Tomoko Akeda

Calcineurin Inhibitors Suppress Cytokine Production from Memory T Cells and Differentiation of Naïve T Cells into Cytokine-Producing Mature T Cells

T cells have been classified as belonging to the Th1 or Th2 subsets according to the production of defining cytokines such as IFN-γ and IL-4. The discovery of the Th17 lineage and regulatory T cells shifted the simple concept of the Th1/Th2 balance into a 4-way mechanistic pathway of local and systemic immunological activity. Clinically, the blockage of cytokine signals or non-specific suppression of cytokine predominance by immunosuppressants is the first-line treatment for inflammatory T cell-mediated disorders. Cyclosporine A (CsA) and Tacrolimus (Tac) are commonly used immunosuppressants for the treatment of autoimmune disease, psoriasis, and atopic disorders. Many studies have shown that these compounds suppress the activation of the calcium-dependent phosphatase calcineurin, thereby inhibiting T-cell activation. Although CsA and Tac are frequently utilized, their pharmacological mechanisms have not yet been fully elucidated.In the present study, we focused on the effects of CsA and Tac on cytokine secretion from purified human memory CD4(+)T cells and the differentiation of naïve T cells into cytokine-producing memory T cells. CsA or Tac significantly inhibited IFN-γ, IL-4, and IL-17 production from memory T cells. These compounds also inhibited T cell differentiation into the Th1, Th2, and Th17 subsets, even when used at a low concentration. This study provided critical information regarding the clinical efficacies of CsA and Tac as immunosuppressants.

Granzyme B is a novel interleukin-18 converting enzyme

BACKGROUND Granzyme B (GrB) is recognized to induce apoptosis; however, little is known about its possible role in other biological events. IL-18, a potent inflammatory cytokine, is produced as an inactive precursor (proIL-18). Several cells, including monocytes/macrophage lineage and non-hematopoietic cells such as keratinocytes, produce proIL-18. ProIL-18 requires appropriate processing to become active. Caspase-1 is the authentic IL-18 processing enzyme and is essential for IL-18 release from monocyte/macrophage lineage cells. However, caspase-1 is absent in non-hematopoietic cells, suggesting that there is another candidate to cleave proIL-18 except for caspase-1. OBJECTIVE GrB can invade and be active in cytoplasm of non-hematopoietic cells via perforin, therefore we investigated whether GrB converts proIL-18 into the biologically active form. METHODS Recombinant proIL-18 (rproIL-18) was produced and purified for protease reaction with GrB; this incubate was evaluated by immunoblotting. Biological activity of the proteolytic fragment cleaved by GrB was determined by IFN-gamma assay using KG-1 cells. IFN-gamma induction was also analyzed between extracts from GrB(+)/caspase-1(-) human CD8+ T cells and proIL-18 from normal human keratinocytes (NHK). RESULTS The proteolytic fragment that GrB cleaved proIL-18 had the same sequence and biological activity compared with mature IL-18 cleaved by caspase-1. Culture extracts from CD8+ T cells was able to cleave proIL-18 into authentic mature IL-18. IFN-gamma induction was also detected in NHK treated with CD8+ T cells. CONCLUSION GrB is a potent IL-18 converting enzyme and suggest that GrB secreted by CTLs and/or NK cells may initiate IL-18 release from target cells, leading to the development of inflammation.

Intratumoral Injection of Propionibacterium acnes Suppresses Malignant Melanoma by Enhancing Th1 Immune Responses

Malignant melanoma (MM) is an aggressive cutaneous malignancy associated with poor prognosis; many putatively therapeutic agents have been administered, but with mostly unsuccessful results. Propionibacterium acnes (P. acnes) is an aerotolerant anaerobic gram-positive bacteria that causes acne and inflammation. After being engulfed and processed by phagocytes, P. acnes induces a strong Th1-type cytokine immune response by producing cytokines such as IL-12, IFN-γ and TNF-α. The characteristic Th2-mediated allergic response can be counteracted by Th1 cytokines induced by P. acnes injection. This inflammatory response induced by P. acnes has been suggested to have antitumor activity, but its effect on MM has not been fully evaluated. We analyzed the anti-tumor activity of P. acnes vaccination in a mouse model of MM. Intratumoral administration of P. acnes successfully protected the host against melanoma progression in vivo by inducing both cutaneous and systemic Th1 type cytokine expression, including TNF-α and IFN-γ, which are associated with subcutaneous granuloma formation. P. acnes-treated tumor lesions were infiltrated with TNF-α and IFN-γ positive T cells. In the spleen, TNF-α as well as IFN-γ producing CD8+T cells were increased, and interestingly, the number of monocytes was also increased following P. acnes administration. These observations suggest that P. acnes vaccination induces both systemic and local antitumor responses. In conclusion, this study shows that P. acnes vaccination may be a potent therapeutic alternative in MM.

