Aged human skin accumulates mast cells with altered functionality that localize to macrophages and vasoactive intestinal peptide-positive nerve fibres.

BACKGROUND: Skin health declines with age and this is partially attributed to immunosenescence. Mast cells (MCs) are innate immune cells that coordinate tissue immune responses integral to skin homeostasis and disease. OBJECTIVES: To understand how MCs contribute to human skin ageing, we investigated how intrinsic ageing impacts MC phenotype and MC relationships with other immune cells and skin structures. METHODS: In photoprotected skin biopsies from young (≤ 30 years) and aged (≥ 75 years) individuals, immunostaining and spatial morphometry were performed to identify changes in MC phenotype, number, distribution and interaction with the vasculature and nerve fibres. Quantitative polymerase chain reaction was used to measure changes in gene expression related to immune cell activity and neuropeptide signalling. RESULTS: Skin MCs, macrophages and CD8+ T cells increased in number in intrinsically aged vs. young skin by 40%, 44% and 90%, respectively (P < 0·05), while CD4+ T cells and neutrophils were unchanged. In aged skin, MCs were more numerous in the papillary dermis and showed a reduced incidence of degranulation (50% lower than in young, P < 0·01), a conserved tryptase-chymase phenotype and coexpression of granzyme B. In aged skin, MCs increased their association with macrophages (~ 48% vs. ~27%, P < 0·05) and nerve fibres (~29% vs. 16%, P < 0·001), while reducing their interactions with blood vessels (~34% vs. 45%, P < 0·001). Additionally, we observed modulation of gene expression of vasoactive intestinal peptide (VIP; increased) and substance P (decreased) with age; this was associated with an increased frequency of VIP+ nerve fibres (around three times higher in aged skin, P < 0·05), which were strongly associated with MCs (~19% in aged vs. 8% in young, P < 0·05). CONCLUSIONS: In photoprotected skin we observed an accumulation of MCs with increasing age. These MCs have both altered functionality and distribution within the skin, which supports a role for these cells in altered tissue homeostasis during ageing.

miRNA-155 controls mast cell activation by regulating the PI3Kγ pathway and anaphylaxis in a mouse model.

BACKGROUND: Mast cells (MCs) play a central role in allergic and inflammatory disorders by rapid degranulation and release of inflammatory mediators upon antigen-driven engagement of the FcεRI. Receptor-mediated MC responses are controlled by the activation of different isoforms of phosphoinositide-3-kinase (PI3K) and the downstream signaling processes. Recent evidence suggests that miRNAs are important molecular players regulating the PI3K/Akt pathway. METHODS: The role of miR-155 in the regulation of MC functions in vivo was studied in the passive cutaneous anaphylaxis (PCA) MC-dependent model. WT and miR-155(-/-) mice were injected intradermally with anti-DNP-IgE and intravenously with the antigen DNP-HSA. Ear swelling was assessed to evaluate the anaphylactic response. All investigations, to characterize miR-155 specific activities in MCs, were conducted comparing WT and miR-155(-/-) bone marrow-derived MCs (BMMCs). RESULTS: We report that miR-155(-/-) mice display enhanced anaphylaxis reactions. Although miR-155(-/-) BMMCs show normal development, proliferation, and survival, miR-155 deficiency enhances FcεRI-mediated degranulation and release of TNF-α, IL-13, and IL-6. Interestingly, the level of Akt phosphorylation on both of its regulatory residues Thr308 and Ser473 was increased in miR-155(-/-) compared to WT BMMCs. Gene expression profiling showed that miR-155(-/-) BMMCs exhibited significantly increased expression of the adapter PI3Kγ subunits Pik3r5 (p101) and Pik3r6 (p84, p87(PIKAP) ). Furthermore, selective blockade of the PI3Kγ pathway inhibited degranulation in miR-155(-/-) BMMCs. CONCLUSIONS: Thus, we suggest that miR-155 plays a critical role in FcεRI-mediated MC responses by modulating components of the PI3Kγ pathway. This newly identified mechanism of miRNA-controlled MC activation may affect the initiation and maintenance of allergic disorders.

