ABSTRACT
Functional forms of the IL-2, IL-4, IL-7, IL-9, and IL-15 receptors require the gamma c receptor component. We have described previously a myeloid cell line called Tf-1beta, which binds IL-2 with intermediate-affinity and proliferates in response to IL-2. In this study, we characterize gamma c expression on Tf-1beta2 cells, a derivative of Tf-1beta cells stimulated exclusively with IL-2. Although Tf-1beta2 cells bind IL-2 with intermediate-affinity and proliferate in response to IL-2, this cell line does not express the p64 gamma c chain at the protein level. This result was surprising because prior studies suggest these cells should not be expected to proliferate in response to IL-2 or IL-15 in the absence of the p64 gamma c chain. A p74 protein was detected by western blot following immunoprecipitation with an anti-gamma c polyclonal antibody, and a p74 protein was identified consistently in complex with IL-2 and IL-15 on these cells. However, the gamma c gene in these Tf-1beta2 cells shows no evidence of mutation by sequence analysis. Furthermore, inhibition of glycosylation of these Tf-1beta2 cells by tunicamycin treatment yields a standard 39-kDa molecule recognized on western blot with anti-gamma c antibody, as seen for the standard 64-kDa isoform of gamma c. These results demonstrate that a 74-kDa gamma c receptor isoform was involved in the response of the Tf-1beta2 cells to cytokines which normally interact with the 64-kDa gamma c chain.
Subject(s)
Interleukin-15/pharmacology , Interleukin-2/pharmacology , Receptors, Interleukin-2/physiology , Alternative Splicing , Blotting, Western , Cell Division/drug effects , Glycosylation , Humans , Interleukin-15/metabolism , Interleukin-2/metabolism , Janus Kinase 3 , Myeloid Progenitor Cells/cytology , Myeloid Progenitor Cells/drug effects , Myeloid Progenitor Cells/metabolism , Phosphorylation/drug effects , Protein Isoforms , Protein-Tyrosine Kinases/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , Receptors, Interleukin-2/genetics , Receptors, Interleukin-2/metabolism , Tumor Cells, CulturedABSTRACT
Although neurotrophic factors have been implicated in several forms of neuroplasticity, little is known concerning their potential role in spinal plasticity. Cervical dorsal rhizotomy (CDR) enhances serotonin terminal density near (spinal) phrenic motoneurons and serotonin-dependent long-term facilitation of phrenic motor output (Kinkead et al., 1998). We tested the hypothesis that selected neurotrophic factors change in a manner consistent with an involvement in this model of spinal plasticity. Brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), glial cell line-derived neurotrophic factor (GDNF), and transforming growth factor-beta(1) (TGF-beta(1)) concentrations were measured (ELISA) in three regions of interest to respiratory control: (1) ventral cervical spinal segments associated with the phrenic motor nucleus (C3-C6), (2) ventral thoracic spinal segments associated with inspiratory intercostal motor output (T3-T6) and (3) the diaphragm. Tissues were harvested from rats 7 d after bilateral CDR and compared with sham-operated and unoperated control rats. CDR increased BDNF (110%; p = 0.002) and NT-3 (100%; p = 0.002) in the cervical and NT-3 in the thoracic spinal cord (98%; p = 0.009). GDNF and TGF-beta(1) were not altered by CDR in any tissue. Immunohistochemistry localized BDNF and NT-3 to motoneurons and interneurons of the ventral spinal cord. These studies provide novel, suggestive evidence that BDNF and NT-3, possibly through their trophic effects on serotonergic neurons and/or motoneurons, may underlie serotonin-dependent plasticity in (spinal) respiratory motor control after CDR.
