Autologous serum skin test reactions in chronic spontaneous urticaria differ from heterologous cell reactions.

BACKGROUND: Autoimmune chronic spontaneous urticaria (CSU) is due to mast cell (MC)-activating autoantibodies, which are screened for by the autologous serum skin test (ASST) and basophil tests (BTs). Many CSU patients are positive in only one of these tests. How often this occurs and why is currently unknown. OBJECTIVES: To characterize the prevalence of mismatched ASST and BTs in CSU patients, and to investigate possible reasons for these mismatches. METHODS: We determined the rates of ASST+/BT- and ASST-/BT+ mismatches in published CSU studies. We assessed sera from 48 CSU patients by ASST, two BTs (basophil histamine release assay, BHRA; basophil activation test, BAT), a MC histamine release assay (MCHRA) and by ex vivo skin microdialysis (SMD). RESULTS: The ASST/BT mismatch rate in published CSU studies was 31% (ASST+/BT-: 22%, ASST-/BT+: 9%). In our patients, the ASST/BHRA and ASST/BAT mismatch rate was 35.4% (ASST+/BHRA-: 18.8% and ASST-/BHRA+: 16.7%) and 31.3% (ASST+/BAT-: 6.3% and ASST-/BAT+: 25.0%), respectively, and the two BTs were significantly correlated (P = 0.0002). The use of heterologous MCs, in vitro and in situ, instead of basophils produced similar results (MCHRA mismatch: 47.9%, ASST+/MCHRA-: 18.8%, ASST-/MCHRA+: 29.2%; SMD mismatch: 40.0%, ASST+/SMD-: 10.0% and ASST-/SMD+: 30.0%), and the MCHRA was highly correlated with SMD results (P = 0.0002). CONCLUSIONS: The ASST and BTs show divergent results in a third of CSU patients. Mismatches cannot be explained by the choice of basophil assay, the type of heterologous cells exposed to CSU serum in vitro (basophils vs. mast cells), nor the experimental setting of heterologous skin mast cells (in vitro vs. in situ). Thus, serum-induced whealing, in CSU patients, seems to involve autologous skin signals modulating MC degranulation.

Functional characterization of Foxp3-specific spontaneous immune responses.

Tumor-infiltrating CD4+CD25+ regulatory T cells (Tregs) are associated with an impaired prognosis in several cancers. The transcription factor forkhead box P3 (Foxp3) is generally expressed in Tregs. Here, we identify and characterize spontaneous cytotoxic immune responses to Foxp3-expressing cells in peripheral blood of healthy volunteers and cancer patients. These immune responses were directed against a HLA-A2-restricted peptide epitope derived from Foxp3. Foxp3-reactive T cells were characterized as cytotoxic CD8+ T cells. These cells recognized dendritic cells incubated with recombinant Foxp3 protein indicating that this protein was indeed internalized, processed and cross-presented in the context of HLA-A2. More importantly, however, Foxp3-specific T cells were able to specifically recognize Tregs. Similarly, Foxp3+ malignant T cells established from a Cutaneous T-cell lymphomas (CTCL) patient were readily killed by the Foxp3-specific cytotoxic T lymphocytes. The spontaneous presence of Foxp3-specific cytotoxic T-cell responses suggest a general role of such T cells in the complex network of immune regulation as such responses may eliminate Tregs, that is, suppression of the suppressors. Consequently, induction of Foxp3-specific cytotoxic T-cell responses appears as an attractive tool to boost spontaneous or therapeutically provoked immune responses, for example, for the therapy of cancer.

IgE-mediated basophil tumour necrosis factor alpha induces matrix metalloproteinase-9 from monocytes.

