CD19, a response regulator of B lymphocytes, regulates wound healing through hyaluronan-induced TLR4 signaling.

Immune cells are critical to the wound-healing process, through both cytokine and growth factor secretion. Although previous studies have revealed that B cells are present within wound tissue, little is known about the role of B cells in wound healing. To clarify this, we investigated cutaneous wound healing in mice either lacking or overexpressing CD19, a critical positive-response regulator of B cells. CD19 deficiency inhibited wound healing, infiltration of neutrophils and macrophages, and cytokine expression, including basic and acidic fibroblast growth factor, interleukin-6, platelet-derived growth factor, and transforming growth factor-beta. By contrast, CD19 overexpression enhanced wound healing and cytokine expression. Hyaluronan (HA), an endogenous ligand for toll-like receptor (TLR)-4, stimulated B cells, which infiltrates into wounds to produce interleukin-6 and transforming growth factor-beta through TLR4 in a CD19-dependent manner. CD19 expression regulated TLR4 signaling through p38 activation. HA accumulation was increased in injured skin tissue relative to normal skin, and exogenous application of HA promoted wound repair in wild-type but not CD19-deficient mice, suggesting that the beneficial effects of HA to the wound-healing process are CD19-dependent. Collectively, these results suggest that increased HA accumulation in injured skin induces cytokine production by stimulating B cells through TLR4 in a CD19-dependent manner. Thus, this study is the first to reveal a critical role of B cells and novel mechanisms in wound healing.

P-selectin glycoprotein ligand-1 contributes to wound healing predominantly as a p-selectin ligand and partly as an e-selectin ligand.

Cell adhesion molecules are critical to wound healing through leukocyte recruitment. Although P-selectin glycoprotein ligand-1 (PSGL-1) regulates leukocyte rolling by binding P-selectin, but also binding E- and L-selectins with lower affinity, little is known about a role of PSGL-1 in wound healing. To clarify a role of PSGL-1 and its interaction with E- and P-selectins in wound healing, we investigated cutaneous wound healing in PSGL-1-deficient (PSGL-1(-/-)) mice in comparison with E-selectin(-/-), P-selectin(-/-), and P-selectin(-/-) mice treated with an anti-E-selectin antibody. PSGL-1 deficiency inhibited early wound healing, which was accompanied by decreased inflammatory cell infiltration and growth factor expression. By contrast, E-selectin deficiency did not affect wound healing. In general, the inhibitory effect of PSGL-1 deficiency on wound healing was similar to that of P-selectin deficiency either alone or with E-selectin blockade. However, early granulation tissue formation, late angiogenesis, and early infiltration of neutrophils and macrophages in PSGL-1(-/-) mice were inhibited beyond the inhibition in P-selectin(-/-) mice, but to a similar level of inhibition in P-selectin(-/-) mice with E-selectin blockade. These results suggest that PSGL-1 contributes to wound healing predominantly as a P-selectin ligand and partly as an E-selectin ligand by mediating infiltration of inflammatory cells.

Increased serum levels of N(epsilon)-(hexanoyl)lysine, a new marker of oxidative stress, in systemic sclerosis.

OBJECTIVE: To determine serum levels of N(epsilon)-(hexanoyl)lysine (HEL), a new marker of oxidative stress, and its clinical association in patients with systemic sclerosis (SSc). METHODS: Serum HEL levels from 26 patients with limited cutaneous SSc (lSSc), 34 with diffuse cutaneous SSc (dSSc), 20 with systemic lupus erythematosus (SLE), 20 with dermatomyositis (DM), and 40 healthy individuals were examined by enzyme linked immunosorbent assay. RESULTS: Serum HEL levels were elevated in patients with SSc compared with healthy controls (n = 40) with similar levels between patients with lSSc and dSSc (p < 0.0001). SSc patients with elevated HEL levels had increased serum levels of anti-agalactosyl IgG antibody, rheumatoid factor (RF), and IgM than those with normal HEL levels (p < 0.05). HEL levels correlated positively with anti-agalactosyl IgG antibody (p = 0.013, r = 0.408) and RF titer (p = 0.0028, r = 0.426). CONCLUSION: Our results suggest that oxidative stress may play an important role in immunological abnormalities of SSc, especially in the production of autoantibodies including anti-agalactosyl IgG antibody and RF.