CD8+ T cell granzyme B activates keratinocyte endogenous IL-18.

IL-18 is a pro-inflammatory cytokine of the IL-1 family involved in Th1/Th2 polarization. IL-18 is produced and stored as an inactive precursor (proIL-18) in several cells including keratinocytes, and thus appropriate processing is required to release its active form. In a previous study using recombinant protein, we demonstrated that granzyme B (GrB) cleaves proIL-18 into its active forms in a similar fashion as caspase-1 and human mast cell chymase. GrB released from cytotoxic T lymphocyte (CTL) and NK cells has roles in apoptosis and cytotoxic activity. In certain inflammatory skin diseases with epidermal cell death, the epidermal keratinocytes are targets of CTL and NK cells. However, IL-18 activation during the direct interaction of CTL/NK with keratinocytes has not been described so far. We investigated the interaction between CTL and keratinocytes, and IL-18 processing by CTL-derived GrB using cultured CD8+ T cells and keratinocyte cell line HaCaT. GrB(+)/caspase-1(−) CD8+ T cells cultivated from healthy human PBMC were co-cultured with interferon(IFN)-γ-treated HaCaT cells. The expression of GrB and caspase-1 in HaCaT cells was analyzed by flow cytometry and PCR. The IL-18 concentration in the culture supernatant was measured by specific ELISA. The interaction between HaCaT cells and CTL co-culture increased the number of cytoplasmic GrB-positive HaCaT cells with limited endogenous GrB mRNA expression. The concentration of mature IL-18 levels increased in the co-culture supernatant. GrB from CTLs acts double roles to keratinocytes: a IL-18 converting enzyme and pro-apoptotic factor in the skin inflammatory diseases.

Neutrophils are not the dominant interleukin-17 producer in psoriasis

Dear Editor, In the psoriasis field, the blockade of interleukin (IL)-17A and its receptor by specific monoclonal antibodies shows dramatic improvement of skin and joint symptoms. However, the main source of IL-17 in psoriasis is still controversial. Neutrophils have been suggested as a major producer of IL-17 as well as T-helper (Th)17, cd T cells, mast cells and, recently, the focus has been on innate lymphoid cell 3 (ILC3). Histopathologically, neutrophils are the dominant skin infiltrating immune cells, but IL-17 production from neutrophils is still a topic of debate. IL-17 immunoreactive neutrophils are detectable in the psoriatic skin lesions as well as in the peripheral blood by histopathological and flow cytometric analysis, which is explained by extracellular trap formation. IL-17 mRNA expression from neutrophils was undetectable by reverse transcription polymerase chain reaction (RT–PCR) in a report, but was slightly detectable in severe psoriasis patients in another report (M. J. Kaplan, unpubl. obs.). Is there a technical issue to address this inconsistency? In the current study, venous blood was drawn after obtaining written informed consent from all subjects, and the investigational protocol was approved by the institutional review board of Mie University Hospital (#2870). This unpublished data is quoted from the Journal of Allergy and Clinical Immunology: “Although our Western blot and

Intratumoral injection of OK-432 suppresses metastatic squamous cell carcinoma lesion inducing interferon-γ and tumour necrosis factor-α

A 79-year-old man presented with a subcutaneous lesion on his upper arm. He had a history of surgically treated squamous cell carcinoma (SCC) on his arm, and axillary lymph-node metastasis 7 years previously. On physical examination, a large subcutaneous tumour was seen on the upper arm, involving the brachial artery and vein (Fig. 1a), which was diagnosed as an in-transit relapse of the SCC. The first-line choice of treatment was amputation of the patient s arm from the shoulder, but he refused. The patient had renal dysfunction, thus chemotherapy was contraindicated. The patient underwent irradiation to the lesion with limited effect. We then started intratumoral injection of OK-432 [2 KE (Klinische Einheit; clinical units)] every 2 weeks. The tumour size gradually diminished (Fig. 1b), and no further metastases have been seen during a followup of 5 years. OK-432 is a biological response modifier (BRM), which is a penicillin G-inactivated and lyophilized preparation of the low-virulence strain Su of Streptococcus pyogenes. OK-432 is an approved adjuvant therapeutic agent for malignancies in Japan. OK-432 exerts its effect by activating the immune system to secrete antitumour agents; however, little is known about its in vivo immunological mechanism. Therefore, we carried out a study to identify the immunological basis of OK-432. We focused on production of the antitumour cytokines interferon (IFN)-c and tumour necrosis factor (TNF)-a in the peripheral blood. The study was approved by the Mie University institutional review board, and informed consent was obtained from the patient. Blood samples were collected before and 24 h after injection of OK-432. Purified peripheral blood mononuclear cells (PBMC) were stimulated with anti-CD3 and CD28 antibodies in the presence of brefeldin A, and cultured for 12 h at 37 C in 5% CO2. Using flow-cytometry analysis, we found that the percentages of CD4 and CD8 T cells producing IFN-c before treatment were higher in this patient than in healthy volunteers matched for age and gender, and these percentages increased further 1 day after treatment. The percentage of CD14+ monocytes producing TNF-a was also increased (Fig. 2). In vitro or in mice experiments, OK-432 activates neutrophils, macrophages, lymphocytes and natural killer cells, inducing multiple inflammatory cytokines including interleukin-12, TNF-a and IFN-c, and polarizes the T-cell response to a T-helper-1 dominant state. OK-432 also activates dendritic cells, which are potent antigen-presenting cells, through Toll-like receptor (TLR)4 signalling, and enhances antigen-specific cytotoxic T-lymphocyte responses. Some successful results after OK-432 therapy

