Search

Summary Fever is a complex physiological response enhancing immune surveillance during infection and inflammation. Fever-range whole-body hyperthermia (WBH) treatment can experimentally mimic the febrile condition in mice. Here, we describe a protocol for the treatment of mice with WBH and normothermia. We describe the isolation of T cells from mouse spleen followed by the evaluation of T-cell adhesion and transmigration. This animal model can be applied to studying the dysfunction of the immune system induced by fever. For complete details on the use and execution of this protocol, please refer to Lin et al. (2019)., Graphical abstract, Highlights • Whole-body hyperthermia (WBH) can mimic the febrile condition in mice • We isolate T cells from WBH- or normothermia-treated mice • T-cell adhesion and transmigration assays show dysfunctions caused by fever, Fever is a complex physiological response enhancing immune surveillance during infection and inflammation. Fever-range whole-body hyperthermia (WBH) treatment can experimentally mimic the febrile condition in mice. Here, we describe a protocol for the treatment of mice with WBH and normothermia. We describe the isolation of T cells from mouse spleen followed by the evaluation of T-cell adhesion and transmigration. This animal model can be applied to studying the dysfunction of the immune system induced by fever.

Summary This protocol describes the comparative proteomic profiling of the spleen of wild type versus mdx-4cv mouse, a model of dystrophinopathy. We detail sample preparation for bottom-up proteomic mass spectrometry experiments, including homogenization of tissue, protein concentration measurements, protein digestion, and removal of interfering chemicals. We then describe the steps for mass spectrometric analysis and bioinformatic evaluation. For complete details on the use and execution of this protocol, please refer to Dowling et al. (2020)., Graphical Abstract, Highlights • Mass spectrometry-based proteomic analyses of spleen tissue samples • Comparative profiling of the spleen from the mdx-4cv model of dystrophinopathy • Protein extraction and protein digestion for peptide mass spectrometric analysis • Bioinformatic analysis of large proteomic datasets for comparative studies, This protocol describes the comparative proteomic profiling of the spleen of wild type versus mdx-4cv mouse, a model of dystrophinopathy. We detail sample preparation for bottom-up proteomic mass spectrometry experiments, including homogenization of tissue, protein concentration measurements, protein digestion, and removal of interfering chemicals. We then describe the steps for mass spectrometric analysis and bioinformatic evaluation.

Two sulfated polysaccharides SCP3 and SCP5 with degree of substitution (DS) 0.45 and 0.13 were obtained by modifying the Cyclocarya paliurus polysaccharide (CP) with chlorosulfonic acid-pyridine method. Results demonstrated that the physicochemical properties, molar ratios of monosaccharide composition, molecular weights, and in vitro immunomodulatory activity were changed after being sulfated. Splenocytes proliferation assay showed that both CP and sulfated polysaccharides have immunomodulatory, while sulfated polysaccharides were stronger. CP mainly stimulate the production of the Th1-type cytokines, SCP3 and SCP5 not only significantly enhanced the secretion level of Th1-type cytokines, but also promote the Th2-type cytokines. Sulfated polysaccharides could significantly stimulate splenic lymphocyte proliferation synergistically with ConA or LPS. SCP3 significantly promoted the proliferation of T lymphocytes, while SCP5 stimulated the proliferation of B lymphocytes, which might be related to the DS. These results indicated that sulfation could improve the immune-enhancing effects of CP and different DS has different immunomodulatory activity.

Publisher Summary This chapter describes quantitative cytotoxic assay using a coulter counter. Damaged cells are more susceptible to protease treatment than intact cells. To quantitate complement-mediated immune cytolysis of lymphosarcoma cells and mouse spleen cells, Stewart and Goldstein combined the procedure of protease digestion with that of electronic counting. In this method, cells are first treated with a protease to completely digest damaged cells and subsequently with a detergent to lyse intact cells as well as erythrocytes. The number of residual nuclei from intact cells is determined electronically. The modified and extended assay method of Stewart and Goldstein is reported. This assay is rapid and reproducible and gives results comparable with those given by a dye-exclusion method. Moreover, this cytotoxicity assay, when appropriately modified, is applicable to the determination of viability of tissue culture cells. A model ZBI coulter counter is used with a 100 μ m orifice aperture tube. To obtain a precise counting of the target cells and an effective rejection of debris, the most important adjustment is the threshold setting.

Publisher Summary This chapter demonstrates the in vitro generation in normal mouse spleen cells of the ability to actively suppress the in vitro induction of cell-mediated cytotoxicity (CML). When spleen cells freshly harvested from normal BALB/c mice are placed in five-day mixed culture with irradiated allogeneic C57BL/6N spleen cells, CML is generated, which is specific for the C57BL/6N alloantigens. If BALB/c spleen cells are first placed in culture alone for at least 72 hours in medium containing fetal calf serum (FCS), then harvested, washed, and recultured together with irradiated C57BL/6N cells, no detectable CML is induced. At a ratio of precultured: fresh BALB/c cells as low as 1:4, suppression or inhibition of CML up to 95% is observed without any measurable effect on cell survival or viability. Kinetic studies indicate that suppressor activity is detectable after 72 hours of preculture but not earlier and that this activity persists for at least 168 hours. The chapter also explains that suppressor cells are active in suppressing CML when added to the in vitro sensitizing cultures.