Anti-CD30 human IL-2 fusion proteins display strong and specific cytotoxicity in vivo.

Although therapy of CD30-positive lymphomas such as classical Hodgkin lymphoma and anaplastic large cell lymphoma has been improved considerably during the last decades, patients suffer from high toxicity of current therapeutic regimens. Since CD30 expression is very restricted, CD30-positive tumors are well suited for immunotherapeutic approaches. Several distinct immunotherapeutic approaches with chimeric, humanized, and bispecific antibodies as well as immunotoxins are already described. In this report, we give a short overview of CD30-targeting approaches in humans. Furthermore, we introduce two novel anti-CD30 fusion proteins consisting of the single chain variable fragment of the CD30 monoclonal antibody Ber-H2 and human interleukin-2, evaluate their biological activity in a human CD30-positive syngeneic murine model, and demonstrate the immunological mechanisms leading to tumor rejection by these reagents. The data indicate that there are several promising approaches in CD30-targeted immunotherapy. The findings of the anti-CD30 IL-2 constructs suggest that these fusion proteins are particularly useful to remove small, residual tumors.

Interleukin-21 stimulates antigen uptake, protease activity, survival and induction of CD4+ T cell proliferation by murine macrophages.

Interleukin (IL)-21 is a T cell-derived cytokine which uses a heterodimeric receptor, composed of the common gamma-chain (CD132) and an IL-21Ralpha-chain. IL-21 activates lymphoid T and B cells, modulates antibody production but also suppresses maturation of myeloid dendritic cells; however, its role in the differentiation and function of other myeloid cells remains less clear. In this study we analysed IL-21/IL-21Ralpha effects on macrophage (MPhi) differentiation and function. MPhi could be generated readily from bone marrow with MPhi-colony-stimulating factor in the presence of IL-21 (designated IL-21MPhi) or from IL-21Ralpha-/- mice. IL-21Ralpha-/- mice had normal MPhi numbers, suggesting a non-essential role of both IL-21 and the IL-21Ralpha for MPhi generation. We could demonstrate that mature MPhi express the IL-21Ralpha and the common gamma-chain. However, short-term IL-21 stimulation did not enhance MPhi proliferation but induced anti-apoptotic cell-cycle regulators p21(waf1)/p27(Kip1) and expression of suppressors of cytokine signalling (SOCS)2/SOCS3. Moreover, IL-21 enhanced phagocytosis by MPhi via IL-21Ralpha signalling and supports protease activity and matrix metalloproteinase 12 expression. Stimulating MPhi with IL-21 enhanced their capacity to induce antigen-specific CD4+ T cell proliferation in dependence from the IL-21Ralpha, which was not the case for CD8+ T cells. Taken together, IL-21 plays a previously unrecognized role in modulating innate and acquired effector mechanisms of murine MPhi by linking these different functions to support CD4+ T cell-mediated immune responses.

Reduced cumulative incidence of diabetes but not insulitis following administration of chimeric human IL-15-murine IgG2b in NOD mice.

BACKGROUND: It has been recently demonstrated that apoptosis is involved in beta-cell destruction in the NOD mouse model of diabetes. The aim of the present study was to investigate whether IL-15, a cytokine involved in the modulation of the apoptotic process, is capable of modifying the natural history of diabetes and/or insulitis in pre-diabetic NOD mice. The rationale for the use of IL-15-IgG2b recombinant cytokine is related to its long half-life (28 +/- 4 h). METHODS: At 10 weeks of age, 2 groups of 24 female mice were treated with single or multiple i.p. doses of IL-15-IgG2b respectively. As control, 2 groups of 24 age- and litter-matched female mice were injected intra-peritoneally with single or multiple doses of IgG2b immunoglobulin. RESULTS: Diabetes incidence at 33 weeks of age was lower in the group of mice treated with multiple doses than in the control group (p = 0.03). The cumulative incidence of diabetes at 33 weeks of age between single-dose treated mice and the control group was similar. No significant differences in the calculated index of insulitis were observed in all treated and control mice. CONCLUSIONS: We conclude that IL-15-IgG2b reduces the cumulative incidence of diabetes, without affecting the extent and severity of the insulitis process. Considering this and the well-defined anti-apoptotic effects of IL-15, we suggest that the reduction of diabetes incidence could be due to a down-regulation of beta-cell apoptosis.