Subject(s)
Brain-Derived Neurotrophic Factor/metabolism , Nerve Growth Factors , Neurotrophin 3/metabolism , Spinal Cord/metabolism , Animals , Diaphragm/metabolism , Enzyme-Linked Immunosorbent Assay , Glial Cell Line-Derived Neurotrophic Factor , Interneurons/metabolism , Male , Motor Neurons/metabolism , Nerve Tissue Proteins/metabolism , Rats , Rats, Sprague-Dawley , Rhizotomy , Spinal Cord/physiology , Transforming Growth Factor beta/metabolismABSTRACT
Many researchers attempt to prepare antipeptide antibodies by immunizing animals with preparations of fusion proteins or conjugates between the target peptide and a larger protein (such as GST or KLH). Often, the immune response to the larger protein dominates. We have engineered a protein to be sparingly soluble in aqueous solution and nonantigenic, and show that fusions of this sparingly soluble non-antigenic protein (SSNAP) to target peptide sequences can be purified easily to a point suitable for immunizations. When animals are immunized with such fusion proteins, the majority of the immune response is to the target peptide. In all three cases tested, the peptide-specific immune response generated using the SSNAP carrier was greater than that obtained with peptides chemically linked to BSA or KLH, or expressed as fusion proteins to GST. The SSNAP carrier induced a very early IgG response with all classes of IgG well represented in the specific antibody response. All of the SSNAP fusion peptide-derived antibodies were capable of recognizing the full-length target protein in both ELISA and Western analysis. Based on the superior performance of the SSNAP antigens, these studies suggest this novel strategy will have broad utility for the generation of peptide antibodies.
Subject(s)
Immunization/methods , Peptides/immunology , Amino Acid Sequence , Animals , Antibody Formation , Antigens/administration & dosage , Antigens/genetics , Base Sequence , Carrier Proteins/administration & dosage , Carrier Proteins/genetics , Carrier Proteins/immunology , Chickens , DNA, Recombinant/genetics , Female , Gene Expression , Genetic Vectors , Mice , Mice, Inbred BALB C , Molecular Sequence Data , Peptides/administration & dosage , Peptides/genetics , Rabbits , Recombinant Fusion Proteins/administration & dosage , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/immunology , SolubilityABSTRACT
PURPOSE: The 2 prominent features of interstitial cystitis are pain and increased numbers of mast cells in the bladder. In this pilot study we determined the concentration of soluble mediators associated with activation of sensory neurons and/or mast cells that were present in the urine. MATERIALS AND METHODS: The study groups included 4 interstitial cystitis patients, 7 kidney donors with no history of bladder disease as negative controls, 6 bladder cancer patients and 7 patients with urinary tract infection as reference controls. Urine samples were assayed for different soluble mediators using immunoassays for tryptase (a marker for mast cell activation), neurotrophic factors (markers of neuronal plasticity) and chemokines (markers of inflammatory cell activity). Results were normalized based on creatinine concentration. RESULTS: There was a marked increase in the average amounts of tryptase and 3 neurotrophic factors in patient urine. Interestingly, the mediator profile in the urine of bladder cancer patients was indistinguishable from that of interstitial cystitis patients with respect to these same 4 proteins. There was no difference between normal control and urinary tract infection urine samples. CONCLUSIONS: These findings may account for several clinical and pathological features found in interstitial cystitis and bladder cancer. Although preliminary due to the limited numbers of patients, they also suggest that increased levels of neurotrophin-3, nerve growth factor, glial cell line-derived neurotrophic factor and tryptase in the urine could serve as a basis for adjunct diagnosis, monitoring and treatment of interstitial cystitis.
Subject(s)
Cystitis, Interstitial/urine , Nerve Growth Factors/urine , Nerve Tissue Proteins/urine , Urinary Bladder Neoplasms/urine , Adult , Aged , Aged, 80 and over , Female , Glial Cell Line-Derived Neurotrophic Factor , Humans , Male , Middle Aged , Neurotrophin 3ABSTRACT
Glial derived neurotrophic factor (GDNF) is essential for the development of the enteric nervous system (ENS). Although previous work has measured GDNF mRNA levels, little is known about the concentration of GDNF protein produced in developing or adult tissues. The aim of this study was to quantitate the concentration of GDNF protein in various tissues of the developing and adult rat and in adult human gut. A two site antibody immunoassay was used to quantitate GDNF using recombinant rat GDNF as a standard. In the adult rat gastrointestinal tract the intestine contained the highest concentration of GDNF while the stomach and esophagus have the lowest concentrations. The isolated muscular wall of the intestine has approximately four times the GDNF concentration of the intact intestine. Other tissues with smooth muscle such as the aorta and urinary bladder contain moderate GDNF concentrations. In contrast, GDNF is barely detectable in the adult kidney and liver. High concentrations of GDNF were also detected in human colon and jejunum. As development proceeds in the rat, there is a tendency for the concentration of GDNF to increase in the intestine but decrease in other tissues. Treatment of the jejunum with the cationic surfactant benzyldimethyltetradecylammonium chloride (BAC) results in an increase in the number of smooth muscle cells, a decrease in myenteric neurons, and an increase in the concentration of GDNF in homogenates of intestine. The observations that GDNF concentrations are high in the adult intestine suggest that this growth factor may be important for the maintenance of the adult ENS.