BACKGROUND: IgE-mediated activation of mast cells has been reported to induce the release of tumour necrosis alpha (TNF-α), which may display autocrine effects on these cells by inducing the generation of the tissue remodelling protease matrix metalloproteinase-9 (MMP-9). While mast cells and basophils have been shown to express complementary and partially overlapping roles, it is not clear whether a similar IgE/TNF-α/MMP-9 axis exists in the human basophil. The purpose of this study was thus to investigate whether IgE-mediated activation of human basophils induces TNF-α and MMP-9 release. METHODS: Human peripheral blood mononuclear cells (PBMC), isolated basophils and monocytes were stimulated up to 21 h with anti-IgE. Mediator releases were assessed by ELISA, and surface expressions of mediators were detected by flow cytometry. Upregulation of cytokine production was detected by Western blot and polymerase chain reaction (PCR). RESULTS: IgE-mediated activation of basophils induced the synthesis and release of both TNF-α and MMP-9 from PBMC. In contrast, IgE-mediated activation of purified basophils induced the release and cellular expression of TNF-α but not MMP-9. Isolated monocytes did not release MMP-9 upon anti-IgE stimulation, but MMP-9 release was induced by stimulating monocytes with supernatants from activated basophils, and this release was inhibited by anti-TNF-α neutralizing antibodies. CONCLUSION: Our results strongly indicate that human basophils release TNF-α following IgE-dependent activation and that this cytokine subsequently stimulates MMP-9 release from monocytes. These findings support a direct involvement of basophils in inflammation as well as suggesting a role for the basophil in tissue remodelling.

Regulatory T cells and immunodeficiency in mycosis fungoides and Sézary syndrome.

Cutaneous T-cell lymphoma (CTCL) is the term for diseases characterized by primary accumulation of malignant T cells in the skin. Patients with the two predominant clinical forms of CTCL called mycosis fungoides (MF) and Sézary syndrome (SS) characteristically develop severe immunodeficiency during disease progression and consequently patients with advanced disease frequently die of infections and not from the tumor burden. For decades, it has been suspected that the malignant T cells actively drive the evolving immunodeficiency to avoid antitumor immunity, yet, the underlying mechanisms remain unclear. The identification of a subset of highly immunosuppressive regulatory T cells (Tregs) triggered a variety of studies investigating if MF and SS are malignant proliferations of Tregs but seemingly discordant findings have been reported. Here, we review the literature to clarify the role of Tregs in MF and SS and discuss the potential mechanisms driving the immunodeficiency.

Oncogenic kinase NPM/ALK induces expression of HIF1α mRNA.

The mechanisms of malignant cell transformation mediated by the oncogenic anaplastic lymphoma kinase (ALK) tyrosine kinase remain only partially understood. In this study, we report that T-cell lymphoma (TCL) cells carrying the nucleophosmin (NPM)/ALK fusion protein (ALK+ TCL) strongly express hypoxia-induced factor 1α (HIF1α) mRNA, even under normoxic conditions, and markedly upregulate HIF1α protein expression under hypoxia. HIF1α expression is strictly dependent on the expression and enzymatic activity of NPM/ALK, as shown in BaF3 cells transfected with wild-type NPM/ALK and kinase-inactive NPM/ALK K210R mutant and by the inhibition of the NPM/ALK function in ALK+ TCL cells by a small-molecule ALK inhibitor. NPM/ALK induces HIF1α expression by upregulating its gene transcription through its key signal transmitter signal transducer and activator of transcription 3 (STAT3), which binds to the HIF1α gene promoter as shown by the chromatin immunoprecipitation assay and is required for HIF1α gene expression as demonstrated by its small interfering RNA-mediated depletion. In turn, depletion of HIF1α increases mammalian target of rapamycin complex 1 activation, cell growth and proliferation and decreases vascular endothelial growth factor synthesis. These results identify a novel cell-transforming property of NPM/ALK, namely its ability to induce the expression of HIF1α, a protein with an important role in carcinogenesis. These results also provide another rationale to therapeutically target NPM/ALK and STAT3 in ALK+ TCL.

Expression of full-length and splice forms of FoxP3 in rheumatoid arthritis.

OBJECTIVE: The aim of our study was to compare the presence of full-length and alternative splice forms of FoxP3 mRNA in CD4 cells from rheumatoid arthritis (RA) patients and healthy controls. METHODS: A quantitative real-time polymerase chain reaction (QRT-PCR) method was used to measure the amount of FoxP3 mRNA full-length and splice forms. CD4-positive T cells were isolated from peripheral blood from 50 RA patients by immunomagnetic separation, and the FoxP3 mRNA expression was compared with the results from 10 healthy controls. RESULTS: We observed an increased expression of full-length FoxP3 mRNA in RA patients when compared to healthy controls, as well as an increase in CD25 mRNA expression, but no corresponding increase in CTLA-4 mRNA expression. The presence of an alternative splice form of FoxP3 lacking exon 2 was confirmed in both RA patients and healthy controls, but with no significant difference in expression between the two groups. There was a positive correlation between the amount of FoxP3 mRNA and the clinical inflammation parameters C-reactive protein (CRP) and erythrocyte sedimentation rate (ESR), and a negative correlation between FoxP3 mRNA and the dose of methotrexate (MTX) given to the patients. CONCLUSION: RA patients express more full-length FoxP3 than healthy controls in peripheral blood CD4-positive cells, suggesting an increased number of regulatory T cells (Tregs). However, no concomitant increase in CTLA-4 expression was seen. We therefore propose that the Tregs are left unable to suppress the ongoing inflammation due to a deficiency in CTLA-4 needed for cell contact-dependent suppression.

