Pia Freyschmidt-Paul

The Importance of Myeloid-Derived Suppressor Cells in the Regulation of Autoimmune Effector Cells by a Chronic Contact Eczema

Induction of a chronic eczema is a most efficient therapy for alopecia areata (AA). We had noted a reduction in regulatory T cells during AA induction and wondered whether regulatory T cells may become recruited or expanded during repeated skin sensitization or whether additional regulatory cells account for hair regrowth. AA could not be cured by the transfer of CD4+CD25high lymph node cells from mice repeatedly treated with a contact sensitizer. This obviously is a consequence of a dominance of freshly activated cells as compared with regulatory CD4+CD25+ T cells. Instead, a population of Gr-1+CD11b+ cells was significantly increased in skin and spleen of AA mice repeatedly treated with a contact sensitizer. Gr-1+CD11b+ spleen cells mostly expressed CD31. Expression of several proinflammatory cytokines as well as of the IFN-γ receptor and the TNF receptor I were increased. Particularly in the skin, Gr-1+ cells expressed several chemokines and CCR8 at high levels. Gr-1+CD11b+ cells most potently suppressed AA effector cell proliferation in vitro and promoted partial hair regrowth in vivo. When cocultured with CD4+ or CD8+ cells from AA mice, the Gr-1+CD11b+ cells secreted high levels of NO. However, possibly due to high level Bcl-2 protein expression in AA T cells, apoptosis induction remained unaltered. Instead, ζ-chain expression was strongly down-regulated, which was accompanied by a decrease in ZAP70 and ERK1/2 phosphorylation. Thus, a chronic eczema supports the expansion and activation of myeloid suppressor cells that, via ζ-chain down-regulation, contribute to autoreactive T cell silencing in vitro and in vivo.

Linear porokeratosis superimposed on disseminated superficial actinic porokeratosis: Report of two cases exemplifying the concept of type 1 segmental manifestation of autosomal dominant skin disorders

A concept of dichotomous types of segmental involvement of autosomal dominant skin disorders has recently been proposed. Among the different types of porokeratosis, disseminated superficial actinic porokeratosis is known to be an autosomal dominant skin disorder, and linear porokeratosis represents the segmental form of the disease. We intended to exemplify the type 2 segmental manifestation within this concept. Clinical and histopathologic aspects of porokeratotic lesions of 2 patients were investigated. The family history was studied in both cases. Linear porokeratosis superimposed on disseminated superficial actinic porokeratosis was observed in both patients. These 2 cases of linear porokeratosis associated with disseminated superficial actinic porokeratosis can be taken as further examples of a type 2 segmental involvement occurring in an autosomal dominant skin disorder.

In vivo CD44-CD49d complex formation in autoimmune disease has consequences on T cell activation and apoptosis resistance

CD44 is involved in leukocyte migration and activation and has recently been reported to contribute to leukocyte extravasation by associating with CD49d. We explored whether similar changes in CD44 activity are seen in vivo using murine alopecia areata (AA) as a chronic, organ‐related autoimmune disease model system. Expression of the activated, hyaluronan‐binding form of CD44, and of CD49d, was elevated in draining lymph node cells (LNC) of AA‐affected mice as compared to control mice. LNC of AA mice displayed increased motility, proliferative activity and apoptosis resistance, which were equally well inhibited by anti‐CD44 and anti‐CD49d. The latter is the sequelae of the association between CD44 and CD49d that is seen in activated lymphocytes. Significantly, due to CD44‐CD49d complex formation, CD44 gains access to focal adhesion kinase and CD49d gains access to CD44‐associated lck and ezrin, such that downstream kinases become activated via CD44 or CD49d engagement. Thus, by their association, CD44 and CD49d mutually avail themselves of the partners signaling pathways and the ligand binding of each one triggers signaling pathways of both. This strongly influences the lymphocytes’ activation state and function.