Publisher Summary This chapter describes the identification, large-scale production, partial purification, and preliminary characterization of an inducible inhibitor of interferon action that is produced by mouse spleen cells treated with T cell mitogens. The mouse spleen cell supernatant fluid preparations that contain the inhibitor of interferon action also contain immune interferon. The inhibitor can be detected and roughly quantitated by taking advantage of the fact that the inhibitor can be measured in undiluted preparations and, except for highly concentrated inhibitor preparations, the inhibitor activity can be diluted out more rapidly than the interferon activity. The purified inhibitor preparations are shown completely to block the antiviral activity of both mouse fibroblast interferon and mouse immune interferon. The inhibitor does not reverse an established antiviral state but can still completely block the development of antiviral activity when added at up to 3 hr after interferon. The inhibitor has also been shown to block the immunosuppressive activity of exogenously added fibroblast interferon in an in vitro plaque-forming cell response.

Publisher Summary This chapter explores the use of antibody-coated polystyrene tissue culture dishes for cell separation, which is a simple, rapid, preparative, inexpensive, and sterile procedure. It has become a routine method in many laboratories for the separation of immunoglobulin-positive (Ig + ) and immunoglobulin-negative (Ig - ) lymphocytes. The chapter reviews that plating can be used as a general method of cell separation for situations where a single cell suspension can be obtained and an antibody is available that binds to a subpopulation of cells in the mixture. This method has been used for the separation of human T lymphocyte subsets, mouse T cells, guinea pig bone marrow neutrophils, lectin-binding T cells, and preparation of monolayers of tumor target cells for microcinematography. The chapter also describes the separation of Ig + and Ig - mouse spleen lymphocytes. It can be readily adapted to other species by using the appropriate anti-Ig. All reagents are sterilized by filtration and sterile procedures are used throughout.

Publisher Summary This chapter discusses the characterization of the mouse lymphocyte receptor for human β2-microglobulin. β2-microglobulin (β2m) is an 11,500 dalton subunit of the HL-A antigen. In a study described in the chapter, human β2m was purified from the urine of several kidney transplant patients. To study the membrane structures that bind β2m, 125I-labeled mouse spleen cells incubated with unlabeled β2m were lysed in NP-40 and the lysates characterized by SDS–PAGE. There was no reactivity between antihuman β2m and mouse spleen cells without added β2m. A treatment of the lysates with an anti-mouse spleen cell antiserum indicated that additional cell-surface structures were involved in the binding of β2m.

Publisher Summary This chapter discusses the different aspects of ethanol and natural killer activity. In a study described in the chapter, natural killer activity of human peripheral blood lymphocytes (HPBL) was studied in a 4 hr 51 Cr release assay using K562 target cells. HPBL preparations derived from a total of 15 normal healthy volunteers and 32 chronic alcoholics were examined for NK activity in 11 experiments. In each experiment, at least one control HPBL preparation was tested along with two to six HPBL preparations from alcoholics. Wide variations in the levels of NK activity were found in HPBL preparations from control and alcoholics, but upon statistical analysis of variance, levels of NK activity in PBL derived from alcoholics were significantly higher than the control levels. The difference in the cytotoxic activity of PBL from control and alcoholic subjects was not abolished as a result of depletion of macrophages, T cells, and B cells, and therefore the cytotoxic activity of PBL from normal and alcoholic subjects resided in the NK population. The NK inhibitory effect of ethanol could be confirmed in the mouse system. Short-term preincubation of mouse spleen cells with ethanol had no effect on their subsequent NK activity.

Publisher Summary This chapter discusses the binding of immune and normal mouse spleen or peritoneal exudate cells (PEL) to a variety of tumor cells. In a study described in the chapter, BALB/c mice were immunized against C57BL/6 leukemia EL4, and the number of spleen cells conjugating with EL4 and with A/J leukemia YAC was determined following centrifugation of mixtures containing spleen and tumor cells, 250 g 10 min at room temperature. The number of cells capable of specifically binding to EL4 increased with time, peaking around day 10. A low but significant level of conjugation was observed with YAC or when nonimmunized spleen cells were examined. To characterize the cell types involved in conjugation, normal and immune spleen cells were stained with fluorescent-labeled antiserum to mouse immunoglobulin before or after conjugating with EL4 or YAC cells. About 29% of immune and 54% of normal conjugated spleen cells were Ig-positive. When the GAMG-F1 treated spleen cells displayed their surface Ig in caps, the caps localized opposite the junctional regions.

Publisher Summary The finding that ushered in the “modern era” of cell culture work in immunology was the report of Mishell and Dutton describing for the first time suitable conditions for the cultivation of dissociated cells that allowed antigenic stimulation to take place and cell multiplication and differentiation to occur. They used spleen cells from unimmunized mice and red blood cells from a variety of sources as antigen. With this system, the number of plaque-forming cells to sheep red blood cells rose to >1000 per 106 mouse spleen cells within 4 days, and this is of the same order as seen in spleens from mice injected with this antigen. Different populations of cells were stimulated in response to two noncross-reacting groups of red cell antigens. The system devised by Mishell and Dutton has proved to be reproducible and is currently used in many laboratories. Although there are numerous methods for assaying antibodies that have a sensitivity considerably less than 1 μg antibody/ml, a major technical advance was the ability to detect and enumerate antibody forming/secreting cells.

Publisher Summary This chapter presents a summary of the workshop focusing largely on the use of allotypes as cell markers in studies of the immune response or in other biological investigations. In the workshop, the regulation of immunoglobulin production and the presence of immunoglobulins on cell surfaces were important topics. Results of a study were discussed in which the production of donor-type immunoglobulins of several classes was followed in the serum of sublethally irradiated allotype congenic mice. The mice had received mouse spleen cells previously incubated for 2 h with rabbit anti-μ sera. Mice receiving antibody-treated cells showed a 70–90% suppression of the development of donor IgG2a, IgG2b, and IgG1 as compared to levels in mice receiving untreated cells. Mice receiving cells treated with anti-IgG were not suppressed. This suggests that there are μ receptors susceptible to suppression on the precursors of IgG-producing cells but gives no evidence for such susceptible γ receptors.