COX-2-dependent PGE(2) acts as a growth factor in mycosis fungoides (MF).

Cancer often originates from a site of persistent inflammation, and the mechanisms turning chronic inflammation into a driving force of carcinogenesis are intensely investigated. Cyclooxygenase-2 (COX-2) is an inducible key modulator of inflammation that carries out the rate-limiting step in prostaglandin synthesis. Aberrant COX-2 expression and prostaglandin E(2) (PGE(2)) production have been implicated in tumorigenesis. In this study we show that COX-2 is ectopically expressed in malignant T-cell lines from patients with cutaneous T-cell lymphoma (CTCL) as well as in situ in lymphocytic cells in 21 out of 22 patients suffering from mycosis fungoides (MF) in plaque or tumor stage. COX-2 is not expressed in lymphocytes of 11 patients with patch-stage MF, whereas sporadic COX-2 staining of stromal cells is observed in the majority of patients. COX-2 expression correlates with a constitutive production of PGE(2) in malignant T cells in vitro. These cells express prostaglandin receptors EP3 and EP4 and the receptor antagonist as well as small interfering RNA (siRNA) directed against COX-2, and specific COX-2 inhibitors strongly reduce their spontaneous proliferation. In conclusion, our data indicate that COX-2 mediated PGE(2) exerts an effect as a tumor growth factor in MF.

Malignant Tregs express low molecular splice forms of FOXP3 in Sézary syndrome.

Sézary syndrome (SS) is an aggressive variant of cutaneous T-cell lymphoma. During disease progression, immunodeficiency develops; however, the underlying molecular and cellular mechanisms are not fully understood. Here, we study the regulatory T cell (Treg) function and the expression of FOXP3 in SS. We demonstrate that malignant T cells in 8 of 15 patients stain positive with an anti-FOXP3 antibody. Western blotting analysis shows expression of two low molecular splice forms of FOXP3, but not of wild-type (wt) FOXP3. The malignant T cells produce interleukin-10 and TGF-beta and suppress the growth of non-malignant T cells. The Treg phenotype and the production of suppressive cytokines are driven by aberrant activation of Jak3 independent of the FOXP3 splice forms. In contrast to wt FOXP3, the low molecular splice forms of FOXP3 have no inhibitory effect on nuclear factor-kappaB (NF-kappaB) activity in reporter assays which is in keeping with a constitutive NF-kappaB activity in the malignant T cells. In conclusion, we show that the malignant T cells express low molecular splice forms of FOXP3 and function as Tregs. Furthermore, we provide evidence that FOXP3 splice forms are functionally different from wt FOXP3 and not involved in the execution of the suppressive function. Thus, this is the first description of FOXP3 splice forms in human disease.

FOXP3+ regulatory T cells in cutaneous T-cell lymphomas: association with disease stage and survival.

FOXP3 is a unique marker for CD4+CD25+ regulatory T cells (Tregs). In solid tumours, high numbers of Tregs are associated with a poor prognosis. Knowledge about the implications of Tregs for the behaviour of haematological malignancies is limited. In this study, skin biopsies from 86 patients with mycosis fungoides (MF) and cutaneous T-cell lymphoma (CTCL) unspecified were analysed for the expression of FOXP3 on tumour cells and tumour-infiltrating Tregs. Labelling of above 10% of the neoplastic cells was seen in one case classified as an aggressive epidermotropic CD8+ cytotoxic CTCL. In the remaining 85 cases, the atypical neoplastic infiltrate was either FOXP3 negative (n=80) or contained only very occasional weakly positive cells (n=5). By contrast, all biopsies showed varying numbers of strongly FOXP3+ tumour-infiltrating Tregs. MF with early or infiltrated plaques had significantly higher numbers of FOXP3+ Tregs than CTCL unspecified or advanced MF with tumours or transformation to large cell lymphoma. An analysis of all patients demonstrated that increasing numbers of FOXP3+ Tregs were associated with improved survival in both MF and CTCL unspecified. In conclusion, our data indicate that the presence of FOXP3+ Tregs in CTCL is associated with disease stage and patient survival.