Dietary soy oil content and soy‐derived phytoestrogen genistein increase resistance to alopecia areata onset in C3H/HeJ mice

Abstract: Alopecia areata (AA) is a complex, multi‐factorial disease where genes and the environment may affect susceptibility and severity. Diet is an environmental factor with the potential to influence disease susceptibility. We considered dietary soy (soya) oil content and the soy‐derived phytoestrogen genistein as potential modifying agents for C3H/HeJ mouse AA. Normal haired C3H/HeJ mice were grafted with skin from spontaneous AA affected mice, a method previously shown to induce AA. Grafted mice were given one of three diets containing 1%, 5% or 20% soy oil and observed for AA development. In a separate study, mice on a 1% soy oil diet were injected with 1 mg of genistein three times per week for 10 weeks or received the vehicle as a control. Of mice on 1%, 5%, and 20% soy oil diets, 43 of 50 mice (86%), 11 of 28 mice (39%), and 2 of 11 mice (18%) developed AA, respectively. Four of 10 mice injected with genistein and 9 of 10 controls developed AA. Mice with AA had hair follicle inflammation consistent with observations for spontaneous mouse AA, but no significant association was observed between the extent of hair loss and diet or genistein injection. Mice that failed to develop AA typically regrew white hair from their skin grafts associated with a moderate macrophage and dendritic cell infiltration. Soy oil and derivatives have previously been reported to modify inflammatory conditions. Hypothetically, soy oil compounds may act on C3H/HeJ mice through modulating estrogen‐dependent mechanisms and/or inflammatory activity to modify AA susceptibility.

Anti‐CD44‐mediated blockade of leukocyte migration in skin‐associated immune diseases

CD44 plays an important role in leukocyte extravasation, which is fortified in autoimmune diseases and delayed‐type hypersensitivity (DTH) reactions. There is additional evidence that distinct CD44 isoforms interfere with the extravasation of selective leukocyte subsets. We wanted to explore this question in alopecia areata (AA), a hair‐follicle centric autoimmune disease, and in a chronic eczema. The question became of interest because AA is treated efficiently by topical application of a contact sensitizer, such that a mild DTH reaction is maintained persistently. Aiming to support the therapeutic efficacy of a chronic eczema in AA by anti‐CD44 treatment, it became essential to control whether a blockade of migration, preferentially of AA effector cells, could be achieved by CD44 isoform‐specific antibodies. Anti‐panCD44 and anti‐CD44 variant 10 isoform (CD44v10) inhibited in vitro migration of leukocytes from untreated and allergen‐treated, control and AA mice. In vivo, both antibodies interfered with T cell and monocyte extravasation into the skin; only anti‐panCD44 prevented T cell homing into lymph nodes. Contributing factors are disease‐dependent alterations in chemokine/chemokine receptor expression and a blockade of CD44 on endothelial cells and leukocytes. It is important that CD44 can associate with several integrins and ICAM‐1. Associations depend on CD44 activation and vary with CD44 isoforms and leukocyte subpopulations. CD44 standard isoform preferentially associates with CD49d in T cells and CD44v10 with CD11b in monocytes. Accordingly, anti‐panCD44 and anti‐CD49d inhibit T cell, anti‐CD11b, and anti‐CD44v10 macrophage migration most efficiently. Thus, allergen treatment of AA likely can be supported by targeting AA T cells selectively via a panCD44‐CD49d‐bispecific antibody.

Interleukin‐6 cytokine family member oncostatin M is a hair‐follicle‐expressed factor with hair growth inhibitory properties

Abstract:  The activation of receptor complexes containing glycoprotein 130 (gp130) identifies the interleukin (IL)‐6 cytokine family. We examined members of this family for their expression and activity in hair follicles. Quantitative polymerase chain reaction using mRNA derived from microdissected, anagen‐stage human hair follicles and comparison to non‐follicular skin epithelium revealed higher levels of IL‐6 (15.5‐fold) and oncostatin M (OSM, 3.4‐fold) in hair follicles. In contrast, expression of all mRNAs coding for IL‐6 cytokine family receptors was reduced. Immunohistology suggested expression of OSM, gp130, leukaemia inhibitory factor receptor (LIFr) and IL‐11r in the hair follicle root sheaths and dermal papilla, while IL‐11, IL‐6r and OSMr were expressed in root sheaths alone. IL‐6 was expressed in the dermal papilla while cardiotrophin‐1 (CT‐1) and LIF were not observed. OSM and to a lesser extent CT‐1 exhibited a dose‐dependent growth inhibition capacity on human hair follicles in vitro. OSM and CT‐1 incubated with agarose beads and injected subcutaneously at 1 μg per mouse into telogen skin of 65‐day‐old mice revealed no capacity to induce anagen hair growth. In contrast, injection of 65‐day‐old mice in which anagen had been induced by hair plucking revealed a moderate hair growth inhibitory capacity for OSM, but no significant effect for CT‐1. The data identify OSM as a modulator of hair follicle growth and suggest other family members may also have some degree of hair growth inhibitory effect. In principle, increased expression of some IL‐6 cytokine family members in cutaneous inflammation might contribute to the promotion of hair loss.

