Follicular lymphoma cells induce changes in T-cell gene expression and function: potential impact on survival and risk of transformation.

PURPOSE: Previous studies have demonstrated the prognostic importance of the immune microenvironment in follicular lymphoma (FL). To investigate the molecular mechanisms during which tumor-infiltrating T cells (TILs) are altered in the FL microenvironment, we studied highly purified CD4 and CD8 TILs from lymph node biopsies at diagnosis in treatment-naive patients with FL compared with reactive tonsils and the peripheral blood of healthy donors. PATIENTS AND METHODS: Gene expression profiling of highly purified CD4 and CD8 TILs was performed on the Affymetrix platform. Diagnostic tissue microarrays from an independent patient set (n = 172) were used to verify protein expression and analyze any impact of TIL-expressed genes on outcome. Time-lapse imaging was used to assess T-cell motility. RESULTS: The most upregulated genes in both CD4 and CD8 TILs were PMCH, ETV1, and TNFRSF9. PMCH is not expressed in peripheral blood T cells, but expression is highly induced on culture with FL. Both CD4 and CD8 TILs from patients with FL have significantly impaired motility compared with those of healthy TILs from reactive tonsils and this can be induced on healthy T cells by FL cells. During multivariate analysis, a model incorporating the number and location of T cells expressing PMCH, NAMPT, and ETV1 showed prognostic significance for overall survival and for time to transformation. CONCLUSION: We showed altered gene expression in TILs in FL and demonstrated that altering the immune microenvironment in FL affects overall survival and time to transformation in this disease.

The hinge region: an important receptor component for GPHR function.

Glycoprotein hormone receptors (GPHRs) are members of the seven-transmembrane-spanning receptor family characterized by a large ectodomain. The hinge region belongs to a part of the GPHR ectodomain for which the three-dimensional structure has not yet been deciphered, leaving important questions unanswered concerning ligand binding and GPHR activation. Recent publications indicate that specific residues of the hinge region mediate hormone binding, receptor activation and/or intramolecular signaling for the three GPHRs, emphasizing the importance of this region. Based on these findings, the hinge region is involved at least in part in hormone binding and receptor activation. This review summarizes functional data regarding the hinge region, demonstrating that this receptor portion represents a link between ligand binding and subsequent GPHR activation.

Melanin-concentrating hormone and immune function.

To date, melanin-concentrating hormone (MCH) has been generally considered as peptide acting almost exclusively in the central nervous system. In the present paper, we revise the experimental evidence, demonstrating that MCH and its receptors are expressed by cells of the immune system and directly influence the response of these cells in some circumstances. This therefore supports the idea that, as with other peptides, MCH could be considered as a modulator of the immune system. Moreover, we suggest that this could have important implications in several immune-mediated disorders and affirm that there is a clear need for further investigation.

Melanin-concentrating hormone and melanin-concentrating hormone receptors in mammalian skin physiopathology.

To date, there is a dearth of evidence to support functions for melanin-concentrating hormone (MCH) and melanin-concentrating hormone receptors (MCH-R) in mammalian skin physiology including pigmentation, inflammation and immune responses and skin cell proliferation. Much research is therefore still needed to define the roles of the hormone and its receptors in mammalian skin. This will be a crucial step to identifying pathogenic mechanisms that may involve the MCH/MCH-R system in the context of inflammatory and autoimmune skin diseases as well as skin cancers. The following review summarizes the studies which have been carried out to examine the expression and function of MCH and MCH-R in mammalian skin. Recent findings with regard to humoral immune responses to the MCH-R1 in patients with the skin depigmenting disease vitiligo are also discussed.

Function-blocking autoantibodies to the melanin-concentrating hormone receptor in vitiligo patients.

