Dimethylfumarate inhibits MIF-induced proliferation of keratinocytes by inhibiting MSK1 and RSK1 activation and by inducing nuclear p-c-Jun (S63) and p-p53 (S15) expression.

OBJECTIVE: Dimethylfumarate (DMF) is used in the treatment of psoriasis. Macrophage migration inhibitory factor (MIF) is elevated in patients with severe psoriasis. We studied the effect of DMF on the MIF-induced activation of the mitogen- and stress-activated kinase 1 (MSK1) and p90 kDa ribosomal S6 kinase (RSK1) signaling pathways which regulate the proliferation of human keratinocytes via transcription factors. METHODS: The effects of DMF on the MIF-induced activation of MSK1, RSK1, cAMP-responsive element-binding protein (CREB), Cox-2 and c-Jun, JunB and p53 were studied by Western blotting using phospho-specific antibodies. RESULTS: DMF inhibited the MIF-induced phosphorylation of MSK1, RSK1, CREB and JunB, and reduced Cox-2 expression and the proliferation of cultured human keratinocytes. The expression of p-p53 (S15) was induced simultaneously with the inhibition of Cox-2. Addition of DMF before MIF induced nuclear expression of p-c-Jun (S63) and c-Jun. Transfection with small interfering MSK1 and RSK1 RNA before MIF incubation stimulated p-p53 (S15) and nuclear p-c-Jun (S63) similarly to DMF. CONCLUSION: Our results indicate that the specific inhibitory effects of DMF on RSK1 and MSK1 activation together with the induction of p-c-Jun (S63) and p-p53 (S15) lead to the inhibition of keratinocyte proliferation, partly explaining the anti-psoriatic effect of DMF.

Expression of the T-helper 2-specific chemokine receptor CCR4 on CCR10-positive lymphocytes in atopic dermatitis skin but not in psoriasis skin.

BACKGROUND: Atopic dermatitis (AD) and psoriasis are inflammatory skin diseases. AD is generally perceived as a T-helper (Th) 2-dominated disease whereas psoriasis is a Th1-dominated disease. The chemokine cutaneous T-cell attracting chemokine (CTACK) and its receptor CCR10 attract skin-homing lymphocytes to inflamed skin, suggesting that CCR10+ cells in AD and psoriasis should be of Th2 and Th1 type, respectively. The chemokine receptor CCR4 is expressed selectively on Th2 lymphocytes and its ligand thymus and activation-regulated chemokine (TARC) is upregulated in AD lesions, suggesting that the CCR10+ cells in AD lesions should also express CCR4. OBJECTIVES: To examine the coexpression of CCR10 and CCR4 on skin-invading lymphocytes in AD and psoriasis lesions as well as the Th1/Th2 cytokine expression of the CCR10+ lymphocytes. METHODS: Skin biopsies from AD and psoriasis patients were double stained with antibodies against CCR10-CCR4, CCR10-CCR5, CCR10-interleukin (IL)-2 and CCR10-IL-4. RESULTS: The CCR10+ cells in AD showed a mixed IL-2/IL-4 expression pattern, and a minor proportion expressed CCR4, whereas a large proportion of the CCR4+ cells did not express CCR10. In psoriasis the CCR10+ cells only expressed IL-2, and no CCR4 expression was detected. CONCLUSIONS: The CCR10+ lymphocytes invading the skin in AD and psoriasis have different Th1/Th2 profiles, as measured by both their cytokine and chemokine receptor expression. This suggests that the CCR10+ subpopulation of lymphocytes is made up of different Th1/Th2 subsets. However, the Th1/Th2 lymphocytes of AD and psoriasis were either CCR10+ or CCR10-, suggesting that both the Th1 and Th2 subpopulation can be subdivided further. CCR4 was found only in AD skin and on both CCR10+ and CCR10- cells, supporting the hypothesis of TARC and CTACK as being independent lymphocyte-attracting chemokines in AD.

Expression of IL-18 mRNA and secretion of IL-18 are reduced in monocytes from patients with atopic dermatitis.