[Alopecia areata. Clinical aspects, pathogenesis and rational therapy of a T-cell-induced autoimmune disease].

ZusammenfassungDie Alopecia areata ist eine T-Zell-vermittelte Autoimmunerkrankung, die sich gegen ein bisher unbekanntes Antigen des Haarfollikels richtet. Es besteht eine genetische Prädisposition für die Erkrankung; auslösende Umweltfaktoren ließen sich bisher nicht nachweisen. Das typische klinische Bild des kreisrunden Haarausfalls einschließlich seiner Maximalformen, der Alopecia areata totalis und universalis, ermöglicht in den meisten Fällen die Diagnose, die durch das Vorliegen von Nagelveränderungen untermauert werden kann. Selten wird eine Histologie erforderlich; alle anderen Laboruntersuchungen sind überflüssig. Wegen der hohen Spontanremissionsrate muss die Wirksamkeit einer rationalen Therapie der Alopecia areata in kontrollierten Studien nachgewiesen werden und im Rahmen der Nutzen-Risiko-Abwägung ein geringes Nebenwirkungsprofil aufweisen. Nach den Regeln der evidenzbasierten Medizin ist die Behandlung mit einem Kontaktallergen derzeit die effektivste und nebenwirkungsärmste Therapie der Alopecia areata; da sie aber sehr aufwändig ist und nicht in allen Fällen wirkt, ist die Entwicklung neuer, spezifischerer Therapieformen notwendig.AbstractAlopecia areata is a T-cell mediated autoimmune disease directed against an unknown auto antigen of the hair follicle. There is a genetic predisposition to develop alopecia areata, whereas environmental triggers have so far not been identified. The diagnosis can be established by characteristic clinical features of alopecia areata including its severe forms alopecia areata totalis and universalis. Nail changes may help confirm the diagnosis. On rare occasions a histopathological examination may be necessary, whereas other laboratory investigations are unnecessary. Because of the high rate of spontaneous remission, the efficacy of a rational treatment of alopecia areata has to be proven in controlled studies and it should be associated with only minor side effects. According to the rules of evidence-based medicine, treatment with a contact sensitizer is at present the most effective treatment of alopecia areata showing only mild side effects. However, it is time-consuming and in some cases ineffective, making it desirable to develop new, more specific forms of treatment.

Delayed type hypersensitivity-induced myeloid-derived suppressor cells regulate autoreactive T cells.

Mild but efficient treatments of autoimmune diseases are urgently required. One such therapy, long‐term maintenance of chronic delayed type hypersensitivity, has been described for alopecia areata (AA), a hair follicle‐affecting autoimmune disease. The molecular mechanisms underlying the therapeutic efficacy are unknown, but may involve myeloid‐derived suppressor cells (MDSCs). AA‐affected mice were treated with squaric acid dibutyl ester (SADBE). The immunoreactivity of SADBE‐treated AA lymphocytes and of AA lymphocytes co‐cultured with SADBE‐induced MDSCs was analyzed. The curative effect of SADBE was abolished by all‐transretinoic acid, which drives MDSCs into differentiation, confirming a central role for MDSCs in therapeutic SADBE treatment. SADBE and SADBE‐induced MDSCs strongly interfered with sustained autoreactive T‐cell proliferation in response to AA skin lysate (autoantigen), which was accompanied by weak ζ‐chain down‐regulation and strongly impaired Lck activation. In contrast, activation of the mitochondrial apoptosis pathway and blockade of the anti‐apoptotic PI3K/Akt pathway by SADBE‐induced MDSCs did not require T‐cell receptor engagement. Apoptosis induction correlated with high TNF‐α expression in SADBE‐induced MDSCs and elevated TNFRI levels in AA lymphocytes. SADBE‐induced MDSCs interfere with persisting autoreactive T‐cell proliferation and promote apoptosis of these T cells, which qualifies MDSCs induced and maintained by chronic delayed type hypersensitivity reactions as promising therapeutics in organ‐related autoimmune diseases.

