ImmunoMethods最新文献

From the original observation that the codons for the hydrophobic and hydrophilic amino acids on one strand of the DNA may be complemented by the codons for the hydrophilic and hydrophobic amino acids, respectively, on the complementary strand, arose the molecular recognition theory which forms the basis for much of the work involving complementary peptides. A number of examples have been documented where peptides with inverted hydropathic profiles have been shown to form complexes in high-affinity chromatography and solid matrix binding assays. Nevertheless, our current understanding of the molecular forces leading to the formation of these complexes is rather rudimentary, and it is highly desirable to have a detailed three-dimensional structure of a complex of interacting complementary peptides. In this article, we provide a brief review of the solution NMR work done by different laboratories in an attempt to study these interactions.

The central nervous system (CNS) affects the immune system through endocrine, paracrine, and neuronal mechanisms. The immune system in turn communicates with the CNS through many of the same mechanisms. Evidence that this bidirectional communication plays a vital role in susceptibility to inflammatory and infectious disease is derived largely from animal models in which the communication has been interrupted or reconstituted surgically, pharmacologlcally, or on a preexisting genetic basis. The advantage of animal models for studying the pathophysiologic relevance of such connections is that the systems can be manipulated at several levels and at different times in relation to development of the inflammatory disease and the outcome of the manipulation can be quantified. While in vitro studies may be used to further define the subcellular and molecular mechanisms of these interactions, only an intact organism, in which the central nervous system is connected to the immune system, can be used to fully define the nature of these interconnections. This review describes approaches to studying CNS-immune system interactions, using relatively inflammatory-susceptible and inflammatory-resistant Lewis and Fischer rats as models for evaluating the role of the HPA axis in susceptibility to inflammatory disease.

The early foundations of both neuroendocrinology and immunology were established by studies that linked the production, secretion, and action of circulating factors to the physiological state of an organism. These studies ultimately identified the cells of the neuroendocrine and immune systems as a rich source of such homeostatic regulatory molecules, and currently they are referred to as neuroendocrine hormones, peptides, and cytokines. More recently, two additional concepts have been added to this model. The first was that immune cells produce neuroendocrine hormones and peptides and that neuroendocrine cells produce cytokines. The second was the notion that both positive and negative factors control a variety of physiological processes. Recently, we have identified a new polypeptide negative regulator of cell proliferation that we have named suppressin (SPN). This negative regulatory molecule is also produced by both neuroendocrine and immune cells. The objective of this article is to provide an example of the biochemical, cellular, and molecular approaches used to characterize SPN and that could be used to characterize similar molecules from neuroendocrine and immune sources.

Autoradiography was performed on mouse brain cryosections to localize interleukin-1 (IL-1) receptors in the mouse brain and pituitary gland and to identify the cell types expressing these receptors. lnterleukin-1 receptor binding sites were mapped in the mouse central nervous system (CNS) with [¹²⁵I]IL-1α and [¹²⁵I]IL-1β. IL-1 receptors were detected in high density in the dentate gyrus of the hippocampus, choroid plexus, meninges, and anterior pituitary. IL-1 receptors are also expressed in the frontoparietal cortex at very low density. Both neurons and glial cells were shown to express IL-1 receptors. An intrahippocampal injection of colchicine, a selective neurotoxin, induced the concurrent disappearance of neuronal cells and [¹²⁵I]IL-1α binding in the hippocampus. This treatment established that IL-1 bound to the neurons of the dentate gyrus. IL-1 receptors on glial cells were not detected in situ in the CNS under basal conditions. However, [¹²⁵I]IL-1α bound to glial cells at the site of astrogliosis induced by a local mechanical injury. These results suggest that activated astrocytes express IL-1 receptors. Furthermore, the results of histoautoradiography of [¹²⁵I]IL-1α binding on astrocyte and microglial cultures showed that astrocytes express IL-1 receptors in vitro. The biochemical characterization of IL-1 binding in the dentate gyrus was achieved by quantitative in situ autoradiography. in the dentate gyrus IL-1 bound to a single class of receptor with characteristics similar to those of the receptor expressed on immune cells (K_d = 0.3 ± 0.2 nM, B_max, 60 ± 10 fmol/mg protein). Competition experiments with IL-1α and IL-1β showed that the neuronal receptor characteristics were similar to those of the type I IL-1 receptor, which binds the two isoforms of IL-1 with the same affinity. Regulation of IL-1 receptor density in the CNS and pituitary was studied after peripheral injection of LPS. Stimulation of IL-1 synthesis by LPS was shown to induce a major decrease in the number of receptors available for IL-1 binding in the CNS. A decrease of 84 ± 9% was observed in the dentate gyrus and in the choroid plexus, but no change occurred in the pituitary gland.

This article presents a detailed overview of the conceptual and technical considerations involved in the measurement of insulin-like growth factor-I (IGF-I) mRNAs in leukocytes. Two different quantitative techniques that take advantage of the in vitro synthesis of antisense and sense synthetic IGF-I RNA, respectively, are described: the ribonuclease protection assay (commonly referred to as solution hybridization) and competitive RT-PCR. We have improved the ribonuclease protection assay by constructing tandem, cassette riboprobes to generate multigene antisense RNAs of varying sizes. This approach permits the simultaneous quantitation of two or more mRNAs in a single RNA sample, one of which can serve as an internal standard for comparison of IGF-I transcripts among various treatments. The second approach of competitive RT-PCR represents an improvement in previous technologies by cloning a competing IGF-I sequence into an RNA expression vector. The resulting synthetic sense competitor IGF-I RNA (1.1 kb) serves as an internal standard during both the reverse transcription and amplification steps. We have used both the ribonuclease protection assay and competitive RT-PCR to define the macrophage as the major cellular source of leukocyte-derived IGF-I and to characterize these macrophage-derived mRNAs as being derived almost exclusively from exon 1. In addition, these techniques have allowed us to study the ontogeny of IGF-I expression in differentiating bone marrow macrophages and show that hematopoietic progenitors are induced to express abundant IGF-I transcripts as they differentiate into macrophages in the presence of CSF-1. These techniques can be readily adapted for measuring steady-state transcripts for a variety of leukocyte-derived hormones.