Vitiligo is a common depigmenting skin disorder resulting from the loss of melanocytes in the cutaneous epidermis. Although the cause of the disease remains obscure, autoimmune mechanisms are thought to be involved. Recently, melanin-concentrating hormone receptor (MCHR)-binding autoantibodies have been identified in vitiligo patients. In the present study, we aimed to determine if MCHR autoantibodies could also affect receptor function either by direct activation or by blocking its response to melanin-concentrating hormone. The results indicated that 10/18 (56%) vitiligo patient IgG samples inhibited the function of MCHR expressed in a Chinese hamster ovary cell line. In contrast, neither control (n=20) nor SLE patient (n=10) IgG samples blocked receptor function. Compared with healthy controls, MCHR function-blocking autoantibodies were found at a significantly increased frequency in the vitiligo patient group (P=0.0004). No MCHR-activating autoantibodies were detected in any of the vitiligo patient, SLE patient or control IgG samples that were analysed. In addition, vitiligo patient IgGs were tested for MCHR autoantibodies that could mediate antibody-dependent cell-mediated cytotoxicity via the receptor. However, this could only be demonstrated in two vitiligo patient sera. Overall, this work has provided additional evidence that MCHR is a B-cell autoantigen in vitiligo and has demonstrated the existence of MCHR function-blocking autoantibodies further to the receptor-binding autoantibodies previously reported.

Autoantibodies in vitiligo patients recognize multiple domains of the melanin-concentrating hormone receptor.

Previously, we reported the identification of autoantibodies against the melanin-concentrating hormone receptor 1 in patients with vitiligo. In this study, the B cell epitopes on melanin-concentrating hormone receptor 1 that are recognized by these autoantibodies have been identified. Deletion derivatives of melanin-concentrating hormone receptor 1 complementary DNA were constructed and then translated in vitro with the concomitant incorporation of [35S]-methionine into the protein products. The [35S]-labeled melanin-concentrating hormone receptor 1 derivatives were subsequently used in radio-binding assays to investigate the reactivity of sera from nine vitiligo patients that were known to contain antibodies to the receptor. Analysis of the results obtained in the radio-binding assays suggested the existence of multiple antibody binding sites on melanin-concentrating hormone receptor 1, including regions between amino acids 1 to 138 and 139 to 298. Several patients exhibited autoantibodies to more than one melanin-concentrating hormone receptor 1 epitope indicating a heterogeneous humoral response to the receptor. Computer prediction of the potential B cell epitopes on melanin-concentrating hormone receptor 1 revealed that the epitope domains identified overlapped, at least in part, with regions predicted to be highly antigenic.

The melanin-concentrating hormone receptor 1, a novel target of autoantibody responses in vitiligo.

Vitiligo is a common depigmenting disorder resulting from the loss of melanocytes in the skin. The pathogenesis of the disease remains obscure, although autoimmune mechanisms are thought to be involved. Indeed, autoantibodies and autoreactive T lymphocytes that target melanocytes have been reported in some vitiligo patients. The objective of this study was to identify pigment cell antigens that are recognized by autoantibodies in vitiligo. Using IgG from vitiligo patients to screen a melanocyte cDNA phage-display library, we identified the melanin-concentrating hormone receptor 1 (MCHR1) as a novel autoantigen related to this disorder. Immunoreactivity against the receptor was demonstrated in vitiligo patient sera by using radiobinding assays. Among sera from healthy controls and from patients with autoimmune disease, none exhibited immunoreactivity to MCHR1, indicating a high disease specificity for Ab's against the receptor. Inhibition of MCH binding to its receptor by IgG from vitiligo patients was also shown.

Desensitization, surface expression, and glycosylation of a functional, epitope-tagged type I PACAP (PAC(1)) receptor.

To study desensitization and glycosylation of the type I pituitary adenylate cyclase-activating polypeptide (PACAP) receptor (PAC(1)R), a hemagglutinin (HA) epitope was inserted within the N-terminal extracellular domain, allowing immunological detection of PAC(1)R both in intact and permeabilized cells. PAC(1)R was tagged without loss of functions in ligand binding and ligand-stimulated cAMP production. In transiently transfected COS-7 cells, PAC(1)R was localized both in the plasma membrane and the cytoplasm around the nucleus. By immunoblot analysis, the immunoreactive bands with relative molecular masses ranging from 45 to 70 kDa were detected in the membrane fractions of PAC(1)R-expressing COS-7 cells. Digestion of the membranes with endoglycosidase F or treatment of the cells with tunicamycin decreased the size of the receptor to major bands of smaller size (approximately 45 and 48 kDa), suggesting that these two forms of PAC(1)R represent core proteins. Flow cytometric analysis indicated that the agonist promoted a disappearance of cell surface receptor. In accordance with this observation, preexposure of cells to PACAP38 induced a desensitization of PAC(1)R to the agonist response, although it did not cause a reduction in PAC(1)R mRNA or protein level and even slightly elevated them. These results suggest that agonist-induced desensitization of PAC(1)R involves the receptor sequestration.