Detection of a functional hybrid receptor gammac/GM-CSFRbeta in human hematopoietic CD34+ cells.

A functional hybrid receptor associating the common gamma chain (gammac) with the granulocyte/macrophage colony-stimulating factor receptor beta (GM-CSFRbeta) chain is found in mobilized human peripheral blood (MPB) CD34+ hematopoietic progenitors, SCF/Flt3-L primed cord blood (CB) precursors (CBPr CD34+/CD56-), and CD34+ myeloid cell lines, but not in normal natural killer (NK) cells, the cytolytic NK-L cell line or nonhematopoietic cells. We demonstrated, using CD34+ TF1beta cells, which express an interleukin (IL)-15Ralpha/beta/gammac receptor, that within the hybrid receptor, the GM-CSFRbeta chain inhibits the IL-15-triggered gammac/JAK3-specific signaling controlling TF1beta cell proliferation. However, the gammac chain is part of a functional GM-CSFR, activating GM-CSF-dependent STAT5 nuclear translocation and the proliferation of TF1beta cells. The hybrid receptor is functional in normal hematopoietic progenitors in which both subunits control STAT5 activation. Finally, the parental TF1 cell line, which lacks the IL-15Rbeta chain, nevertheless expresses both a functional hybrid receptor that controls JAK3 phosphorylation and a novel IL-15alpha/gammac/TRAF2 complex that triggers nuclear factor kappaB activation. The lineage-dependent distribution and function of these receptors suggest that they are involved in hematopoiesis because they modify transduction pathways that play a major role in the differentiation of hematopoietic progenitors.

Enhanced inhibition of tumour growth and metastasis, and induction of antitumour immunity by IL-2-IgG2b fusion protein.

Cytokine-immunoglobulin (Ig)-fusion proteins have attracted increasing interest as antitumour agents. Here, we have investigated the antimetastatic and antitumour responses elicited in vivo by mammary adenocarcinoma cells (TS/A) engineered to secrete interleukin (IL)-2-IgG fusion proteins. TS/A cells were transfected with DNA coding for IL-2-IgG2b, IgG2b or IL-2, and injected subcutaneously into syngeneic mice. Animals injected with TS/A-IL-2 or TS/A-IL-2-IgG2b both efficiently rejected tumours, whereas treatment with parental cells or TS/A-IgG2b was lethal. Interestingly, only mice vaccinated with IL-2-IgG2b fusion protein-secreting cells showed a long-lasting protective immunity against a later challenge with parental tumour cells. Moreover, the metastatic potential of TS/A-IL-2-IgG2b-transfected cells was dramatically decreased compared with TS/A-IL-2-cells, with a virtual absence of lung metastases after intravenous injection. Adenocarcinomas secreting IL-2-IgG2b exhibited a more prominent, early and persistent infiltration of CD4+, CD8+ and natural killer (NK) cells than TS/A-IL-2 cells. Therefore, upon transfection into adenocarcinoma cells, the IgG2b part of IL-2 fusion protein exerts intriguing added antitumour properties over IL-2 alone, thus contributing to a long-lasting tumour immunity, probably by the recruitment of specific immune effector cells. These findings suggest a promising new oncotherapeutic strategy for poorly immunogenic tumours: vaccination with tumour cells engineered to secrete IL-2-IgG2b fusion protein.

[Immunomodulation after keratoplasty by CTL4-Ig and anti-CD154 antibodies]. / Immunmodulation nach Keratoplastik durch CTLA4-Ig und anti-CD154-Antikörper.