Subject(s)
Digestive System/metabolism , Enteric Nervous System/metabolism , Nerve Growth Factors/metabolism , Nerve Tissue Proteins/metabolism , Adult , Animals , Digestive System/growth & development , Digestive System/innervation , Enzyme-Linked Immunosorbent Assay , Glial Cell Line-Derived Neurotrophic Factor , Humans , Hydrogen-Ion Concentration , Male , Muscle, Smooth/cytology , Muscle, Smooth/innervation , Muscle, Smooth/metabolism , Proteins/metabolism , Rats , Rats, Sprague-Dawley , Tissue DistributionABSTRACT
Glial cell line-derived neurotrophic factor (GDNF), a distant member of the transforming growth factor-beta (TGFbeta) family, is a protein that is essential for the survival of dopaminergic, motor, and peripheral neurons. To facilitate its study, we and others have developed sensitive (low pg/ml) enzyme-linked immunosorbant assays (ELISA) to quantitate endogenous concentrations of GDNF, along with neurotrophin-3 (NT-3) and nerve growth factor (NGF). However, endogenous tissue levels of GDNF in adult animals are not readily detected by ELISA and do not correlate well with message RNA. Based upon previously described methods for the extraction of TGFbeta from tissue samples, we have developed an acid-treatment procedure to allow the quantification of total endogenous GDNF. This procedure also was evaluated for use when measuring total endogenous levels of NT-3 and NGF from biological samples. The acid-treatment procedure increases the detectable amounts of GDNF, NT-3, and NGF in all tissue samples and most of the serum samples tested. Moreover, these values were as much as 35 times greater than those detected using traditional extraction buffers. Such elevated concentrations likely resulted from the acid treatment promoting the dissociation of ligands from receptors or binding proteins, thereby making more of the analyte available to be measured in the ELISA. These findings indicate that appropriate sample treatment is essential for the measurement of total endogenous neurotrophic factors.
Subject(s)
Acids/pharmacology , Nerve Tissue Proteins/analysis , Neuroprotective Agents/analysis , Animals , Antibody Specificity , Cattle , Enzyme-Linked Immunosorbent Assay/methods , Female , Glial Cell Line-Derived Neurotrophic Factor , Goats , Horses , Humans , Mice , Nerve Growth Factors/analysis , Neurons/chemistry , Neurotrophin 3 , Rats , Sheep , Species SpecificityABSTRACT
Bovine gamma/delta T cells proliferate in response to stimulation with gamma-irradiated autologous monocytes in the autologous mixed leukocyte reaction (AMLR). Flow cytometric analyses indicated that the proliferating cells included three major subpopulations of bovine gamma/delta T cells, distinguished by the differential expression of the gamma/delta T cell receptor epitopes N6 and N7. Interleukin-2 and acid-labile interferon were produced in AMLR cultures but the cultured cells did not lyse any of a large variety of target cells, including monocytes, allogeneic lymphoblasts, transformed bovine B cells (BL3), bovine fibroblast and the natural killer cell targets D17 and K562, even in the presence of lectins or after co-stimulation in the AMLR with antibodies to WC1, the gamma/delta T cell lineage-specific cell-surface differentiation antigen. Ex vivo gamma/delta T cells did not display lymphokine-activated killing whereas populations of peripheral blood mononuclear cells containing alpha/beta T cells did.
