and manipulate this process. Most of those growth factors, extracellular matrix molecules, and cell types, recently discovered and considered as crucial factors in blood vessel formation, have been identified and analyzed during skin repair and the process of wound angiogenesis. This article will review cellular and molecular mechanisms controlling angiogenesis in cutaneous tissue repair in light of recent reports and data from our laboratories. In this article we will discuss the contribution of growth factors, basement membrane molecules, and mural cells in wound angiogenesis. The article provides a rationale for targeting the angiogenic response in order to modulate the outcome of the healing response.     

The essential role of the UA-rich sequence in endotoxin-induced cachectin/TNF synthesis

Using a series of reporter constructs in which a CAT coding sequence is constitutively transcribed under the influence of the SV40 late promoter but followed by varying portions of the 3'-untranslanted region of TNF, we have shown that the UA-rich element, present in the distal third of the 3'-unstranslated region of TNF, is absolutely required for translational activation of TNF synthesis. Replacement of the UA-rich element by an unrelated sequence of similar length completely abolishes the response. However, the context within which the UA-rich element is presented also appears to be essential, since replacement of flanking portions of 3'-untranslated sequence also blocks the response to LPS. These findings suggest that the primary role of the UA-rich element may consist in its ability to mediate translational activation in response to specific inducing signals.     

Activation of macrophage CD8: pharmacological studies of TNF and IL-1 beta production

Previously, we demonstrated that rat macrophages express CD8 and that Ab to CD8 stimulates NO production. We confirm that CD8 is expressed by rat macrophages and extend understanding of its functional significance. Activation of CD8 alpha (OX8 Ab) on alveolar macrophages stimulated mRNA expression for TNF and IL-1 beta and promoted TNF and IL-1 beta secretion. Similarly, OX8 Ab (CD8 alpha) stimulated NR8383 cells to secrete TNF, IL-1 beta, and NO. Activation of CD8 beta (Ab 341) on alveolar macrophages increased mRNA expression for TNF and IL-1 beta and stimulated secretion of TNF, but not IL-1 beta. Interestingly, anti-CD8 Abs did not stimulate IFN-gamma or PGE2 production, or phagocytosis by macrophages. OX8 (CD8 alpha)-induced TNF and IL-1 beta production by macrophages was blocked by inhibitors of protein tyrosine kinase(s), PP1, and genistein, but not by phosphatidylinositol-3 kinase inhibitor, wortmannin. Moreover, OX8 stimulated protein tyrosine kinase activity in NR8383 cells. Further analysis of kinase dependence using antisense to Syk kinase demonstrated that TNF, but not IL-1 beta, stimulation by CD8 alpha is Syk dependent. By contrast, protein kinase C inhibitor Ro 31-8220 had no effect on OX8-induced TNF production, whereas OX8-induced IL-1 beta production was blocked by Ro 31-8220. Thus, there are distinct signaling mechanisms involved in CD8 alpha (OX8)-induced TNF and IL-1 beta production. In summary, macrophages express CD8 molecules that, when activated, stimulate TNF and IL-1 beta expression, probably through mechanisms that include activation of Src and Syk kinases and protein kinase C. These findings identify a previously unknown pathway of macrophage activation likely to be involved in host defense and inflammation.     

Carnosine enhances diabetic wound healing in the db/db mouse model of type 2 diabetes

Diabetes mellitus (DM) is a progressive disorder with severe late complications. Normal wound healing involves a series of complex and well-orchestrated molecular events dictated by multiple factors. In diabetes, wound healing is grossly impaired due to defective, and dysregulated cellular and molecular events at all phases of wound healing resulting in chronic wounds that fail to heal. Carnosine, a dipeptide of alanine and histidine and an endogenous antioxidant is documented to accelerate healing of wounds and ulcers. However, not much is known about its role in wound healing in diabetes. Therefore, we studied the effect of carnosine in wound healing in db/db mice, a mice model of Type 2 DM. Six millimeter circular wounds were made in db/db mice and analyzed for wound healing every other day. Carnosine (100?mg/kg) was injected (I.P.) every day and also applied locally. Treatment with carnosine enhanced wound healing significantly, and wound tissue analysis showed increased expression of growth factors and cytokines genes involved in wound healing. In vitro studies with human dermal fibroblasts and microvascular-endothelial cells showed that carnosine increases cell viability in presence of high glucose. These effects, in addition to its known role as an antioxidant and a precursor for histamine synthesis, provide evidence for a possible therapeutic use of carnosine in diabetic wound healing.     