Jak3- and JNK-dependent vascular endothelial growth factor expression in cutaneous T-cell lymphoma.

Biopsies from patients with cutaneous T-cell lymphoma (CTCL) exhibit stage-dependent increase in angiogenesis. However, the molecular mechanisms responsible for the increased angiogenesis are unknown. Here we show that malignant CTCL T cells spontaneously produce the potent angiogenic protein, vascular endothelial growth factor (VEGF). Dermal infiltrates of CTCL lesions show frequent and intense staining with anti-VEGF antibody, indicating a steady, high production of VEGF in vivo. Moreover, the VEGF production is associated with constitutive activity of Janus kinase 3 (Jak3) and the c-Jun N-terminal kinases (JNKs). Sp600125, an inhibitor of JNK activity and activator protein-1 (AP-1) binding to the VEGF promoter, downregulates the VEGF production without affecting Jak3 activity. Similarly, inhibitors of Jak3 inhibit the VEGF production without affecting JNK activity. Downregulation of Stat3 with small interfering RNA has no effect, whereas curcumin, an inhibitor of both Jak3 and the JNKs, almost completely blocks the VEGF production. In conclusion, we provide evidence of VEGF production in CTCL, which is promoted by aberrant activation of Jak3 and the JNKs. Inhibition of VEGF-inducing pathways or neutralization of VEGF itself could represent novel therapeutic modalities in CTCL.

Constitutive SOCS-3 expression protects T-cell lymphoma against growth inhibition by IFNalpha.

Signal transducer and activator of transcription (Stat)3 is constitutively activated in cutaneous T-cell lymphoma (CTCL), where it protects tumour cells against apoptosis. The constitutive activation of Stat3 leads to a constitutive expression of suppressor of cytokine signalling (SOCS)-3. In healthy cells, SOCS-3 is transiently expressed following cytokine stimulation and functions as a negative feedback inhibitor of the Stat3-activating kinases. Here, we attempt to resolve the apparent paradox of a simultaneous SOCS-3 expression and Stat3 activation in the same cells. We show that (i) SOCS-3 expression in tumour cells is equal to or higher than in cytokine-stimulated nonmalignant T cells, (ii) SOCS-3 is not mutated in CTCL, (iii) overexpression of SOCS-3 blocks IFNalpha-mediated growth inhibition without affecting Stat3 activation, growth, and apoptosis, and (iv) inhibition of SOCS-3 by a dominant negative Stat3 (Stat3D) increases the IFNalpha-mediated growth inhibition. Taken together, these data show that SOCS-3 does not inhibit Stat3 activation, growth, and survival in CTCL. In contrast, SOCS3 protects tumour cells against growth inhibition by IFNalpha. Unlike SOCS-1, SOCS-3 is therefore not a tumour suppressor but rather a protector of tumour cells.

In vivo activation of STAT3 in cutaneous T-cell lymphoma. Evidence for an antiapoptotic function of STAT3.

A characteristic feature of neoplastic transformation is a perpetual activation of oncogenic proteins. Here, we studied signal transducers and activators of transcription (STAT) in patients with mycosis fungoides (MF)/cutaneous T-cell lymphoma (CTCL). Malignant lymphocytes in dermal infiltrates of CTCL tumors showed frequent and intense nuclear staining with anti-PY-STAT3 antibody, indicating a constitutive activation of STAT3 in vivo in tumor stages. In contrast, only sporadic and faint staining was observed in indolent lesions of patch and plaque stages of MF. Moreover, neoplastic lymphocytes in the epidermal Pautrier abscesses associated with early stages of MF did not express activated STAT3. To address the role of STAT3 in survival/apoptosis, CTCL tumor cells from an advanced skin tumor were transfected with either wild-type STAT3 (STAT3wt) or dominant-negative STAT3 (STAT3D). Forced inducible expression of STAT3D triggered a significant increase in tumor cells undergoing apoptosis, whereas forced expression of STAT3wt or empty vector had no effect. In conclusion, a profound in vivo activation of STAT3 is observed in MF tumors but not in the early stages of MF. Moreover, STAT3 protects tumor cells from apoptosis in vitro. Taken together, these findings suggest that STAT3 is a malignancy factor in CTCL.