Heat‐killed bacillus Calmette–Guérin and Mycobacterium kansasii antigen 85B combined vaccination ameliorates dermatitis in a mouse model of atopic dermatitis by inducing regulatory T cells

Background  Atopic dermatitis (AD) is a recurrent inflammatory skin disease characterized by dominant T‐helper (Th) 2 cytokine response. Bacillus Calmette–Guérin (BCG) has been used for preventing tuberculosis, and is regarded as a strong Th1 cytokine inducer. Antigen (Ag) 85B is a secretory protein present in Mycobacterium species that induces Th1 cytokine production.

Single step modified ink staining for Tzanck test: Quick detection of herpetic giant cells in Tzanck smear

Tzanck test has been recently re‐evaluated as a method for the diagnosis of herpes virus infection. Giemsa staining for the Tzanck test is time‐consuming and laborious. There is a need to develop simple and quick staining methods for bedside diagnosis of this disease. We report a single step and quick method for staining herpes giant cells in Tzanck smears using routinely available inks and physiological saline. A keratinocyte cell line (HaCaT) was cultured on a slide glass and stained with various commercially available blue, blue–black and black inks serially diluted with physiological saline. Clinical smear samples from herpes lesions were also stained with these solutions without specific pretreatment. The nuclei of HaCaT were clearly stained showing high contrast with the cytoplasm using 5% Parker–Quink blue–black ink saline solution. Concentration of ink solution higher or lower than 5% resulted in less contrast. Blue or black inks or other manufacturers’ inks can also be used, but staining of the cultured keratinocytes was less clear. Smear of clinical samples from herpes lesions were also stained with 5% ink solution. The nuclei of the multinucleated giant cells were clearly stained, and the sample could be immediately used for microscopic examination. One step staining of Tzanck smear using this diluted ink solution is an inexpensive and a convenient bedside diagnostic tool for the dermatologist.

The Plasma Levels of ADAMTS-13, von Willebrand Factor, VWFpp, and Fibrin-Related Markers in Patients With Systemic Sclerosis Having Thrombosis:

This study aimed to examine the hemostatic abnormalities in patients with systemic sclerosis (SSc) and the relationship between these abnormalities and thrombotic events (THEs), focusing on the difference in diffuse cutaneous SSc (dcSSc) and limited cutaneous SSc (lcSSc). The plasma levels of ADAMTS-13 (a disintegrin-like and metalloproteinase with thrombospondin type 1 motifs 13), von Willebrand factor (VWF), VWF propeptide (VWFpp), d-dimer, and soluble fibrin (SF) were measured in 233 patients with SSc. The relationship between their levels and organ involvement, including THEs and interstitial lung disease (ILD), was evaluated. The plasma levels of VWF and VWFpp were significantly elevated and ADAMTS-13 activity was significantly decreased in patients with SSc compared to healthy participants. The VWFpp in dcSSc was significantly higher than in lcSSc. Twelve patients with SSc were complicated with acute THE, and 25 patients with SSc were complicated with past THE. The plasma levels of d-dimer and SF were significantly elevated in patients with SSc having THE. The plasma levels of VWF and VWFpp were significantly elevated in patients with SSc having ILD. The plasma levels of d-dimer were elevated in patients with SSc having other connective tissue diseases (CTDs). The plasma levels of ADAMTS-13 were significantly decreased and VWF, VWFpp, and SF were increased in patients with a d-dimer level of ≥1 μg/mL. Systemic sclerosis carries a high risk of THE, especially in patients with other CTDs. Plasma hemostasis-related markers are closely related to ILD and THE. These markers are important as markers of organ involvement as well as THE.