Pituitary adenylate cyclase-activating polypeptide promotes the survival of basal forebrain cholinergic neurons in vitro and in vivo: comparison with effects of nerve growth factor.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a member of the vasointestinal polypeptide gene family for which neurotrophic activity has been postulated. PACAP mRNA is expressed in the developing and adult hippocampus, which is the principal target region of septal cholinergic neurons. We therefore studied the effects of PACAP on septal cholinergic neurons. In primary cultures from septum of embryonic and postnatal rats, PACAP increased the number of neurons immunohistochemically stained for the low-affinity nerve growth factor (NGF) receptor p75 and for the enzyme choline acetyltransferase (ChAT). PACAP also caused a corresponding increase in ChAT activity. In comparison, NGF had a greater effect than PACAP on the number of p75- and ChAT-positive neurons in these cultures. In vivo, following fimbria fornix transection, the number of immunohistochemically stained septal cholinergic neurons fell significantly to 18% in rats given continuous intracerebroventricular infusion of vehicle, whereas in rats given NGF the number of these neurons did not differ significantly from unoperated controls. In PACAP-treated rats the number was 48% of unoperated values, which represented a significant increase compared with vehicle-treated rats and a significant decrease compared with NGF-treated rats or unoperated controls. Double-staining experiments revealed that most ChAT-positive neurons in rat medial septum also express PACAP receptor 1. Together the results show that PACAP promotes the survival of septal cholinergic neurons in vitro, and after injury in vivo, suggesting that PACAP acts as a neurotrophic factor influencing the development and maintenance of these neurons.

Immunohistochemical localization and distribution of VIP/PACAP receptors in human lung.

VIP and PACAP are distributed in nerve fibers throughout the respiratory tract acting as potent bronchodilators and secretory agents. By using RT-PCR and immunoblotting techniques, we have previously shown the expression of common VIP/PACAP (VPAC(1) and VPAC(2)) and specific PACAP (PAC(1)) receptors in human lung. Here we extend our aims to investigate by immunohistochemistry their localization and distribution at this level. A clear immunopositive reaction was obtained in human lung sections by using either anti-VPAC(1) or -VPAC(2) receptor antibodies but not with anti-PAC(1) receptor antibody. However, PAC(1) receptor (and VPAC(1) and VPAC(2) receptors) could be identified in lung membranes by immunoblotting which supports that the PAC(1) receptor is expressed at a low density. Both VPAC(1) and VPAC(2) receptors showed similar immunohistochemical patterns appearing in smooth muscle cells in the wall of blood vessels and in white blood cells (mainly in areas with inflammatory responses). The results agree with previous evidence on the importance of both peptides in the immune system and support their anti-inflammatory and protective roles in lung.

The distribution of the mRNA and protein products of the melanin-concentrating hormone (MCH) receptor gene, slc-1, in the central nervous system of the rat.

Melanin-concentrating hormone (MCH), a 19 amino acid cyclic peptide, is largely expressed in the hypothalamus. It is implicated in the control of general arousal and goal-orientated behaviours in mammals, and appears to be a key messenger in the regulation of food intake. An understanding of the biological actions of MCH has been so far hampered by the lack of information about its receptor(s) and their location in the brain. We recently identified the orphan G-protein-coupled receptor SLC-1 as a receptor for the neuropeptide MCH. We used in situ hybridization histochemistry and immunohistochemistry to determine the distribution of SLC-1 mRNA and its protein product in the rat brain and spinal cord. SLC-1 mRNA and protein were found to be widely and strongly expressed throughout the brain. Immunoreactivity was observed in areas that largely overlapped with regions mapping positive for mRNA. SLC-1 signals were observed in the cerebral cortex, caudate-putamen, hippocampal formation, amygdala, hypothalamus and thalamus, as well as in various nuclei of the mesencephalon and rhombencephalon. The distribution of the receptor mRNA and immunolabelling was in good general agreement with the previously reported distribution of MCH itself. Our data are consistent with the known biological effects of MCH in the brain, e.g. modulation of the stress response, sexual behaviour, anxiety, learning, seizure production, grooming and sensory gating, and with a role for SLC-1 in mediating these physiological actions.