BACKGROUND: IL-18 has been found to be an IFN-gamma-inducing factor that plays an important role in T(H)1 cell activation. Recently, IL-18 has also been found to enhance a T(H)2 cellular response in a specific setting. OBJECTIVE: The aim of this study was to elucidate the role of monocytes and soluble factors, with special focus on IL-18, in the pathogenesis of atopic dermatitis (AD). METHODS: The release of cytokines from PBMCs and purified monocytes was measured through use of ELISA; mRNA expression was evaluated by RT-PCR. The results from patients with AD were compared with those from healthy controls. RESULTS: IL-18 secretion was reduced in both unstimulated and lipopolysaccharide-stimulated monocytes from patients with AD. The mRNA expression of IL-18 and IL-1 beta-converting enzyme was significantly reduced in unstimulated monocytes from patients with AD (P <.03 and P <.01, respectively). Patients with AD had an elevated secretion of prostaglandin E(2) (PGE(2)) from unstimulated PBMCs (P <.001). The anti-PGE(2) antibody reversed the suppressive effect of PGE(2) on IL-18 secretion in unstimulated PBMCs from patients with AD. CONCLUSIONS: Decreased IL-18 production, together with a significantly reduced IL-18 and ICE mRNA expression in unstimulated monocytes and elevated PGE(2) secretion from PBMCs, was associated with the pathogenesis of AD.

Cytokine expression of skin T-lymphocytes from patients with atopic dermatitis.

We analysed the cytokine profile of skin T cells by establishing 11 T-cell lines from adult patients with moderate-to-severe atopic eczema using T-cell growth factors interleukin-2 and interleukin-4. We compared T-cell lines from lesional skin of atopic dermatitis patients with those from non-atopic skin of patients with other skin diseases, observing that T-cell lines of patients with atopic dermatitis unstimulated cultures expressed a Th1 profile. After stimulation with anti-CD3 and anti-CD28 monoclonal antibodies, the cytokine expression showed rapid initial upregulation of Th2 followed by a Th1 profile. Furthermore, strong upregulation of interleukin-10 was observed after 24 h stimulation. Our findings suggest that skin T-lymphocytes from atopic dermatitis patients seem to consist of a heterogenous population of Th1 and Th2 or Th0 cells and the results for secreted cytokines indicate that T-cell lines from each inflammatory skin disease showed the corresponding disease-specific original cytokine profile.

IL-10 augments the IFN-gamma and TNF-alpha induced TARC production in HaCaT cells: a possible mechanism in the inflammatory reaction of atopic dermatitis.

The CC-chemokine TARC is known to be a ligand for the CCR4 receptor which in turn is known to be expressed selectively on the Th(2)-subset of lymphocytes. Atopic dermatitis is generally believed to be a Th(2)-type disease, and TARC has been shown to be expressed in the skin lesions of a murine model of AD. IL-10 is an interleukine generally known for its ability to inhibit cytokine production, however it has been found to be highly expressed in the skin from AD patients. We show in this report that IL-10 is able to augment the TARC inducing effects of TNFalpha and IFNgamma in HaCaT cells, a property that may be important in the determination of the composition of the cells of the inflammation in the skin of AD patients. In addition, we show that the IL10 agonist IT 9302, a nona-peptide from the carboxylic end of IL-10, has the same effect on TARC production from HaCaT cells.

A Th2 chemokine, TARC, produced by keratinocytes may recruit CLA+CCR4+ lymphocytes into lesional atopic dermatitis skin.

Atopic dermatitis is an inflammatory skin disease in which the inflammation is characterized by the influx of lymphocytes into the dermis. It is generally believed that atopic dermatitis is a Th2-type disease, i.e., the T lymphocytes produce interleukin-4, interleukin-5, interleukin-10, and interleukin-13, although it has become evident in recent years that the cytokine profile in the skin changes during the course of the disease towards a Th1-Th2 mixed cytokine profile (interferon-gamma, tumor necrosis factor alpha, and interleukin-2). The lymphocytes that home into the skin express cutaneous lymphocyte-associated antigen, and it has recently been shown that most of the lymphocytes in this population express the chemokine receptor CCR4. CCR4 is the receptor for the CC chemokine TARC (thymus and activation regulated chemokine), and this chemokine is expressed predominantly by keratinocytes in the basal layer of the epidermis of lesional atopic dermatitis skin in mice. In humans, however, it was shown to be expressed in the endothelial cells of the dermis. We have examined the peripheral blood mononuclear cells of atopic dermatitis patients for the expression of cutaneous lymphocyte-associated antigen and CCR4 and compared them with peripheral blood mononuclear cells from normal controls. We found that the proportion of CLA+CCR4+ lymphocytes is upregulated in atopic dermatitis patients. In addition we have examined skin biopsies of lesional and non-lesional skin from atopic dermatitis patients and found that the keratinocytes, but not the endothelial cells, produce TARC in the lesional but not in the nonlesional skin. To gain insight in the stimulatory mechanisms for TARC production in keratinocytes, as previously observed in mice, we cultured HaCaT cells and found that interferon-gamma and tumor necrosis factor alpha work synergistically to induce TARC production. These observations suggest that the induction of TARC production in keratinocytes plays an important role in the late phase skin invasion by CCR4+CLA+ Th2-type lymphocytes in atopic dermatitis.