Protein A and other surface components of Staphylococcus aureus stimulate production of IL-1 alpha, IL-4, IL-6, TNF and IFN-gamma.

Studies were carried out on the ability of protein A (PA) and of muramic acid (MA) from S. aureus to induce the release of cytokines both from monocytes and lymphocytes in vitro. Results show that protein A induces the greatest activity, compared to the activity already known for the theicoic acid (TA) and for muramyl dipeptide (MDP). At concentration of 10 micrograms/ml; PA induces roughly +180% release of TNF with respect to controls, while release of IL-1 alpha is about 500% control values, and is higher than those obtained when cells are treated with TA and MDP; IL-6 release is higher than that stimulated by Con A, used as standard challenge. At PA concentrations of 5 micrograms/ml, IL-4 release is about five times higher than that induced by Con A. Release of IFN-gamma showed similar dose-dependent stimulations. Muramic acid (MA) is particularly active in inducing the release of cytokines from target cells, inducing TNF release of about +75% with respect to the controls. This increase is less than that obtained with PA. Also IL-4 and IFN-gamma are released by PA in quantities higher than those induced by TA and MDP. Our results lead us to believe that during infections by Gram-positive bacteria, their surface components are able to induce a series of chain reactions ranging from the inflammatory to the immunologic responses which are also conditioned by release of cytokines.     

Cell and molecular regulation of endothelin-1 production during hepatic wound healing

During hepatic wound healing, activation of key effectors of the wounding response known as stellate cells leads to a multitude of pathological processes, including increased production of endothelin-1 (ET-1). This latter process has been linked to enhanced expression of endothelin-converting enzyme-1 (ECE-1, the enzyme that converts precursor ET-1 to the mature peptide) in activated stellate cells. Herein, we demonstrate up-regulation of 56- and 62-kDa ECE-1 3'-untranslated region (UTR) mRNA binding proteins in stellate cells after liver injury and stellate cell activation. Binding of these proteins was localized to a CC-rich region in the proximal ECE-1 3' UTR base pairs (the 56-kDa protein) and to a region between 60 and 193 base pairs in the ECE-1 3' UTR mRNA (62 kDa). A functional role for the 3' UTR mRNA/protein interaction was established in a series of reporter assays. Additionally, transforming growth factor-beta1, a cytokine integral to wound healing, stimulated ET-1 production. This effect was due to ECE-1 mRNA stabilization and increased ECE-1 expression in stellate cells, which in turn was a result of de novo synthesis of the identified 56- and 62-kDa ECE-1 3' UTR mRNA binding proteins. These data indicate that liver injury and the hepatic wound healing response lead to ECE-1 mRNA stabilization in stellate cells via binding of 56- and 62-kDa proteins, which in turn are regulated by transforming growth factor-beta. The possibility that the same or similar regulatory events are present in other forms of wound healing is raised.     

Induction of the chemokine beta peptides, MIP-1 alpha and MIP-1 beta, by lipopolysaccharide is differentially regulated by immunomodulatory cytokines gamma-IFN, IL-10, IL-4, and TGF-beta.

The macrophage occupies a central role in the host response to invasion, exerting its control over the developing inflammatory response largely through the elaboration of an assortment of endogenous mediators including many cytokines. The beta chemokine peptides, macrophage inflammatory protein [MIP]-1 alpha and MIP-1 beta, are two such effectors markedly up-regulated in macrophages following exposure to bacterial lipopolysaccharide (LPS). These highly homologous peptides, like the other members of the beta chemokine family, exhibit diverse but partially overlapping biological activity profiles, suggesting that the cellular participants and intensity of an inflammatory response may in part be regulated by selective expression of these chemokines. Studies reported here demonstrate that, in contrast to the "balanced" MIP-1 alpha/MIP-1 beta chemokine responses of LPS-stimulated macrophage cultures in vitro, circulating levels of MIP-1 beta are significantly higher than those of MIP-1 alpha following LPS administration in vivo. Further studies have revealed that several immunomodulatory cytokines known to be up-regulated in vivo as a consequence of exposure to an invasive stimulus (gamma-IFN, IL-10, IL-4, and transforming growth factor [TGF]-beta) down-regulated the LPS-induced release of MIP-1 alpha by macrophages in vitro, but spared the MIP-1 beta response. This altered pattern of secretion may explain, at least in part, the high circulating levels of MIP-1 beta relative to MIP-1 alpha observed in vivo in response to LPS challenge.     