PACAP-38 is a chemorepellent and an agonist for the lysozyme receptor in tetrahymena thermophila.

Pituitary adenylate cyclase activating peptide (PACAP-38) is a peptide hormone which functions in many mammalian systems, including the nervous and digestive systems. Using in vivo behavioral studies, we have found that this hormone functions as a chemore-pellent in Tetrahymena thermophila with an EC50 of 10 nM. Cells previously adapted to PACAP-38 were found to be adapted to lysozyme and vice versa. Furthermore, the in vivo behavioral activity of PACAP-38 was blocked by addition of the anti-lysozyme receptor antibody, 5545. Chemorepellent activity of PACAP-38 was also inhibited by the addition of neomycin sulfate (inhibition constant Ki = 0.080 micromol x l(-1)), a competitive inhibitor of lysozyme binding to its receptor. PACAP-38 is a more potent and specific agonist for the lysozyme receptor than either intact lysozyme or CB2, a 24-amino acid fragment of lysozyme.

Production and characterization of monoclonal antibodies to pituitary adenylate cyclase activating polypeptide type I receptor.

Pituitary adenylate cyclase activating polypeptide type I receptor (PACAPr) belongs to the novel subfamily of the G-protein coupled receptors with a long extracellular N-terminus, which functions as a major binding site for the PACAP. Three different N-terminal fragments of rat PACAPr were overexpressed in Escherichia coli and purified using His-tags or maltose-binding protein as anchors for affinity chromatography. The purified and refolded proteins were used for the production and screening of monoclonal antibodies (MAbs) to PACAPr. Fifteen hybridoma cell lines producing MAbs specific to PACAPr were generated and characterized. Epitope analysis by competitive enzyme-linked immunoadsorbent assay (ELISA) indicated the presence of two groups of overlapping epitopes in the N-terminal fragment of PACAPr. Reactivity of MAbs with SDS-denaturated and native rat PACAPr was demonstrated by immunoblotting and flow cytometric analysis using transiently transfected COS cells and stably transfected CHO cells expressing rat PACAPr. Each antibody was examined by immunoblotting for the ability to cross react with the human PACAPr in human neuroblastoma NB-OK cells and most of them were shown to recognize human PACAPr as effectively as rat PACAPr. MAbs against the N-terminal extracellular domain of PACAPr can be used for the immunochemical study of the receptor-ligand interaction and for the investigation of PACAPr distribution in normal and tumor tissues.

Presynaptic localization of the PACAP-typeI-receptor in hippocampal and cerebellar mossy fibres.

The distribution of PACAP-typeI-receptor (PACAP-I-R) mRNA and protein was studied in mouse using probes and a newly developed antiserum recognizing all known splice variants. RNase protection assays revealed highest expression levels of PACAP-I-R mRNA in brain, in particular the hypothalamus and hippocampus. At the cellular level, in situ hybridization analysis demonstrated widespread distribution of PACAP-I-R mRNA in neurons throughout the brain, while glial cells did not express the gene. Highest expression levels of PACAP-I-R mRNA were observed in three regions: the limbic system, the hypothalamus, and the brainstem. In accordance with data obtained from in situ hybridization analysis, immunohistochemistry showed widespread distribution of PACAP-I-R like immunoreactivity in the neuropil. Rather strong immunoreactivity was found in cerebellar and hippocampal mossy fibres where double immunolabelling revealed the presynaptic localization of the receptor protein. At the ultrastructural level, PACAP-I-R like immunoreactivity was observed around synaptic vesicles and close to the presynaptic grid in hippocampal mossy fibre terminals. This finding is in contradiction to the described postsynaptic localization of the PACAP-I-R in dendritic processes of hippocampal granule cells in rat. Due to their presynaptic induction, mossy fibre LTPs are distinctly different from LTPs in all other hippocampal regions. Therefore, the presynaptic localization of the PACAP-I-R in mossy fibre terminals may implicate this gene in influencing the synaptic strength of the mossy fibre pathway and hence memory consolidation.