Pretreatment with granulocyte colony-stimulating factor attenuates the inflammatory response but not the bacterial load in cerebrospinal fluid during experimental pneumococcal meningitis in rabbits.

A possible immunomodulatory role of granulocyte colony-stimulating factor (G-CSF) was investigated in an experimental pneumococcal meningitis model in rabbits. Animals were pretreated with G-CSF (10 micrograms/kg subcutaneously twice a day) starting 48 h before in vivo and ex vivo experiments, causing a five- to six-fold increase in the peripheral leukocyte level. Meningitis was induced by intracisternal inoculation of approximately 4 x 10(5) CFU of Streptococcus pneumoniae type 3. Neutrophil pleocytosis and interleukin-8 (IL-8) levels were significantly attenuated in G-CSF-pretreated animals compared to untreated animals (P < 0.05). Furthermore, G-CSF pretreatment significantly delayed alterations in cerebrospinal fluid (CSF) tumor necrosis factor alpha and IL-1beta levels, as well as protein and glucose levels (P < 0.05). No difference in CSF bacterial concentrations was found, whereas the blood bacterial concentration was significantly decreased in G-CSF-pretreated animals (P < 0.05). Ex vivo chemotaxis of neutrophils isolated from G-CSF-pretreated animals was significantly decreased compared to that of neutrophils from untreated animals (P < 0.05). In conclusion, G-CSF pretreatment attenuates meningeal inflammation and enhances systemic bacterial killing. Further preclinical studies are required to investigate whether this may affect the clinical course of meningitis and thus whether G-CSF treatment may have a beneficial role in pneumococcal meningitis.

Systemic and cell-mediated immune response after laparoscopic and open cholecystectomy in patients with chronic liver disease. A randomized, prospective study.

BACKGROUND: As impaired immune function observed in cirrhotic patients is known to increase the risk of postoperative complications, the immunological response to surgery was investigated. METHODS: Twenty-eight patients with postnecrotic liver cirrhosis or chronic hepatitis C and symptomatic gallstone disease were randomly allocated to laparoscopic (LC) or open cholecystectomy (OC). Changes in concentrations of cytokines (TNF-alpha, IL-1beta, IL-6, IL-8 and IL-10) were followed and the effect of surgical trauma on the distribution of lymphocyte subpopulations (CD3, CD4, CD8, CD16 and CD19) and NK cell cytotoxicity were measured. RESULTS: After OC a decrease in circulating CD3 (p < 0.05) and CD4 (p < 0.05) and an increase in CD19 (p < 0.05) cells were detected in contrast to LC after which only CD16 cells decreased (p = 0.05). The number of CD3 cells was higher after LC than after OC (p < 0.01), whereas the number of CD19 cells was higher after OC than after LC (p < 0.01). NK cell cytotoxicity was reduced after LC (p < 0.05). In cirrhotic patients circulating cytokines were unaffected by OC, whereas TNF-alpha (p < 0.05) and IL-1beta (p < 0.05) were reduced after LC. In chronic hepatitis IL-1beta decreased after OC (p = 0.05) and IL-10 was significantly higher after LC than following OC (p < 0.05). CONCLUSION: The immune response is less pronounced after a laparoscopic procedure compared to a conventional approach in patients with chronic liver disease.

Tirilazad inhibits surgically induced edema and interleukin-1 production. An experimental study in rats.