BACKGROUND: The immunoreaction after corneal transplantation is caused by the T cell receptor interacting with the major histocompatibility complex (MHC) receptor of the antigen-presenting cell. The signal is amplified by the CD4 receptor and the costimulatory signal interactions of CD28-B7 and CD40-CD154. We investigated the influence of costimulatory signal blocking on corneal transplant survival in mice. METHODS: Seven groups of 6 BALB/c mice received an orthotopic corneal transplant from C3H mice differing in minor and major MHC and were postoperatively treated as follows: (1) 80 micrograms of CTLA4 fusion protein intraperitoneally (i.p.) for 6 days; (2) 50 microliters of PBS i.p. for 6 days; (3) 1 mg of Solu-Decortin H i.p. for 5 days + dexamethasone AT 0.1% for 35 days; (4) therapy (3) + 50 micrograms of CTLA4 fusion protein i.p. for 6 days; (5) CTLA4-Ig as in (1) + 15 micrograms of anti-CD154 subconjunctivally (s.c.) on days 0, 2, 4, 6, and 8; (6) CTLA4-Ig as in (1) + 25 micrograms of anti-CD154 s.c. for 9 days; and (7) 25 microliters of PBS s.c. for 9 days. RESULTS: All animals had an immunoreaction on the following days: (1) day 18 +/- 3.1; (2) day 13.6 +/- 1.6; (3) day 48 +/- 6.6; (4) day 65 +/- 41; (5) day 23.5 +/- 8.5; (6) day 16.2 +/- 3.6; (7) day 13.8 +/- 2.7. CONCLUSION: The significant prolongation of transplant survival achieved by corticosteroids alone (P < 0.001) is again significantly increased by combining them with CTLA4-Ig (P < 0.001). Specific immunotherapy combined with nonspecific steroid therapy may also improve clinical corneal transplantation results. Compared to the two control groups, CTLA4-Ig and anti-CD154 only influenced transplant survival at a low dosage (P < 0.001).

The IL-15R alpha chain signals through association with Syk in human B cells.

The alpha-chain of the IL-15R (IL-15Ralpha) serves as the specific, high-affinity receptor for IL-15. It is expressed by lymphoid and nonlymphoid cells, including B cell lymphoma lines. In this study, we have further explored IL-15Ralpha-mediated signaling in activated primary B cells and in Raji cells, a human B-lymphoblastoid cell line which expresses the IL-15Ralpha and IL-2Rgamma chains, but lacks the IL-2Rbeta chain. Stimulation of Raji cells with IL-15 induces their proliferation and rescues them from C2-ceramide-induced apoptosis. By immunoprecipitation and Western blotting, we show that treatment of Raji cells and activated primary B cells with IL-15 induces coprecipitation of Syk kinase with the IL-15Ralpha chain. Upon association, the activated Syk kinase phosphorylates the IL-15Ralpha chain as well as phospholipase Cgamma, which coprecipitates with Syk. Furthermore, transfection of Raji cells with stem-loop Syk antisense oligonucleotides prevents IL-15Ralpha and phospholipase Cgamma phosphorylation as well as the inhibition of apoptosis by IL-15. Mutation of a defined region of the intracellular signaling portion of IL-15Ralpha (Tyr227) abrogates both the IL-15Ralpha/Syk association and IL-15Ralpha phosphorylation. Taken together, this suggests that Syk kinase physically and functionally associates with the IL-15Ralpha chain in B cells and that Syk plays a key role in mediating IL-15-induced signal transduction, thus accounting for the distinct functional consequences of IL-15 vs IL-2 binding to B cells.

Structure of the Hodgkin's lymphoma-associated human CD30 gene and the influence of a microsatellite region on its expression in CD30(+) cell lines.

The CD30 antigen is a member of the tumor necrosis factor receptor (TNFR) family which is overexpressed on the surface of the tumor cells of Hodgkin's lymphoma, anaplastic large cell lymphoma (ALCL), and embryonal carcinoma of the testis. In this study the entire cd30 gene which is more than 24000 bp long and organized in eight exons was characterized by analyzing cosmid and phage lambda clones from human placental libraries with long-range polymerase chain reaction (PCR) and sequencing. Differences to other genes of the TNFR family were detected in the region encoding the extracellular domain of the cd30 gene. In nearly all other TNFR genes, the coding region of each cysteine-rich repeat is interrupted by one intron, i.e., the 3-4 cysteine-rich repeats of these receptors are encoded by at least 4-5 exons, whereas the six cysteine-rich repeats of the cd30 gene are encoded by two exons, i.e., each of these exons encode three cysteine-rich repeats. In addition, we also found a genetic polymorphism of tetranucleotide ATCC-repeats in the 5' part of the CD30 promoter. This region was amplified by PCR from seven CD30 overexpressing human lymphoid cell lines and five human tissues with an absent or very low CD30 expression. The amplification products showed length differences of more than 550 bp. The number of the ATCC-repeats was higher in CD30(+) cell lines than in normal tissues. Comparison of the individual PCR products in reporter gene assays revealed that the CD30 promoter activity increased with the length of this polymorphic region up to eightfold. The data suggest that the number of ATCC-repeats in the 5' region of the CD30 promoter modulates the regulation of CD30 expression.