Specific antibody recognition of rat pituitary adenylate cyclase activating polypeptide receptors.

Pituitary adenylate cyclase activating polypeptide (PACAP) is a new member of the secretin/VIP family of peptides. The specific receptor for PACAP has been cloned in rat, human, and bovine tissues. The distribution of the transcripts of PACAP receptor genes has been studied in various tissues using in situ hybridization. However, the unavailability of a specific antibody against the PACAP receptor has hampered further study of the expression of receptor proteins. In the present study, rabbit antisera were generated against a synthetic 25-residue peptide corresponding to the C-terminal intracellular domain of the rat PACAP receptor. To validate the specificity of the antisera, CHO cells and cells stably transfected with rat PACAP receptor cDNA were prepared. Using one of these antisera, the membrane and soluble fractions of the transformants were examined by Western blot analysis. Three bands were observed in subcellular fractions from the transfected CHO cells, but no bands were found in similar preparations from the nontransfected cells. A distinct 57-kDa band, which corresponds to the size of cloned rat PACAP receptor, was detected. In addition, a less intense band, larger than 57 kDa, and a very weakly stained band, smaller than 57 kDa, were demonstrated. All of these bands disappeared or were considerably diminished when the antiserum was preabsorbed with the synthetic immunogen peptide. This suggests that these bands are PACAP receptor-related proteins. The membranes from the transfected CHO cells bound to [125I]PACAP27. The size of the ligand/protein crosslinked product approximated 60 kDa, corresponding to the combined size of the PACAP receptor and PACAP27. No additional bands were observed, indicating that the immunopositive proteins larger or smaller than 57 kDa do not bind to the ligand and are not functional. Unlabeled PACAP27 and PACAP38, but not VIP, displaced the binding, suggesting that the receptors expressed in CHO cells are specific for PACAP. Solubilized membrane fractions prepared from rat brains were used for an immunoprecipitation study with [125I]PACAP27 and [125I]VIP. The PACAP receptor antiserum recognized [125I]PACAP-, but not [125I]VIP-bound proteins in the solubilized brain membrane fractions. Immunohistochemistry using this antiserum showed a distribution of PACAP receptor-like immunoreactivities similar to the distribution of the mRNA of PACAP receptor in the rat brain. Thus, the PACAP receptor antiserum is sufficiently specific to be used as a tool for studying the expression of PACAP receptors and related proteins.

Adrenocorticotropic hormone receptor-blocking immunoglobulins in serum from patients with Addison's disease: a reexamination.

The presence of antibodies inhibitory to ACTH-stimulated cortisol secretion was investigated in patients with Addison's disease. In preliminary experiments, immunoglobulin G (IgG) samples from patients with Addison's disease and control subjects were prepared using diethylaminoethyl-cellulose ion exchange chromatography. Twelve of 29 Addison's IgGs and 1 of 3 control IgGs inhibited ACTH-stimulated cortisol secretion in an in vitro guinea pig adrenal cell bioassay. All but 1 of the samples that inhibited ACTH-stimulated cortisol secretion also inhibited dibutyryl cAMP-stimulated steroidogenesis. IgGs purified by protein-G immunoaffinity were similarly screened for inhibition of ACTH-stimulated cortisol secretion; samples from 5 controls and 5 patients with Graves' disease were without effect, and IgGs from only 2 of 25 patients with Addison's disease decreased ACTH bioactivity (by < 12%). The effects of the IgGs on ACTH bioactivity in vitro were not concentration dependent. Our results suggest that Addison's disease is not caused by circulating antibodies to the ACTH receptor. The inhibitory effects seen with IgG prepared by diethylaminoethyl ion exchange chromatography were most likely attributable to the effects of non-IgG components in the preparations.

A monoclonal anti-peptide antibody mimics adrenocorticotropic hormone activity.