The aim of this study was to evaluate the anti-inflammatory effect of tirilazad mesylate on edema and interleukin-1 (IL-1) levels in serum following standardized surgical procedures. Four groups, each containing eight rats, were randomized for treatment as follows: A) no medication, B) low-dose tirilazad, C) high-dose tirilazad, and D) corticosteroids. The animals were examined by nuclear magnetic resonance imaging (NMRI) 24 and 72 hours after surgery and the NMRI data were used in the determination of soft tissue edema. In addition, serum was obtained for analysis of IL-1 levels. Four other groups of animals were subjected to the same treatment regimen as groups A-D), respectively, and 24 hours after surgery the animals were killed, whereafter serum was obtained for analysis of IL-1 levels. The present study demonstrated that low-dose tirilazad significantly reduces soft tissue edema compared with all other treatment regimens 24 hours postoperatively. At 72 hours postoperatively significant reduction of soft tissue edema was achieved at low-dose tirilazad when compared to high-dose tirilazad and steroids. In addition, a significant suppression of the expression of IL-1 was observed at 24 and 72 hours when comparing low-dose tirilazad and the control group.

IT 9302, a synthetic interleukin-10 agonist, diminishes acute lung injury in rabbits with acute necrotizing pancreatitis.

BACKGROUND: Proinflammatory cytokines (eg, tumor necrosis factor [TNF]-alpha, interleukin [IL]-1 and Il- 8) are believed to play an important role in the pathogenesis of acute necrotizing pancreatitis (ANP) and its systemic complications. Recently, IL-10 has emerged as a major anti-inflammatory cytokine, inhibiting the secretion and activities of inflammatory cytokines. Further, a protective effect of IL-10 has recently been shown in experimental acute pancreatitis. The purpose of this study was to test the potential role of a newly developed IL-10 agonist, IT 9302, in a model of ANP in rabbits. METHODS: ANP was induced in 18 rabbits by retrograde injection of 5% chenodeoxycholic acid in the pancreatic duct, followed by duct ligation. The rabbits were allocated to pretreatment with intravenous physiologic saline solution or IT 9302 (200 micrograms/kg) 30 minutes before the induction of ANP. RESULTS: Injection of IT 9302 resulted in a significant reduction in the blood levels of TNF-alpha and IL-8 from 3 to 6 hours. IT 9302 also reduced the amount of ascitic fluid and significantly inhibited neutrophil infiltration and margination, as well as the number of CD11b- and CD18-positive cells in the lung tissues. By contrast, the local pancreatic necrosis, as well as the biochemical changes such as serum amylase, lipase, and calcium, was sever and similar in both groups. Survival was improved significantly after treatment with IT 9302. CONCLUSIONS: As expected, IT 9302 cannot change the degree of ANP induced by 5% bile acid but does reduce mortality rates and the development of acute lung injury, probably through the inhibition of circulating levels of TNF-alpha, IL-8, and the expression of the adhesion molecule complex CD11b/CD18.

A monoclonal anti-interleukin 8 antibody (WS-4) inhibits cytokine response and acute lung injury in experimental severe acute necrotising pancreatitis in rabbits.

BACKGROUND: Interleukin 8 (IL-8) has recently been proposed to have an important role in mediating the development of the systemic sequelae associated with severe acute pancreatitis. AIMS: To define the role of IL-8 in acute pancreatitis by neutralising its effects with a monoclonal anti-IL-8 antibody (WS-4), in a rabbit model of severe acute pancreatitis. METHODS: Acute pancreatitis was induced by retrograde injection of 5% chenodeoxycholic acid into the pancreatic duct and duct ligation. Twenty rabbits were divided equally into two groups: acute pancreatitis controls received physiological saline and the treated group received WS-4, 30 minutes before induction of acute pancreatitis. RESULTS: Pretreatment of animals with WS-4 resulted in significant down regulation of serum IL-8 and tumour necrosis factor alpha (TNF-alpha) from three to six hours after induction of acute pancreatitis (p = 0.011 and 0.047 for IL-8 and 0.033 and 0.022 for TNF-alpha, respectively). In addition, a significant reduction in the CD11b and CD18 positive cells and the amount of interstitial neutrophil infiltration in the lungs from WS-4 treated animals was seen. In contrast, WS-4 did not alter the amount of pancreatic necrosis and the serum concentrations of amylase, lipase, calcium, and glucose. CONCLUSION: WS-4 cannot change the amount of pancreatic necrosis induced by injection of 5% bile acid, but does reduce the acute lung injury, presumably through inhibition of circulating IL-8 and TNF-alpha, and CD11b/CD18 in lung tissue. Therefore, a role of IL-8 in the progression of acute pancreatitis and the development of its systemic complications is suggested.