In this report a procedure for the analysis of mRNA expression in cells of limited availability by the reverse transcriptase-polymerase chain reaction (RT-PCR) method is described. The cells are lysed with Nonidet P-40, and the mRNA in the lysate is used directly as template for the cDNA synthesis reaction. Target cDNA is then amplified by PCR, and the products can be analyzed that same day by agarose gel electrophoresis. The oligonucleotide primers used for amplification are designed to include restriction sites to facilitate cloning for subsequent sequencing. We have demonstrated that luteinizing hormone-releasing hormone mRNA can be amplified from the hypothalamus and thymus of a 7-day rat pup, in which the starting cell number was limited. Furthermore, exon usage by target cDNA in different cell types can be easily determined by amplifying with exon-specific primers. Proopiomelanocortin (POMC) mRNA expressed in the pituitary utilizes all three exons, while a majority of POMC mRNA expressed in lymphocytes lacks exons 1 and 2. Thus, this provides an extremely rapid and sensitive means not only for analyzing mRNA expression but also for differential exon usage.

Prolactin (PRL) Is involved In a wide range of physiological effects in several species and its immunoregulatory role has already been well documented. The PRL receptor has been cloned from various species. There are at least two receptor isoforms (short and long) in rats and mice, which differ only in their cytoplasmic domains, generated by alternative splicing of a single gene, although in human only the long form exists. Using the reverse transcriptase-polymerase chain reaction (RT-PCR), we detected transcripts encoding both forms of PRL receptor in all lymphoid tissues examined in human, mouse, and rat, but in mouse and rat the ratio between the two forms was variable from animal to animal. Concerning the transcript encoding the PRL itself, a clear signal was always found in human lymphocytes and occasionally in rat thymus. We also developed a quantitative PCR (Q-PCR) in order to measure the absolute number of transcripts in thymus and spleen from rats at two stages of estrous cycle. The level of expression of the two forms was about equal. Finally, we identified the tyrosine kinase JAK2, which is constitutively associated with the PRLR, using the Nb2 rat lymphoma cell line as a model system with which to study the action of PRL on cell mitogenesis. We also showed that, after stimulation by PRL, the dimerization process is a prerequisite step for the phosphorylatlon of the PRLR and JAK2, which represents the earliest event in the signal transduction pathway.

In this paper we demonstrate several aspects of the mechanisms of action of the neurotransmitter Substance P in the immune system. We describe how Substance P can activate T cells, B cells, monocytes, and granulocytes to, respectively, proliferation, immunoglobulin synthesis, cytokine production, and chemotaxis. However, the neurotransmitter does not trigger cells of the immune system only via the well-characterized neurokinin-1 receptor, which mediates the signaling by Substance P In the neuroendocrine system. We show that Substance P can activate T cells receptor-independently. The receptor-independent activation of T cells leads to the activation of heterotrimeric G proteins and calcium-influx into the T cell, followed by an increase in proliferation of the cell. Apart from the receptor-independent activation pathway, Substance P can also activate monocytes and B cells via a nonneurokinin Substance P receptor. Activation of this novel receptor leads to the activation of MAP kinase, which is an important second messenger in the cascade leading to cytokine production by monocytes. In contrast to the nonneurokinin Substance P receptor, triggering of the NK-1 receptor, transfected in Jurkat cells, or triggering of T cells via receptor independent pathways does not lead to activation of MAP kinase. Combining the data, we can conclude that the interaction between the neuroendocrine system and the immune system with regard to Substance P clearly indicates that the immune system does not necessarily mirror the communication pathways that are used in the neuroendocrine system. Substance P is capable of signaling the immune system via multiple activation pathways.

The generation of an effective immune response requires the selection and proliferation of antigen-specific lymphocytes. Activation of at least some T lymphocytes involves the sequential stimulation of cell surface receptors, including the T-cell receptor for antigen, the interleukin 2 receptor, and the prolactin receptor. Prolactin signaling in T lymphocytes appears to utilize a classical receptor-mediated kinase cascade and a novel peptide hormone activation pathway involving nuclear translocation.

A number of anatomical studies have demonstrated the presence of peptidergic nerve fibers infiltrating mucosal lymphoid tissues. The exact mechanisms of how neuropeptides are released to affect these lymphoid sites are unclear, but radiolabeled binding studies have shown that mucosal leukocytes bear a number of neuropeptide receptors on their cell surfaces capable of responding to neural signals. The presence of neuropeptide-containing fibers and the ability to receive neural signals suggest that mucosal lymphocytes can be influenced by neurogenic mediators. The objectives set forth in this review are to provide what is currently known about the ability of substance P and vasoactive intestinal peptide to promote mucosal IgA responses in the gastrointestinal tract via Th2 mechanisms and to discuss how these neuropeptides contribute to the exacerbation of the inflammatory diseases of the gastrointestinal tract. We describe how immune responses develop in the gastrointestinal immune system and emphasize how neuropeptides may influence the differentiation of lymphocytes in mucosal inductive tissues and their subsequent expression in mucosal effector sites. Finally, we discuss new techniques developed by the Mucosal Immunization Research Group that have enabled the study of mucosal immune responses.