Inhibition of protein phosphatase 2A induces serine/threonine phosphorylation, subcellular redistribution, and functional inhibition of STAT3.

Signal transducers and activators of transcription (STATs) are rapidly phosphorylated on tyrosine residues in response to cytokine and growth factor stimulation of cell surface receptors. STATs hereafter are translocated to the nucleus where they act as transcription factors. Recent reports suggest that serine phosphorylation of STATs also is involved in the regulation of STAT-mediated gene transcription. Here, we studied the role of serine/threonine phosphatases in STAT3 signaling in human antigen-specific CD4(+) T cell lines and cutaneous T cell lymphoma lines, expressing a constitutively activated STAT3. We show that an inhibitor of protein phosphatases (PPs) PP1/PP2A, calyculin A, induces (i) phosphorylation of STAT3 on serine and threonine residues, (ii) inhibition of STAT3 tyrosine phosphorylation and DNA binding activity, and (iii) relocation of STAT3 from the nucleus to the cytoplasm. Similar results were obtained with other PP2A inhibitors (okadaic acid, endothall thioanhydride) but not with inhibitors of PP1 (tautomycin) or PP2B (cyclosporine A). Pretreatment with the broad serine/threonine kinase inhibitor staurosporine partly blocked the calyculin A-induced STAT3 phosphorylation, whereas inhibitors of serine/threonine kinases, such as mitogen-activated protein kinase-1 extracellular-regulated kinase-kinase, mitogen-activated protein p38 kinase, and phosphatidylinositol 3-kinase, did not. In conclusion, we provide evidence that PP2A plays a crucial role in the regulation of STAT3 phosphorylation and subcellular distribution in T cells. Moreover, our findings suggest that the level of STAT3 phosphorylation is balanced between a staurosporine-sensitive kinase(s) and PP2A.

Inhibition of constitutively activated Stat3 correlates with altered Bcl-2/Bax expression and induction of apoptosis in mycosis fungoides tumor cells.

The Jak/Stat signaling pathway transmits signals from many cytokine and growth factor receptors to target genes in the nucleus. Constitutive activation of Stat3 has recently been observed in many tumor cells and dysregulation of the Stat signaling pathway has been proposed to be implicated in malignant transformation. In a previous study, we found constitutively tyrosine phosphorylated Stat3 in mycosis fungoides tumor cells. Here, we show that the Jak kinase inhibitor, Ag490, inhibits the constitutive binding of Stat3 to an oligonucleotide representing the Stat-binding sequence from the ICAM promotor. The decreased ability of Stat3 to bind DNA precedes dynamic alterations in the expression of anti-apoptotic Bcl-2 and pro-apoptotic Bax proteins (decreased Bcl-2 expression and increased Bax expression) and induction of apoptosis. Thus, our data suggest that the involvement of Stat3 in oncogenic transformation could be mediated through regulation of survival signals.

The role of caspase 3 and BclxL in the action of interleukin 7 (IL-7): a survival factor in activated human T cells.

The effects of interleukin 7 (IL-7) on apoptosis in interleukin 2 (IL-2)-dependent, activated, primary, human T lymphocytes (hT cells) was examined. IL-7 (like IL-2) rescued cells from apoptosis, as measured by their cellular DNA profile and fragmentation. IL-2 also acted as a mitogen in these T cells. Both cytokines abrogated the dexamethasone-induced stimulation of Caspase 3 and prevented the cleavage of poly (ADP-ribose) polymerase (PARP), a substrate for the Caspase 3. IL-7 upregulated the expression of Bc1xL and counteracted the downregulation of this anti-apoptotic protein by the synthetic glucocorticoid, dexamethasone. Bcl-2 protein expression was uupregulated by IL-7 with or without dexamethasone, but Bc1-2 was expressed at a much lower level than BclxL in these cells. Levels of Bax did not markedly change on either cytokine stimulation or dexamethasone treatment. An unidentified 23-kDa band, which was recognized by the anti-Bc1-2 antibody, was induced by dexamthasone and suppressed by IL-7 and IL-2. This protein was subject to independent regulation as compared to the p26 Bc1-2 protein, suggesting that it may be a novel factor, possibly involved in the regulation of apoptosis. A clear role for IL-7 as a survival factor for cytokine withdrawal and glucocorticoid induced apoptosis in activated primary hT cells is implicated. In addition, regulation of BclxL and downstream inhibition of Caspase 3 activity may mediate this rescue signal.