Increased levels of urinary nitrite and nitrotyrosine in Yusho victims 40 years after accidental poisoning with polychlorinated biphenyls in Nagasaki, Japan.

Forty years have passed since the accidental poisoning with polychlorinated biphenyls (PCB) in Japan in 1968, named Yusho. High concentrations of PCB are still detected in the serum of the Yusho victims. PCB produces superoxide (O(2) (-)) in the metabolic process and we reported high concentrations of serum nitrite, a stable metabolite reflecting nitric oxide (NO), in the Yusho victims. NO reacts with O(2) (-) and immediately produces peroxynitrite (ONOO(-)). ONOO(-) causes nitration of tyrosine residues and produces nitrotyrosine (NT). Therefore, we measured urinary concentrations of nitrite and NT in the victims and age-matched controls. The mean urinary concentrations of nitrite and NT were significantly higher than in the controls. There was a positive correlation between urinary nitrite and NT in the Yusho victims. Furthermore, there was a positive correlation between the ratio of urinary NT to nitrite and serum PCB concentrations in the Yusho victims. It was considered that the emergence of some ailments could be presumed to have been caused by high levels of urinary nitrite and NT in the Yusho victims.

Lipid peroxidation is enhanced in Yusho victims 35 years after accidental poisoning with polychlorinated biphenyls in Nagasaki, Japan.

Thirty-five years have passed since the accidental poisoning with polychlorinated biphenyls (PCB) in Japan and yet high concentrations of PCB are still detected in the serum of the victims. PCB produces superoxide and thus victims are considered to be in a persistent state of oxidative stress. Urinary concentrations of 8-isoprostane (8IP) in the victims and age-matched controls were measured to assess this hypothesis. The mean urinary concentration of 8IP was significantly higher than that in the controls. There was a positive correlation between urinary 8IP and serum concentrations of cholinesterase. It was considered that Yusho is an oxidative stress and 8IP is a useful tool for checking the oxidative condition in Yusho victims.

Lipoteichoic acid-related molecule derived from the streptococcal preparation, OK-432, which suppresses atopic dermatitis-like lesions in NC/Nga mice.

Bacterial stimulation may serve to control atopic disorders such as atopic dermatitis (AD) through inducement of Th1 cell-mediated immune response. The lipoteichoic acid (LTA)-related molecule (okLTA) from streptococcal preparation, OK-432, has been shown to be a potent Th1 inducer through the action of IL-12. Examination was made of the therapeutic effects of this okLTA injected intra- and/or subcutaneously into AD-like lesions in NC/Nga mice, particularly in the vicinity of the suppressor of cytokine signaling (SOCS) regulatory pathways. Using immunohistochemical staining with IL-4/IL-12p40 and phosphorylated STAT6/p-STAT4 and RT-PCR for IL-4/IL-12p40, STAT6/STAT4 and mRNA expression and in situ hybridization of SOCS3 and 5, evaluation was made of the immunoregulatory effects of this okLTA in the treatment of spontaneous AD-like lesions in NC/Nga mice. Following the injection of okLTA, remarkable improvement in the lesions of NC/Nga mice was noted. In okLTA-treated skin, IL-12p40/p-STAT4 positive cellular infiltration was extensive while IL-4/p-STAT6 positive cell infiltration was seen to diminish considerably, compared to untreated NC mice. SOCS3 in situ expression in okLTA-treated mice was noted to be significantly less compared to untreated NC mice, in which the expression was prominent. SOCS5 in situ expression was rather, though not significantly, strong in okLTA-treated mice. okLTA treatment is clearly shown to induce Th1 cellular response and down-regulate immune response in the Th2 pathway through SOCS3 reduction in AD-like lesions of NC/Nga mice. The present results demonstrate that bacterial wall components such as okLTA should serve as an effective new therapeutic approach for treating AD.