High-dose proinflammatory cytokines induce apoptosis of hair bulb keratinocytes in vivo.

BACKGROUND: Hair loss following skin inflammation may in part be mediated by keratinocyte (KC) apoptosis. While the effects of different cytokines or other apoptosis stimulating agents such as interferon (IFN)-gamma or tumour necrosis factor (TNF)-alpha on KC apoptosis in vitro have been addressed in several studies, little is known about the effects of proinflammatory cytokines on KC apoptosis in vivo. OBJECTIVES: To study the effects of intradermally injected TNF-alpha, interleukin (IL)-1beta and IFN-gamma on KC apoptosis in the back skin of C57BL/6 mice. METHODS: Apoptosis in epidermal and hair bulb KCs was analysed by immunohistology using TUNEL staining. RESULTS: Injection of TNF-alpha induced a significantly higher number of apoptotic cells within the epidermis than vehicle; all three proinflammatory cytokines together further increased their number. Intrafollicular hair bulb KCs were much more susceptible to apoptosis induction by TNF-alpha or IL-1beta; their injection significantly upregulated apoptosis after 6 h, which was further increased after 24 h. The combination of all cytokines together accelerated intrafollicular apoptosis after 6 h by doubling the number of apoptotic cells per hair bulb, compared with the effects of TNF-alpha or IL-1beta alone. CONCLUSIONS: These data suggest that programmed cell death of proliferating KCs in vivo can be induced by proinflammatory cytokines.

The restricted expression pattern of the Hodgkin's lymphoma-associated cytokine receptor CD30 is regulated by a minimal promoter.

One of the most peculiar immunohistological characteristics of the tumour cells of Hodgkin's lymphoma, anaplastic large cell lymphoma (ALCL), and embryonal carcinoma of the testis is the expression of the CD30 antigen. Physiologically, CD30 expression is restricted to a few activated lymphocytes in normal lymphoid tissue and a small population of decidual cells. To clarify the reasons behind this highly restricted expression pattern and to learn about the combination of transcription factors involved in this regulation in Hodgkin's lymphoma and other CD30(+) malignancies, the 5'-flanking regulatory region of the cd30 gene was analysed. The major transcription start site was determined to be 270 bases upstream of the translational start codon in the Hodgkin's lymphoma-derived cell lines L591 and L428. Reporter gene assays revealed that the CD30 promoter (-413 to 84) induces a 50- to 1000-fold higher luciferase expression in CD30(+) human lymphoid cell lines (Co, Jurkat, and the Hodgkin's lymphoma-derived cell line L540) than in CD30(-) human lymphoid cell lines (DG75, SUP-T1, and U698M), CD30(-) human carcinoma cell lines (HeLa and MCF-7), or COS1 cells. Deletion analysis defined a TATA-less, minimal promoter sequence from -164 to 84. The transcription factor Sp1 and members of the Ets family induce CD30 expression, whereas the transcription factor Sp3 diminishes its induction. These data suggest that a high Sp1/Sp3 expression ratio and a peculiar expression pattern of the Ets transcription factors are involved in the overexpression of CD30 and might contribute to the transformation of CD30(+) tumour cells.

Inhibition of keratinocyte apoptosis by IL-15: a new parameter in the pathogenesis of psoriasis?