A monoclonal antibody that specifically recognizes the adrenocorticotropic receptor (ACTH) on rat adrenal cells was tested for hormonal activity. The antibody behaved as an agonist based on three different biological activities associated with ACTH. An antibody concentration of 16 micrograms/ml stimulated isolated rat adrenal cells to secrete 800 ng/10(4) cells of corticosterone with a concomitant 10-fold increase of cAMP to 30 pmol/10(5) cells. Antibody concentrations above 16 micrograms/ml inhibited mitotic activity in mouse Y-1 adrenal cells. A radio-immunoassay using an anti-ACTH antibody showed that the monoclonal anti-adrenocorticotropic receptor antibody and ACTH are antigenically related. These findings suggest that the anti-receptor antibody recognizes the ligand binding domain of the ACTH receptor.

Antisense peptides: tools for receptor isolation? Lack of antisense MSH and ACTH to interact with their sense peptides and to induce receptor-specific antibodies.

The use of antisense peptides for receptor isolation as proposed by Blalock and his colleagues (e.g. TIBTECH 8, 140-144, 1990) was tested for human ACTH as well as alpha- and beta-MSH. We synthesized the corresponding antisense peptides HTCAh, HSM-alpha and HSM-beta and analyzed them for specific interaction with the sense peptides using several types of binding assay and bioassay. Similarly HTCAh antibodies were tested for binding to ACTH receptors and ACTH antibodies. All these experiments were negative, i.e. there was no specific interaction between sense and antisense peptides nor between the corresponding antibodies. Receptor binding of the sense peptides was not affected by the antisense peptides or HTCAh antibodies. Unexpectedly, HTCAh but not HSM-alpha or HSM-beta was a weak MSH agonist acting through a site independent of the MSH receptor. A detailed analysis of the concept of antisense peptides revealed that the theoretical background of the hypothesis of the 'molecular recognition theory' is rather weak, explaining the failure of various attempts to obtain specific receptor antibodies.

Differentiation and the tumorigenic and metastatic phenotype of murine melanoma cells.

Using B16 F10 murine melanoma cells and sublines generated from the JB/MS melanoma which exhibit various degrees of melanogenesis, the relationships among differentiation, tumorigenicity, and metastatic potential were examined. The effect of melanocyte-stimulating hormone (MSH), which specifically stimulates differentiation of melanocytes, was also studied. All melanoma lines tested were capable of growing as experimental pulmonary metastases but, surprisingly, the undifferentiated and amelanotic JB/MS-w cells failed to grow as primary subcutaneous tumors. JB/MS-w cells, which had few surface MSH receptors, did not respond to MSH with an increase in melanin production, unlike the other cell lines. Although in vitro treatment with MSH did not change the rates of growth of primary tumors by these cell lines, such treatment decreased the number of pulmonary metastases from B16 F10, JB/MS cells, JB/MS-b1 cells and JB/MS-w cells. Conversely, MSH treatment significantly increased the rates of pulmonary metastases from JB/MS-p cells. The expression of surface melanoma antigens, urokinase-type plasminogen activity and susceptibility to natural killer cells were examined. MSH did not significantly alter surface melanoma antigen expression, but increased the natural killer cell susceptibility of B16 F10, JB/MS and JB/MS-b1 cells, cells which possess abundant surface MSH receptors. There was an inverse correlation between differentiation (pigmentation) and proliferation in vitro, and the more pigmented melanoma cells (B16 F10, JB/MS and JB/MS-b1) expressed relatively lower levels of class-I MHC, relatively higher levels of class-II MHC and the highest metastatic capacity. These results demonstrate that MSH possesses the capacity to regulate not only melanogenesis, but also other factors critical to the metastatic growth of the cells.

A monoclonal anti-peptide antibody recognizes the adrenocorticotropic receptor.

We have produced a monoclonal antibody that specifically recognizes the adrenocorticotropic receptor on rat adrenal cells. The immunogen was designed from an RNA sequence complementary to the mRNA coding for ACTH1-24. This complementary peptide, termed HTCA, has been shown to specifically bind ACTH and was proposed to mimic the ACTH binding site of the hormone receptor. The monoclonal anti-HTCA antibody recognized a restricted domain of the HTCA peptide, bound to Y-1 adrenal cells with a KD of 1.8 nM, and blocked the binding of 125I-ACTH to rat adrenal cells. These findings show that anti-HTCA competes with ACTH for binding to the ACTH receptor.