Subject(s)
Lymphocyte Activation , Monocytes/immunology , Receptors, Antigen, T-Cell, gamma-delta/biosynthesis , T-Lymphocyte Subsets/immunology , Animals , Cattle , Cell Line , Cells, Cultured , Female , Flow Cytometry , Fluorescent Antibody Technique, Indirect , Gamma Rays , Humans , Interleukin-2/pharmacology , Killer Cells, Natural/immunology , Kinetics , Lymphocyte Culture Test, Mixed , Monocytes/radiation effects , Receptors, Antigen, T-Cell, gamma-delta/analysis , Recombinant Proteins/pharmacology , T-Lymphocyte Subsets/drug effectsABSTRACT
Bovine gamma/delta T cells have been shown previously to proliferate when cocultured with gamma-irradiated bovine monocytes in the 'autologous mixed leucocyte reaction' (AMLR). It was suggested that the response may be to culture-derived or culture-induced antigenic epitopes. Data presented here indicate that the gamma/delta T-cell stimulatory activity is attributable to a self-derived cell-surface molecule of mononuclear phagocytes that is constitutively expressed in vivo. The ability to induce an AMLR did not require in vitro culture or stress associated with in vitro isolation of cells or increased temperature since it could be induced by monocytes fixed by paraformaldehyde during blood collection from normal animals. Furthermore, stimulation by monocytes did not depend upon secreted molecules since fixed monocytes that had been incubated overnight at 37 degrees to allow secretion of preformed molecules, or subjected to hypotonic shock in H2O for 10 min before addition to the cultures, induced an AMLR as did plasma membranes prepared from ex vivo monocytes. In contrast, enzymatic treatment of monocytes to digest surface molecules followed by fixation destroyed their ability to stimulate an AMLR. The ability of monocytes to stimulate proliferation of gamma/delta T cells was distinguishable from their ability to stimulate alpha/beta T cells, since the former was destroyed by glutaraldehyde fixation whereas stimulation of alpha/beta T cells by major histocompatibility complex (MHC)-presented antigenic epitopes is not. Moreover, induction of proliferation of bovine gamma/delta T cells was not MHC-restricted. Finally, bovine alveolar macrophages, sheep monocytes and transformed bovine monocytes stimulated proliferation of bovine gamma/delta T cells whereas none of the following did so: human monocytes, murine macrophages, bovine myeloid cells other than mononuclear phagocytes, other nucleated cells found in bovine blood including activated MHC class II-bearing B cells, and a variety of species of bacteria. Thus, the stimulatory epitope is unique to and conserved among mononuclear phagocytes of ruminants. Demonstration of stimulation of bovine gamma/delta T cells by self-derived molecules is consistent with reports for murine gamma/delta T cells.
Subject(s)
Monocytes/immunology , Receptors, Antigen, T-Cell, gamma-delta/analysis , T-Lymphocyte Subsets/immunology , Animals , Antigens, Bacterial/immunology , Cattle , Cell Division/immunology , Cell Membrane/immunology , Female , Fixatives , Formaldehyde , Humans , Lymphocyte Activation , Lymphocyte Culture Test, Mixed , Mice , Polymers , Sheep , Species Specificity , Tissue FixationABSTRACT
Gamma-irradiated ex vivo bovine monocytes induce proliferation of gamma/delta T cells in the autologous mixed lymphocyte reaction (AMLR), whereas when not irradiated they prevent this response. In contrast, non-irradiated autologous monocytes have no effect on bovine alpha/beta T-cell proliferation in the allogenic MLR suggesting that the regulation is specific for gamma/delta T-cell responses. Here, we showed that the inhibition was not mediated by inducing cell death and that the ability of ex vivo monocytes to prevent proliferation of gamma/delta T cells was not generalized in that gamma/delta T cells still responded to mitogenic stimulation. Inhibition of the AMLR by non-irradiated monocytes could not be overcome by addition of interleukin-2 to the cultures or by costimulation with antibodies to WC1, a gamma/delta T-cell-specific cell-surface differentiation antigen shown elsewhere by us to be involved in activation of gamma/delta T cells. Furthermore, we showed that monocytes inhibited gamma/delta T-cell responses via a soluble product since inhibition occurred even when monocytes and gamma/delta T cells were separated by membranes of transwells or when supernatants from monocyte cultures were added to AMLR cultures. Maximal secretion of the inhibitory product by the monocytes occurred during the first 6 hr of in vitro culture at 37 degrees, rapidly decreased thereafter, and did not occur when monocytes were incubated at 4 degrees. The inhibition was not attributable to nitric oxide, reactive oxygen intermediates, prostaglandin E2 or transforming growth factor-beta (TGF-beta) but the ability of monocyte supernatants to mediate inhibition was sensitive to heating at 65 degrees. Lipopolysaccharide and granulocyte-macrophage colony-stimulating factor activation of monocytes temporarily abrogated their ability to inhibit proliferation. In contrast, heat-shocking had no effect on their ability to inhibit. We hypothesize that non-irradiated monocytes produce the inhibitory material in vivo in order to regulate gamma/delta T-cell responses to self-derived monocyte membrane components, but that when monocytes are altered by infection, transformation, irradiation, or cytokine activation, production of the inhibitor is temporarily suspended allowing stimulation of gamma/delta T cells to occur.