Keratinocytes (KC) are important source of and targets for several cytokines. Although KC express IL-15 mRNA, the functional effects of IL-15 on these epithelial cells remain to be dissected. Investigating primary human foreskin KC and HaCaT cells, we show here by semiquantitative RT-PCR and flow cytometric analysis that both translate IL-15 and IL-15R mRNA and express IL-15 and IL-15Ralpha protein on the cell surface, suggesting that human KC can employ IL-15 for juxtacrine signaling. While IL-15 exerted no significant effect on KC proliferation and IL-6 or IL-8 secretion, IL-15 inhibited both anti-Fas and methylcellulose-induced KC apoptosis in vitro. This is in line with the recognized potent anti-apoptotic effects of IL-15. IL-2, whose receptor shares two components with the IL-15R, failed to inhibit KC apoptosis. Together with the role of IL-15 in sustaining chronic immune reactions, this invited the question of whether a reduction of KC apoptosis by IL-15 may be involved in the pathogenesis of psoriasis, a chronic hyperproliferative inflammatory skin disease characterized by abnormally low KC apoptosis in the epidermis. Remarkably, compared with nonlesional psoriatic skin and skin of healthy volunteers, lesional psoriatic epidermis showed high IL-15 protein expression in the epidermis and enhanced binding activity for IL-15. Therefore, antagonizing the inhibitory effects of IL-15 on KC apoptosis deserves exploration as a novel therapeutic strategy in psoriasis management.

The effect of corticosteroid and cyclosporin A on murine corneal allograft rejection.

BACKGROUND: The immunomodulatory T-helper type 1 (Th1) cytokine interferon-gamma (IFN-gamma) was measured in serum and cornea to ascertain its general contribution to corneal graft rejection and to establish a rational basis for the decision for or against systemic therapy. METHODS: Eight groups of differently treated BALB/c (H-2d) mice received a C3H (H-2 k) corneal graft. There was one saline-treated control group and two groups that received intramuscular cyclosporin A (CsA) for 14 or 40. Three groups received systemic or topical, systemic plus topical corticosteroid treatment, which was combined with CsA in two further groups. To measure the IFN-gamma level by enzyme-linked immunosorbent assay (ELISA), blood was taken by heart puncture and corneae were excised at the limbus. RESULTS: Five days of systemic corticosteroid and 14 days of CsA had no significant effect on graft survival. A 40-day CsA treatment and a 40-day combined corticosteroid treatment significantly prolonged graft survival. An 80-day topical corticosteroid treatment produced additional prolongation. IFN-gamma could not be detected (limit of detection 25 pg/ml) in any of the serum samples, while significantly increased amounts of IFN-gamma were detected in the supernatants of the corneal tissue 13 or 14 days after allogeneic but not syngeneic corneal graft, corresponding to 9.5 pg, 5.1 pg and 1.8 pg per cornea. CONCLUSION: The detection of Th1 cytokines in the cornea but not the serum of mice at the time of allograft rejection is in accordance with the finding of long-lasting dose-dependent immunosuppression of topical steroids and the inefficacy of short-term systemic CsA and corticosteroids.

The HTLV-I tax protein transcriptionally modulates OX40 antigen expression.

OX40 is a member of the TNF receptor family, expressed on activated T cells. It is the only costimulatory T cell molecule known to be specifically up-regulated in human T cell leukemia virus type-I (HTLV-I)-producing cells. In a T cell line, OX40 surface expression was shown to be induced by HTLV-I Tax alone. To understand molecular mechanisms of OX40 gene regulation and modulation by HTLV-I Tax, we have cloned the human OX40 gene and analyzed its 5'-flanking region. By reporter gene analysis with progressive 5' deletions from nucleotides -1259 to -64, we have defined a 157-bp DNA fragment as a minimal promoter for constitutive expression. In addition, we show that in the OX40+ cell line, Co, Tax is able to further increase OX40 surface expression. Up-regulation of OX40 promoter activity by Tax requires two upstream NF-kappaB sites, which are not active in the constitutive OX40 expression. Their deletion abrogates Tax responsiveness in reporter gene analysis. The site-directed mutagenesis of each NF-kappaB site demonstrates that cooperative NF-kappaB binding is a prerequisite for Tax-directed activity as neither site alone is sufficient for a full Tax responsiveness of the OX40 promoter. Upon Tax expression, both sites bind p65 and c-Rel. These data provide new insight into the direct regulation of OX40 by Tax and add to our understanding of the possible role of the OX40/OX40 ligand system in the proliferation of HTLV-I+ T cells.