Monocyte chemotactic and activating factor (MCAF/MCP-1) has an autoinductive effect in monocytes, a process regulated by IL-10.

MCAF (MCP-1) a member of the chemokine-beta-family known to be chemotactic for monocytes is believed to play a significant role in several inflammatory processes, both immuno-pathological disorders, such as atherosclerosis, psoriasis, chronic inflammatory diseases of the liver and lungs, and during the normal immune response against microorganisms. This chemokine is produced spontaneously by monocytes, and in the present article we also demonstrate that MCAF induces its own production in monocytes. The methods used are two dimensional SDS-PAGE gel electrophoresis. Western-blotting and ELISA quantification of supernatant from monocyte cultures stimulated with MCAF (1, 10, 100 ng ml). Also, we found that this process is regulated by IL-10 (100 ng ml). Our results suggest that monocytes migrating to a site of inflammation due to the local production of the chemokine MCAF/MCP-1 further enhance the focal accumulation of monocytes by producing and releasing bioactive MCAF MCP-1.

Identification of functional domains on human interleukin 10.

Interleukin 10 (IL-10) is a recently described natural endogenous immunosuppressive cytokine that has been identified in human, murine, and other organisms. Human IL-10 (hIL-10) has high homology with murine IL-10 (mIL-10) as well as with an Epstein-Barr virus genome product BCRFI. This viral IL-10 (vIL-10) shares a number of activities with hIL-10. IL-10 significantly affects chemokine biology, because human IL-10 inhibits chemokine production and is a specific chemotactic factor for CD8+ T cells. It suppresses the ability of CD4+ T cells, but not CD8+ T cells, to migrate in response to IL-8. A nonapeptide (IT9302) with complete homology to a sequence of hIL-10 located in the C-terminal portion (residues 152-160) of the cytokine was found to possess activities that mimic some of those of hIL-10. These are: (i) inhibition of IL-1beta-induced IL-8 production by peripheral blood mononuclear cell, (ii) inhibition of spontaneous IL-8 production by cultured human monocytes, (iii) induction of IL-1 receptor antagonistic protein production by human monocytes, (iv) induction of chemotactic migration of CD8+ human T lymphocytes in vitro, (v) desensitization of human CD8+ T cells resulting in an unresponsiveness toward rhIL-10-induced chemotaxis, (vi) suppression of the chemotactic response of CD4+ T human lymphocytes toward IL-8, (vii) induction of IL-4 production by cultured normal human CD4+ T cells, (viii) down-regulation of tumor necrosis factor-alpha production by CD8+ T cells, and (ix) inhibition of class II major histocompatibility complex antigen expression on IFN-gamma-stimulated human monocytes. Another nonapeptide (IT9403) close to the NH2-terminal part of hIL-10 did not reveal cytokine synthesis inhibitory properties, but proved to be a regulator of mast cell proliferation. In conclusion, we have identified two functional domains of IL-10 exerting different IL-10 like activities, an observation that suggests that relatively small segments of these signal proteins are responsible for particular biological functions.

Psoriasin: a novel chemotactic protein.

Inflammatory skin disorders such as psoriasis show a preferential epidermal infiltration of neutrophils and T lymphocytes. This observation raises a question as to which factors determine the appearance and composition of leukocyte tissue infiltrations. Previously, we described a low molecular mass calcium-binding protein (psoriasin, molecular mass 11,457 Da, pI 6.77) belonging to the S1OO family that is highly upregulated in psoriatic keratinocytes and whose expression patterns implied a role in the inflammatory response. Here we report that human psoriasin is a potent and selective chemotactic inflammatory protein for CD4+ T lymphocytes and neutrophils at concentrations of about 10(-11) M. Psoriasin is not structurally related to the alpha or the beta chemokine subfamilies or to lymphotactin, a member of a newly described class of chemokines. Thus, we have observed a chemotactic protein outside the chemokine subfamilies that could be an important new inflammatory mediator.