Tolerance induction by hair-specific keratins in murine alopecia areata

AA is a presumptive autoimmune disease, severely damaging the hair follicle. Hair‐ and nail‐specific keratins are discussed as potential candidates, which we controlled in C3H/HeJ mice that develop AA spontaneously or after skin transplantation. From nine keratins, K71 and K31 peptides supported T cell activation when presented by DCs to syngeneic naive T cells, and young C3H/HeJ mice receiving s.c. injections of peptide‐loaded DC developed AA. The frequency of K71‐ and K31‐specific CD4+ and CD8+ T cells increased four‐ to fivefold by vaccination, which corresponds with the frequency seen in skin transplantation‐induced AA mice. Also, accessory molecule expression, the cytokine profile with a dominance of IFN‐γ‐expressing T cells, the proliferative response against AA lysate or peptide‐loaded DCs, as well as peptide‐specific cytotoxic T cells were similar in keratin peptide‐ and skin transplantation‐induced AA. Instead, vaccination with soluble K71 or K31 peptides significantly retarded AA induction and prevented progression. Soluble peptide vaccination did not provoke immunosuppression but induced long‐lasting T cell anergy with unresponsiveness to DC‐presented K71 and K31 peptides. Thus, keratins K71 and K31 contribute to AA induction, and peptide application in a nonimmunogenic form serves as an efficient therapeutic.

Alopecia areata im Tiermodell – Neue Einblicke in Pathogenese und Therapie einer T-Zell-vermittelten Autoimmunerkrankung

Die Möglichkeiten zur Erforschung der Pathogenese der Alopecia areata und zur Entwicklung neuer Therapieansätze sind beim Menschen aus ethischen Gründen begrenzt. Daher ist die Entwicklung geeigneter Tiermodelle für die Alopecia areata sinnvoll. Mit der Dundee experimental bald rat (DEBR) und der C3H/HeJ‐Maus stehen mittlerweile zwei gut charakterisierte Tiermodelle der Alopecia areata zur Verfügung, an denen Studien zur Genetik, Pathogenese und Therapie der Alopecia areata durchgeführt werden können. Ein besonderer Vorteil der C3H/HeJ‐Maus besteht in der Möglichkeit, die Alopecia areata durch Hauttransplantation experimentell zu induzieren, und dann den Einfluß verschiedener Faktoren auf den Verlauf der Erkrankung zu untersuchen. Durch Untersuchungen an der C3H/HeJ‐Maus und der DEBR konnte die T‐Zell‐vermittelte Autoimmunpathogenese der Alopecia areata bestätigt werden, und es ließen sich einzelne Mechanismen dieser T‐Zell‐vermittelten Immunreaktion analysieren. Ausgehend von diesen Erkenntnissen ergeben sich verschiedene neue Optionen für die Therapie der Alopecia areata wie die Blockade des Lymphozytenhomings durch Applikation eines monoklonalen Anti‐CD44v10‐Antikörpers oder die Hemmung der T‐Zell‐Aktivierung durch Blockade kostimulatorischer Moleküle. Therapeutische Studien am Maus‐ und Rattenmodell sprechen dafür, daß die topische Applikation von Tacrolimus zur Behandlung der Alopecia areata des Menschen im Prinzip geeignet ist, sofern sich die Galenik verbessern läßt. Zukünftige Untersuchungen zum Wirkmechanismus des allergischen Kontaktekzems in der Behandlung der Alopecia areata sollen dazu dienen, eine auf dieser Behandlung aufbauende, spezifischere Therapie zu entwickeln.