An interleukin-2-IgG-Fas ligand fusion protein suppresses delayed-type hypersensitivity in mice by triggering apoptosis in activated T cells as a novel strategy for immunosuppression.

BACKGROUND: Cell-mediated immune responses can be down-regulated by induction of apoptosis of immunoreactive lymphocytes. In the present study, we have tested the feasibility of a strategy for immunosuppression by the selective induction of apoptosis in activated, interleukin (IL)-2 receptor-positive lymphocytes, using a triple IL-2-IgG-FasL fusion protein. The IL-2-IgG-FasL fusion protein combines IL-2 for the selection of activated T cells, with the extracellular domain of the FasL molecule for inducing T-cell apoptosis. These components were separated by the Fc part of IgG1 serving as a spacer as well as for half-life prolongation. METHODS: The gene for the chimeric protein was created by fusing DNA sequences encoding for the three functional components: human IL-2, the Fc part of human IgG1, and the extracellular domain of murine FasL. When the fusion gene was expressed in murine J558L cells, we obtained soluble dimeric immunoglobulin-like proteins in the supernatant. After analyzing the function of the IL-2 and FasL portions individually in vitro, a delayed-type hypersensitivity (DTH) reaction to sheep red blood cells as model for cell-mediated immune responses was investigated to evaluate the IL-2-IgG-FasL-mediated immunosuppression in vivo. RESULTS: In vitro, the IL-2-IgG-FasL fusion protein supported IL-2-dependent proliferation of Fas-resistant CTLL-2 cells, whereas concanavalin A-T blasts were induced to undergo apoptosis by the FasL portion. In vivo, this fusion protein potently inhibited a murine DTH. This was associated with an increased rate of apoptosis in activated lymphocytes in the spleen, even at very low doses of the fusion protein. Furthermore, a second antigen challenge 10 days after IL-2-IgG-FasL treatment still failed to elicit a DTH response. CONCLUSION: The abrogation of a standard T cell-dependent immune response in vivo demonstrates that IL-2-IgG-FasL can be successfully exploited to trigger the death of deleterious T cells, presenting a potentially useful strategy in the management of autoimmune diseases and allotransplant rejections.

Intercellular adhesion molecule-1 and hair follicle regression.

Although the intercellular adhesion molecule-1 (ICAM-1) is recognized for its pivotal role in inflammation and immune responses, its role in developmental systems, such as the cyclic growth (anagen) and regression (catagen) of the hair follicle, remains to be explored. Here we demonstrate that ICAM-1 expression in murine skin is even more widespread and more developmentally regulated than was previously believed. In addition to endothelial cells, selected epidermal and follicular keratinocyte subpopulations, as well as interfollicular fibroblasts, express ICAM-1. Murine hair follicles express ICAM-1 only late during morphogenesis. Thereafter, morphologically identical follicles markedly differ in their ICAM-1 expression patterns, which become strikingly hair cycle-dependent in both intra- and extrafollicular skin compartments. Minimal ICAM-1 and leukocyte function-associated (LFA-1) protein and mRNA expression is observed during early anagen and maximal expression during late anagen and catagen. Keratinocytes of the distal outer root sheath, fibroblasts of the perifollicular connective tissue sheath, and perifollicular blood vessels exhibit maximal ICAM-1 immunoreactivity during catagen, which corresponds to changes of LFA-1 expression on perifollicular macrophages. Finally, ICAM-1-deficient mice display significant catagen acceleration compared to wild-type controls. Therefore, ICAM-1 upregulation is not limited to pathological situations but is also important for skin and hair follicle remodeling. Collectively, this suggests a new and apparently nonimmunological function for ICAM-1-related signaling in cutaneous biology.

Protection of trinitrobenzene sulfonic acid-induced colitis by an interleukin 2-IgG2b fusion protein in mice.