IL-8 induces T cell chemotaxis, suppresses IL-4, and up-regulates IL-8 production by CD4+ T cells.

Interleukin-8 (IL-8), a neutrophil-activating cytokine, also activates certain T cell functions such as chemotaxis. We additionally find (n = 6) that recombinant (rIL-8; 1-100 ng/ml), when added to 24 h culture of human CD4+ T cells, suppressed the spontaneous production of IL-4 (50-85%). Steady state production of Il-4 was typically around 30 pg/ml, determined by use of a solid- phase immunoabsorbant assay. De novo synthesis of IL-4 from CD4+ T cells cultured for 3 days was also evaluated by use of detection of [35S]methionine incorporation, as visualized by autoradiography of 2-D gels, and showed that IL-8 suppressed IL-4 production. This suppression of IL-4 production was confirmed in the cytosol fraction by use of Western blotting. The effect of IL-8 (100 ng/ml) was comparable to that of 10 ng/ml recombinant interferon-gamma, both strongly suppressing IL-4 production. The regulatory effect of IL-8 on IL-4 production was also indicated by the fact that addition of a neutralizing monoclonal anti-IL-8 antibody (WS.4) enhanced the spontaneous IL-4 production when added to the culture of CD4+ T cells, thereby probably inactivating the effect of IL-8 originating from the cultured T cells. Also, we observed that IL-4 mRNA expression was down-regulated when the CD4+ T cells were cultured for 12 h in the presence of 100 ng/ml IL-8. The suppression of IL-4 mRNA expression could be prevented by adding anti-IL-8 (20 microgram/ml) or IL-10 (100 ng/ml) l h before adding rIL-8. Thus, IL-8 may be an important regulator of CD4+ T cell-derived IL-4, thereby possibly regulating the balance between humoral and cellular T cell-dependent responses.

Heterogeneous nuclear ribonucleoproteins H, H', and F are members of a ubiquitously expressed subfamily of related but distinct proteins encoded by genes mapping to different chromosomes.

Molecular cDNA cloning, two-dimensional gel immunoblotting, and amino acid microsequencing identified three sequence-unique and distinct proteins that constitute a subfamily of ubiquitously expressed heterogeneous nuclear ribonucleoproteins corresponding to hnRNPs H, H', and F. These proteins share epitopes and sequence identity with two other proteins, isoelectric focusing sample spot numbers 2222 (37.6 kDa; pI 6.5) and 2326 (39.5 kDa; pI 6.6), indicating that the subfamily may contain additional members. The identity between hnRNPs H and H' is 96%, between H and F 78%, and between H' and F 75%, respectively. The three proteins contain three repeats, which we denote quasi-RRMs (qRRMs) since they have a remote similarity to the RNA recognition motif (RRM). The three qRRMs of hnRNP H, with a few additional NH2-terminal amino acids, were constructed by polymerase chain reaction amplification and used for ribohomopolymer binding studies. Each qRRM repeat bound poly(rG), while only the NH2-terminal qRRM bound poly(rC) and poly(rU). None of the repeats bound detectable amounts of poly(rA). The expression levels of hnRNPs H and F were differentially regulated in pairs of normal and transformed fibroblasts and keratinocytes. In normal human keratinocytes, the expression level of H was unaffected by treatment with several substances tested including two second messengers and seven cytokines. Likewise the expression level of F was independent of these substances, although it was strikingly down-regulated by long term treatment with 4 beta-phorbol 12-myristate 13-acetate, indicating that the protein kinase C signaling pathway regulates its expression. No effect of 4 beta-phorbol 12-myristate 13-acetate was observed on the expression of hnRNP H. The genes coding for hnRNPs H, H', and F were chromosome-mapped to 5q35.3 (HNRPH1), 6q25.3-q26, and/or Xq22 (HNRPH2) and 10q11.21-q11.22 (HNRPF), respectively.

The delayed-type hypersensitivity reaction is dependent on IL-8. Inhibition of a tuberculin skin reaction by an anti-IL-8 monoclonal antibody.

