Toyoko Kikuchi

Psoriasis Vulgaris Lesions Contain Discrete Populations of Th1 and Th17 T Cells

The importance of T helper 17 (Th17) cells in inflammation and autoimmunity is now being appreciated. We analyzed psoriasis skin lesions and peripheral blood for the presence of IL-17-producing T cells. We localized Th17 cells predominantly to the dermis of psoriasis skin lesions, confirmed that IL-17 mRNA increased with disease activity, and demonstrated that IL-17 mRNA expression normalized with cyclosporine therapy. IL-22 mRNA expression mirrored IL-17 and both were downregulated in parallel with keratin 16. Th17 cells are a discrete population, separate from Th1 cells (which are also in psoriasis lesions), and Th2 cells. Our findings suggest that psoriasis is a mixed Th1 and Th17 inflammatory environment. Th17 cells may be proximal regulators of psoriatic skin inflammation, and warrant further attention as therapeutic targets.

Interleukin-11 therapy selectively downregulates type I cytokine proinflammatory pathways in psoriasis lesions

Psoriasis is a chronic inflammatory skin disease in which epidermal hyperplasia results from skin infiltration by type I T lymphocytes and release of associated cytokines. A multifunctional cytokine, rhIL-11, modulates macrophage and type I T-lymphocyte function in cell culture and shows anti-inflammatory activity in animal models. We are testing subcutaneous delivery of rhIL-11 to patients with psoriasis in a phase 1 open-label dose-escalation clinical trial. Tissue was obtained from lesional and uninvolved skin before and during treatment with rhIL-11 and was examined by histology/immunohistochemistry and quantitative RT-PCR. Expression of over 35 genes was examined in all patients, and multiple genetic markers of psoriasis were identified. Expression of numerous proinflammatory genes was elevated in psoriatic tissue compared with nonlesional skin. Seven of 12 patients responded well to rhIL-11 treatment. Amelioration of disease by rhIL-11, as shown by reduced keratinocyte proliferation and cutaneous inflammation, was associated with decreased expression of products of disease-related genes, including K16, iNOS, IFN-gamma, IL-8, IL-12, TNF-alpha, IL-1beta, and CD8, and with increased expression of endogenous IL-11. We believe that this is the first study in humans to indicate that type I cytokines can be selectively suppressed by an exogenous immune-modifying therapy. The study highlights the utility of pharmacogenomic monitoring to track patient responsiveness and to elucidate anti-inflammatory mechanisms.

Human Langerhans cells induce distinct IL-22-producing CD4+ T cells lacking IL-17 production

IL-22 is a cytokine that acts mainly on epithelial cells. In the skin, it mediates keratinocyte proliferation and epidermal hyperplasia and is thought to play a central role in inflammatory diseases with marked epidermal acanthosis, such as psoriasis. Although IL-22 was initially considered a Th17 cytokine, increasing evidence suggests that T helper cells can produce IL-22 even without IL-17 expression. In addition, we have shown the existence of this unique IL-22-producing T cell in normal skin and in the skin of psoriasis and atopic dermatitis patients. In the present study, we investigated the ability of cutaneous resident dendritic cells (DCs) to differentiate IL-22-producing cells. Using FACS, we isolated Langerhans cells (LCs; HLA-DR+CD207+ cells) and dermal DCs (HLA-DRhiCD11c+BDCA-1+ cells) from normal human epidermis and dermis, respectively. Both LCs and dermal DCs significantly induced IL-22-producing CD4+ and CD8+ T cells from peripheral blood T cells and naive CD4+ T cells in mixed leukocyte reactions. LCs were more powerful in the induction of IL-22-producing cells than dermal DCs. Moreover, in vitro-generated LC-type DCs induced IL-22-producing cells more efficiently than monocyte-derived DCs. The induced IL-22 production was more correlated with IFN-γ than IL-17. Surprisingly, the majority of IL-22-producing cells induced by LCs and dermal DCs lacked the expression of IL-17, IFN-γ, and IL-4. Thus, LCs and dermal DCs preferentially induced helper T cells to produce only IL-22, possibly “Th22” cells. Our data indicate that cutaneous DCs, especially LCs, may control the generation of distinct IL-22 producing Th22 cells infiltrating into the skin.