BACKGROUND & AIMS: We have shown in previous studies that an interleukin 2 (IL-2)-IgG2b fusion protein suppresses both humoral and cellular immune reactions in a murine model of DTH reaction. We now analyze the effects of IL-2-IgG2b in a model of intestinal inflammation in mice induced by the hapten reagent 2,4, 6-trinitrobenzene sulfonic acid (TNBS) that mimics immunologic characteristics of human Crohn's disease. METHODS: In TNBS-induced colitis, colonic and splenic T-cell subsets were characterized by immunohistochemistry and flow cytometry. Cytokine synthesis was studied by semiquantitative reverse-transcription polymerase chain reaction and intracellular cytokine staining in CD4(+) T cells. RESULTS: When mice were treated with IL-2-IgG2b, improvement in both wasting disease and histopathologic signs of colonic inflammation was observed. An increase in the number of colonic CD4(+)/CD25(+) T cells and increased synthesis of the immunosuppressive cytokine IL-10 also occurred. The protective role of IL-10 was demonstrated by the finding that neutralization of IL-10 in vivo using IL-10-specific antibodies inhibited the IL-2-IgG2b effects in TNBS-induced colitis. CONCLUSIONS: These studies show for the first time that the IL-2-IgG2b fusion protein can abrogate experimental colitis by local induction of IL-10-secreting T cells.

Death deflected: IL-15 inhibits TNF-alpha-mediated apoptosis in fibroblasts by TRAF2 recruitment to the IL-15Ralpha chain.

Interleukin-15 (IL-15) is a potent inhibitor of several apoptosis pathways. One prominent path toward apoptosis is the ligand-induced association of TNF receptor 1 (TNFR1) with death domain adaptor proteins. Studying if and how IL-15 blocks TNFR1-mediated apoptosis in a murine fibroblast cell line (L929), we show here that IL-15 blocks TNFR1-induced apoptosis via IL-15Ralpha chain signaling. The intracellular tail of IL-15Ralpha shows sequence homologies to the TRAF2 binding motifs of CD30 and CD40. Most important, binding of IL-15 to IL-15Ralpha successfully competes with the TNFR1 complex for TRAF2 binding, which may impede assembly of key adaptor proteins to the TNFR1 complex, and induces IkappaBalpha phosphorylation. Thus, IL-15Ralpha chain stimulation is a powerful deflector of cell death very early in the apoptosis signaling cascade, while TNF-alpha and IL-15 surface as major opponents in apoptosis control.

Human monocytes constitutively express membrane-bound, biologically active, and interferon-gamma-upregulated interleukin-15.

Interleukin-15 (IL-15) is a potent regulator of T-, B-, and natural killer cell proliferation and displays unusually tight controls of secretion. Even though IL-15 mRNA is constitutively expressed in monocytes/macrophages and is upregulated by a variety of stimuli, evidence for IL-15 cytokine secretion is only found exceptionally, eg, conditions of pathological, chronic inflammation. This raises the possibility that monocytes express membrane-bound IL-15 rather than secrete it. The current study explores this hypothesis. We demonstrate here that biologically active IL-15 is indeed detectable in a constitutively expressed, membrane-bound form on normal human monocytes, as well as on monocytic cell lines (MONO-MAC-6, THP-1, and U937), but not on human T or B cells (MT4, M9, C5966, JURKAT, DAUDI, RAJI, and Epstein-Barr virus-immortalized B-cell clones). Furthermore, cell surface-bound IL-15 is upregulated upon interferon-gamma stimulation. Interestingly, monocyte/macrophage inhibitory cytokines such as IL-4 and IL-13 fail to downregulate both constitutive and induced cell-surface expression of IL-15. Membrane-bound IL-15 does not elute with acetate buffer or trypsin treatment, suggesting that it is an integral membrane protein and that it is not associated with the IL-15 receptor complex. Finally, membrane-bound IL-15 stimulates T lymphocytes to proliferate in vitro, indicating that it is biologically active. These findings enlist IL-15 in the fairly small family of cytokines for which the presence of a biologically active membrane-bound form has been demonstrated (eg, IL-1, tumor necrosis factor-alpha, and IL-10) and invites the speculation that most of the biological effects of IL-15 under physiological conditions are exerted by the cell surface-bound form.