Cell-mediated immune reactions are essential to our immune response toward foreign organisms such as microorganisms, or in the response toward foreign tissue Ags, as seen in the rejection of allogeneic transplanted organs. Similar reactions form the basis for the development and the progression of delayed-type hypersensitivity (DTH) reactions. We found that the alpha-chemokine IL-8 plays an important pathophysiologic role for the development of a DTH reaction because infusion of a neutralizing anti-IL-8 mAb (WS-4) was able to suppress the development of a tuberculin skin reaction in rabbits, as judged by histologic, biochemical, and clinical examinations. Thus, the number of neutrophil granulocytes and lymphocytes at the site of tuberculin injection was decreased considerably, and the clinical signs of inflammation were suppressed almost completely at 24 h after intracutaneous injection of tuberculin, as judged by the size of the infiltrates. In contrast, we did not see any effect on the visible erythema of the skin. We found that the tissue content of myeloperoxidase (MPO), reflecting the number of infiltrating neutrophils, was lowered significantly. Furthermore, immunohistochemical analysis confirmed that IL-8 immunoreactivity is actually enhanced in the skin of positive tuberculin reactions. The results indicate that IL-8 plays an important role for the early accumulation of leukocytes in the skin and for the clinical signs of a DTH reaction.

Interleukin-8 induces its own production in CD4+ T lymphocytes: a process regulated by interleukin 10.

The neutrophil and T cell chemotactic factor interleukin 8 (IL-8) is believed to play a pathophysiological role in the development of various inflammatory disorders. So far no other effects of IL-8 on T cells have been observed. We observed that purified CD4+ T cells in particular, but also CD8+ T cells, spontaneously synthesize IL-8 mRNA and secrete IL-8 protein. The culture supernatants of CD4+ T cells contained T cell chemotactic activity as well as IL-8 protein. In addition, we confirmed the ability of CD4+ T cells to produce IL-8 by double immunofluorescence staining and by the demonstration of IL-8 mRNA expression. Further, IL-8 induced its own production in CD4+ T cells, while its synthesis by CD8+ T cells was low and not always auto-stimulatory. Both the spontaneous, as well as the IL-8 induced IL-8 production, could be inhibited in the presence of human interleukin 10 (100 ng/ml). This observation suggests that IL-10 plays a homeostatic role in regulating the IL-8 circuit in CD4+ T cells.

Regulation of human T lymphocyte chemotaxis in vitro by T cell-derived cytokines IL-2, IFN-gamma, IL-4, IL-10, and IL-13.

There has been a number of conflicting reports regarding the T lymphocyte chemotactic activities of several cytokines. IL-2 and IFN-gamma are known to promote augmentation of immune inflammation, whereas IL-4, IL-10, and IL-13 display immunomodulatory effects on inflammatory cells including inhibition of cytokine production. Their effects on chemotaxis of inflammatory cells are unknown. We observed that IL-1 alpha could induce chemotaxis both in overnight cultured and anti-CD3 mAb-activated T lymphocytes and that overnight culture and anti-CD3 activation increase the number of IL-1R on T lymphocytes. In contrast, IL-8 selectively attracts freshly isolated T lymphocytes. Staurosporine inhibits freshly isolated T lymphocyte chemotaxis toward IL-8, whereas tyrphostin 23 inhibits chemotaxis of overnight cultured and anti-CD3-activated T lymphocytes toward IL-1 alpha. We have found that IL-2 and IL-13 inhibit the chemotactic migration of both CD4+ and CD8+ T lymphocytes toward IL-8, and RANTES. IL-4 inhibits only CD8+ T lymphocyte chemotaxis toward RANTES, IL-8 and IL-10. IL-10 inhibits only CD4+ T lymphocytes in their chemotactic response toward RANTES and IL-8. IFN-gamma does on the other hand augment the sensitivity of human T lymphocytes to chemotactic stimuli. Thus, our results demonstrate that different proinflammatory cytokines will induce chemotactic migration of T lymphocytes under different circumstances acting through different signaling pathways. The T cell-derived cytokines IL-2, IL-4, IL-10, and IL-13 are able to block further T lymphocyte chemotaxis, thus leading to a focusing of T lymphocytes in an area of T lymphocyte activation. These mechanisms seem relevant in our understanding of the specific and continuous localization of T lymphocytes in allergic and autoimmune disorders.