Potential Role of the Chemokine Receptors CXCR3, CCR4, and the Integrin |[agr]|E|[bgr]|7 in the Pathogenesis of Psoriasis Vulgaris

Various adhesion molecules have been implicated in T lymphocyte binding to dermal vascular endothelium in psoriasis vulgaris, but the chemotactic signals that promote subsequent homing into the adjacent dermis and overlying epidermis are poorly defined. We studied chemokine receptor (CCR1–CCR5, CXCR1–CXCR3), chemokine (interferon-γ inducible protein 10 [IP-10]), monokine induced by interferon-γ (MIG), thymus and activation-regulated chemokine (TARC), macrophage-derived chemokine (MDC), and adhesion molecule (cutaneous lymphocyte antigen [CLA], E-selectin, lymphocyte function-associated antigen-1 [LFA-1], intercellular adhesion molecule-1 [ICAM-1], very late antigen 4 [VLA-4], vascular cell adhesion molecule-1 [VCAM-1], αEβ7, and E-cadherin) expression in psoriasis by immunohistology, flow cytometry, and molecular techniques. CXCR3 and CCR4 were expressed by dermal CD3+ lymphocytes, and their chemokine ligands, IP-10, MIG, TARC, and MDC, were up-regulated in psoriatic lesions. Keratinocytes stimulated with tumor necrosis factor-α and interferon-γ up-regulated expression of IP-10, MIG, and MDC mRNA, whereas dermal endothelial cells, similarly stimulated, up-regulated expression of IP-10, MDC, and TARC mRNA, suggesting that these cell types were sources of the chemokines detected in biopsies. There was enhanced expression of E-selectin, CLA, LFA-1, ICAM-1, VLA-4, VCAM-1, and αEβ7 in psoriatic lesions versus nonlesional skin. Finally, intra-epidermal CLA+ and αEβ7+ T lymphocytes selectively expressed the chemokine receptor CXCR3. Collectively, these data suggest that CXCR3 and CCR4 may be involved in T lymphocyte trafficking to the psoriatic dermis and that CXCR3 is selectively involved in subsequent T cell homing to the overlying epidermis.

Molecular classification of psoriasis disease-associated genes through pharmacogenomic expression profiling

Psoriasis is recognized as the most common T cell-mediated inflammatory disease in humans. Genetic linkage to as many as six distinct disease loci has been established but the molecular etiology and genetics remain unknown. To begin to identify psoriasis disease-related genes and construct in vivo pathways of the inflammatory process, a genome-wide expression screen of multiple psoriasis patients was undertaken. A comprehensive list of 159 genes that define psoriasis in molecular terms was generated; numerous genes in this set mapped to six different disease-associated loci. To further interpret the functional role of this gene set in the disease process, a longitudinal pharmacogenomic study was initiated to understand how expression levels of these transcripts are altered following patient treatment with therapeutic agents that antagonize calcineurin or NF-κB pathways. Transcript levels for a subset of these 159 genes changed significantly in those patients who responded to therapy and many of the changes preceded clinical improvement. The disease-related gene map provides new insights into the pathogenesis of psoriasis, wound healing and cellular-immune reactions occurring in human skin as well as other T cell-mediated autoimmune diseases. In addition, it provides a set of candidate genes that may serve as novel therapeutic intervention points as well as surrogate and predictive markers of treatment outcome.

Alefacept (anti-CD2) causes a selective reduction in circulating effector memory T cells (Tem) and relative preservation of central memory T cells (Tcm) in psoriasis

BackgroundAlefacept (anti-CD2) biological therapy selectively targets effector memory T cells (Tem) in psoriasis vulgaris, a model Type 1 autoimmune disease.MethodsCirculating leukocytes were phenotyped in patients receiving alefacept for moderate to severe psoriasis.ResultsIn all patients, this treatment caused a preferential decrease in effector memory T cells (CCR7- CD45RA-) (mean 63% reduction) for both CD4+ and CD8+ Tem, while central memory T cells (Tcm) (CCR7+CD45RA-) were less affected, and naïve T cells (CCR7+CD45RA+) were relatively spared. Circulating CD8+ effector T cells and Type 1 T cells (IFN-γ-producing) were also significantly reduced.ConclusionAlefacept causes a selective reduction in circulating effector memory T cells (Tem) and relative preservation of central memory T cells (Tcm) in psoriasis.

Blockade of CD11a by Efalizumab in Psoriasis Patients Induces a Unique State of T-Cell Hyporesponsiveness

Efalizumab (anti-CD11a) interferes with LFA-1/ICAM-1 binding and inhibits several key steps in psoriasis pathogenesis. This study characterizes the effects of efalizumab on T-cell activation responses and expression of surface markers on human circulating psoriatic T cells during a therapeutic trial. Our data suggest that efalizumab may induce a unique type of T-cell hyporesponsiveness, directly induced by LFA-1 binding, which is distinct from conventional anergy described in animal models. Direct activation of T cells through different activating receptors (CD2, CD3, CD3/28) is reduced, despite T cells being fully viable. This hyporesponsiveness was spontaneously reversible after withdrawal of the drug, and by IL-2 in vitro. In contrast to the state of anergy, Ca(+2) release is intact during efalizumab binding. Furthermore, lymphocyte function-associated antigen-1 (LFA-1) blockade resulted in an unexpected downregulation of a broad range of surface molecules, including the T-cell receptor complex, co-stimulatory molecules, and integrins unrelated to LFA-1, both in the peripheral circulation and in diseased skin tissue. These observations provide evidence for the mechanism of action of efalizumab. The nature of this T-cell hyporesponsiveness suggests that T-cell responses may be reduced during efalizumab therapy, but are reversible after ceasing efalizumab treatment.