Uncontrollable prurigo nodularis effectively treated by roxithromycin and tranilast.

Treatment of prurigo nodularis (PN) is often very difficult even with strong corticosteroid dressing and other available means. Macrolide roxithromycin (RXM) is used in consideration of its immunosuppressive effects in treating several skin disorders. Tranilast (N-(3,4-dimethoxycinnamoyl) is useful for treating atopic disorders and hypertrophic scars as well, suggesting its capacity to inhibit fibroblast proliferation. More adequate and effective therapy for this disorder has been requested. We report 3 cases of uncontrollable PN treated with 300 mg/day roxithromycin and 200 mg/day tranilast. Complete and/or remarkable regression of PN was observed on treatment with roxithromycin and tranilast in combination within 4 to 6 months. The 2 agents in combination can be used effectively for the treatment of uncontrollable PN.

Topical glucocorticoid augments scratching behaviour in dinitrofluorobenzene-sensitized mice by the induction of substance P.

Topical glucocorticoid (GC) is commonly applied in atopic dermatitis treatment. However, the chronic use of GC may be associated with significant side effects. In this study, we investigated whether long-term epicutaneous application of GC modulates scratching behaviour in dinitrofluorobenzene (DNFB) contact-sensitized mice. After challenge with DNFB, scratching behaviour was increased in DNFB-sensitized mice treated with GC in contrast to control mice. In addition, reverse transcriptase-polymerase chain reaction analysis demonstrated that the expression of preprotachykinin-A (PPT-A) mRNA, a precursor of substance P (SP), and inducible nitric oxide synthase (iNOS) mRNA in mice, to which GC was applied, was only observed. In order to evaluate the factors responsible for the augmented scratching behaviour, we injected various cytokines (interleukin-1alpha (IL-1alpha), IL-2, IL-3 and tumour necrosis factor-alpha (TNF-alpha)) subcutaneously into the ear of DNFB contact-sensitized mice before DNFB challenge. Among the cytokines, only IL-3 and TNF-alpha significantly increased scratching behaviour in DNFB contact dermatitis mice. Furthermore, PPT-A mRNA was only expressed in mice pre-injected with IL-3 before challenge, but not in those pre-injected with other cytokines. Taken together, our results suggest that topical GC may augment the itching sensation in DNFB-sensitized mice through modulation of iNOS and SP induced by IL-3.

Therapeutic effects of streptococcal preparation OK-432 on atopic dermatitis-like lesions in NC/Nga mice: possible shift from a Th2- to Th1-predominance.

BACKGROUND: The inducement of Th1 cell-mediated immune response, possibly brought about through bacterial stimulation, may serve to control atopic disorders such as atopic dermatitis (AD). The streptococcal preparation, OK-432, has been shown a potent Th1 inducer through the action of IL-12. NC/Nga mice under ordinary conditions have been found to contract dermatitis similar to human AD. OBJECTIVE: Examination was made of the therapeutic effects of OK-432 local intra- and/or subcutaneous injections on AD-like lesions in NC/Nga mice. METHODS: Immunohistochemical staining with IL-4/IL-12p40 and CD80/86 and phosphorylated STAT4/p-STAT6 and RT-PCR for IL-4/IL-12p40 and STAT6/STAT4 mRNA was conducted for the evaluation of OK-432 treatment of spontaneous AD-like lesions in NC/Nga mice. RESULTS: At 5 weeks following injection of OK-432, for treating head and back lesions in NC/Nga mice, 10 of 12 OK-432 treated NC mice were found to have clinically improved quite considerably. On the head and back skin of OK-432-treated mice, IL-12p40/CD80 positive cellular infiltration was conspicuous, in contrast to non-treated mice. IL-4/CD86 positive cellular infiltrates in OK-432-treated mice had decreased significantly more than in non-treated mice and IL-4 mRNA expression was virtually absent in OK-432-treated mice. P-STAT4 positive cells could be seen abundantly present in OK-432-treated mice, and p-STAT6 positive cells were much fewer than in non-treated mice. CONCLUSIONS: OK-432-treatment appears to induce Th1 cellular response and to down-regulate that of the Th2 pathway in AD-like lesions of NC/Nga mice. The present results demonstrate bacterial components from such Streptococcus to likely constitute an effective new therapeutic approach in the treatment of AD.