Regulation of Staphylococcus epidermidis-induced IFN-gamma in whole human blood: the role of endogenous IL-18, IL-12, IL-1, and TNF

Interleukin 12 (IL-12) and IL-18 act synergistically to stimulate interferon gamma (IFN-gamma) production; moreover, IL-1 and tumor necrosis factor (TNF) may also augment IFN-gamma synthesis. We have investigated the relative contributions of these cytokines in the production of IFN-gamma and TNF by the Gram-positive bacterium Staphylococcus epidermidis, using the specific cytokine inhibitors IL-18 binding protein (IL-18BP), IL-1 receptor antagonist (IL-1Ra), anti-IL-12 antibodies (anti-IL-12 Ab), and TNF binding protein. Inhibition of caspase-1 reduced IFN-gamma and IL-1beta levels (by 80 and 67%, respectively) when heat-killed S. epidermidis was added to whole human blood cultures. IL-18BP reduced S. epidermidis-induced IFN-gamma (77% maximal suppression). In contrast, blocking IL-1 receptors by IL-1Ra had no effect on IFN-gamma production. Blocking endogenous IL-12 and TNF reduced IFN-gamma production by 69 and 36%. S. epidermidis-induced TNF-alpha was inhibited by IL-18BP and IL-1Ra, but not anti-IL-12 Ab, whereas IL-8 production was unaffected by any of the specific cytokine blocking agents. In conclusion, S. epidermidis stimulates IFN-gamma which is IL-18, IL-12 and TNF-dependent, but IL-1 independent.     

The involvement of TNF,IL-1 and IL-6 in the immune response to protozoan parasites

One early reaction of the host to infection with protozoan parasites is the secretion of an array of potent cytokines including tumor necrosis factor (TNF), interleukin 1 (IL-1) and IL-6. The combined action of these cytokines causes fever, leukocytosis and the production of acute phase proteins such as C-reactive protein (CRP). These early responses contribute significantly to the outcome of infection by influencing the course of infection directly and by regulating the specific immune response to the parasite.     

七氟烷吸入麻醉对单肺通气患者血清IL-6、IL-10、MIP-2、SP-D及HMGB1水平的影响

目的:探讨七氟烷吸入麻醉对单肺通气患者血白细胞介素-6(IL-6)、白细胞介素-10(IL-10)、巨噬细胞炎性蛋白-2(MIP-2)、人肺表面活性特异蛋白(SP-D)及高迁移率族蛋白1(HMGB1)水平的影响。方法:选择2014年9月~2016年9月于我院行单肺通气的患者108例,参照抽签法分为两组,每组各54例。对照组行常规麻醉,实验组采用七氟烷麻醉,比较两组通气前后血清IL-6、IL-10、MIP-2、SP-D、HMGB1、丙二醇(MDA)、超氧化物歧化醇(SOD)、平均动脉压(MAP)、心率(HR)水平的变化及并发症的发生情况。结果:通气前,两组血清IL-6、IL-10、MIP-2、SP-D、HMGB1、MDA、SOD、MAP、HR水平比较差异均无统计学意义(P0.05)。通气后,两组血清IL-6、IL-10、MIP-2、SP-D、HMGB1、MDA水平均较治疗前显著上升,而实验组以上指标均明显低于对照组;两组血清SOD、MAP、HR均较治疗前显著降低,且研究组低于对照组,差异均有统计学意义(P0.05)。结论:七氟烷吸入麻醉能够抑制单肺通气患者血清IL-6、IL-10、MIP-2、SP-D及HMGB1水平上升,改善机体的氧化应激状态,利于血流动力学的稳定,发挥肺部保护作用。