Novel Insight into the Agonistic Mechanism of Alefacept In Vivo: Differentially Expressed Genes May Serve as Biomarkers of Response in Psoriasis Patients

Alefacept is an LFA3-Ig fusion protein that binds to CD2 and is thought to inhibit T cell activation by antagonism of CD2 signaling or by lysis of CD2+ cells. Alefacept is potential future therapeutic for organ transplant recipients or graft-vs-host disease and is an approved therapeutic for psoriasis vulgaris, which is a T cell-mediated inflammatory disease. However, alefacept improves psoriasis in only ∼50% of patients treated for 12 wk. We studied the immunologic effects of alefacept in a group of psoriasis patients during treatment. We found that T cells, especially CD8+ T cells, were rapidly decreased in the peripheral circulation. Decreases in circulating T cells were not associated with induced apoptosis. Unexpectedly, in addition to suppression of inflammatory genes, we found a marked induction of mRNAs for STAT1, IL-8, and monokine induced by IFN-γ during the first day of treatment in PBMC. We confirmed the agonistic effects of alefacept in PBMC in vitro, which were similar to CD3/CD28 ligation on T cells. These data establish that alefacept activates gene expression programs in leukocytes and suggest that its therapeutic action may be as a mixed agonist/antagonist. Furthermore, responding patients to alefacept treatment show unique patterns of gene modulation. Whereas alefacept down-regulated TCRs CD3D and CD2 in responders, nonresponders reveal a higher expression of T cell activation genes such as CD69 in pretreatment PBMC. These finding suggest a potential basis for categorizing responders vs nonresponders at an early time point in treatment or before treatment of a broad range of proinflammatory diseases. This study 1) establishes alefacept as a novel CD2 agonist molecule for induction of leukocyte activation genes (prior work proposed its mechanism as a CD2 antagonist) and 2) that differential activation of genes may categorize clinical responders to this agent, critical for cost-effective use of this drug.

Post-therapeutic relapse of psoriasis after CD11a blockade is associated with T cells and inflammatory myeloid DCs.

To understand the development of new psoriasis lesions, we studied a group of moderate-to-severe psoriasis patients who experienced a relapse after ceasing efalizumab (anti-CD11a, Raptiva, Genentech). There were increased CD3+ T cells, neutrophils, CD11c+ and CD83+ myeloid dendritic cells (DCs), but no increase in CD1c+ resident myeloid DCs. In relapsed lesions, there were many CD11c+CD1c−, inflammatory myeloid DCs identified by TNFSF10/TRAIL, TNF, and iNOS. CD11c+ cells in relapsed lesions co-expressed CD14 and CD16 in situ. Efalizumab induced an improvement in many psoriasis genes, and during relapse, the majority of these genes reversed back to a lesional state. Gene Set Enrichment Analysis (GSEA) of the transcriptome of relapsed tissue showed that many of the gene sets known to be present in psoriasis were also highly enriched in relapse. Hence, on ceasing efalizumab, T cells and myeloid cells rapidly enter the skin to cause classic psoriasis. Trial registration Clinicaltrials.gov NCT00115076

Multiple Pathways Lead to Activation of the Survival Mechanism in Quiescent BALB/c-3T3 Cells

Abstract The survival of density-arrested quiescent murine BALB/c-3T3 cells in serum-free Dulbeccos medium requires the presence of cell growth factors or second messenger agonists. The protein synthesis inhibitor anisomycin blocks the survival-mediating action of the basic fibroblast growth factor (bFGF) and of 12-O-tetradecanoylphorbol 13-acetate (TPA), but has little or no effect on the protective action of platelet-derived growth factor or 8-bromoadenosine 3′:5′-cyclic monophosphate (Br-cAMP). The effects of anisomycin are concentration dependent in the range from 2.5 to 25 μM and show that the survival-enhancing abilities of bFGF and TPA critically require protein synthesis, whereas those of platelet-derived growth factor and Br-cAMP do not. The survival-mediating action of bFGF and TPA can also be blocked with the RNA synthesis inhibitors actinomycin D and 5,6-dichloro-1-β-d-ribofuranosylbenzimidazole (DRB), whereas the action of platelet-derived growth factor and Br-cAMP is largely resistant. Results on the time course of action of DRB, a selective inhibitor of the synthesis of mRNA precursor molecules, suggest that the RNA required for the survival-enhancing action of bFGF and TPA is present in cells at the time of serum withdrawal and addition of the survival factor and has a half-life greater than 3 h. The new evidence provides further support for the hypothesis that protection of serum-deprived, density-arrested BALB/c-3T3 fibroblasts against death can be achieved either via pathways that entail the synthesis of protein and RNA (e.g., via diacylglycerol-protein kinase C) or via pathways that do not involve de novo biosynthesis (e.g., via cAMP-protein kinase A).