The role of RANTES in a murine model of food allergy.

Food allergy is an important and common health issue, and there is a need to identify and characterize the sensitizing mechanisms. One of the common causes of food allergy is ovalbumin (OVA), a dietary antigen from eggs. We hypothesized that OVA-induced food allergy in the gut involves the activation of the chemokine regulated on activation, normal T cell expressed and secreted (RANTES), which then recruits eosinophils to lesioned tissue. The purpose of this study was to clarify whether RANTES expression correlates with eosinophil infiltration in the gut of OVA-sensitized BALB/c mice in response to oral OVA challenge. BALB/c mice were immunized with OVA 1 microg and sensitized after 2 weeks by intragastric administration of OVA. Sensitization to the oral OVA challenge was analyzed by examining eosinophil infiltration into the gut tissue (immunohistochemistry), mucosal eosinophil cationic protein (ECP) concentration, and RANTES mRNA expression (reverse-transcriptase polymerase chain reaction and Southern blotting) at 3, 6, 12, and 24 h after the challenge. There was marked edema of the intestinal villi, and eosinophil infiltration to the lamina propria peaked at 6 h in OVA-sensitized mice. RANTES mRNA expression peaked at 3 h and 6 h and declined thereafter. The expression of RANTES mRNA in the allergic mice was much higher than in the nonallergic, normal, or unsensitized control mice. Tissue eosinophilia and intestinal ECP levels were significantly correlated with the RANTES mRNA level. We conclude that RANTES may play a central role in the pathogenesis of food-mediated gastrointestinal allergy.

Substance P induced preprotachykinin-a mRNA, neutral endopeptidase mRNA and substance P in cultured normal fibroblasts.

In certain skin diseases, stress can modulate the induction and/or progression of cutaneous manifestations. However, little is known about the circuit in neuroendocrine and in the immune systems of the skin. To address this question, we have analyzed the regulatory mechanisms of autocrine induction of substance P (SP) by cultured normal human fibroblasts that compose the major population of the skin and might augment stress-induced skin inflammatory responses. In nonstimulated conditions, normal fibroblasts express a moderate amount of preprotachykinin-A (PPT-A), a precursor of SP mRNA, and exogenous SP significantly upregulated PPT-A mRNA expression. Maximum response of SP peptide and SP mRNA in fibroblasts was observed 1-3 h after stimulation with SP. In contrast, the expression of neutral endopeptidase (NEP), a cell surface peptide with hydrolyzing activity of SP, was increased in fibroblasts stimulated with SP after 24 h. The administration of NEP inhibitor (phosphoramidon) to the fibroblasts induced higher SP production. In addition, the neurokinin (NK) receptor antagonists (spantide, FK224 and FK888) and protein synthesis inhibitor (cycloheximide) inhibited SP production by 30-40% of control response. In immunostaining study, specific cytoplasmic staining of SP was observed in fibroblasts stimulated with SP. Finally, we confirmed that the nucleotide sequence of the PPT-A expressed in fibroblasts perfectly corresponded to the gene bank human PPT-A cDNA. This is the first report that SP mRNA, NEP mRNA and SP peptide can be induced by normal human skin fibroblasts in response to exogenous SP, and that fibroblast-derived SP might play an important role in the induction and acceleration of certain cutaneous diseases.