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• W2104976130 abstract "Interplay between T-helper-1 (Th1) and T-helper-2 (Th2) cells is considered important in the development of acute allograft rejection and many other immune-mediated disease processes. Existing methods for evaluating expression of Th1 and Th2 cytokines, including reverse transcriptase polymerase chain reaction (RT-PCR), RNase protection assay (RPA), immunohistochemistry, and enzyme-linked immunosorbent assay (ELISA) all have limitations; alternate techniques to quantify cell populations expressing specific cytokine proteins, generate statistically analyzable data, and allow simultaneous identification of cytokine-secreting cell type are needed. To this end, we adapted a flow cytometric technique for intracellular cytokine immunofluorescence staining for use with cells isolated from solid tissue. To demonstrate the utility of the method, we determined the number of CD4+ and CD8+ cells secreting the prototypical Th1 and Th2 cytokines, interferon (IFN)-γ, and interleukin (IL)-4 in acutely rejecting murine cardiac allografts. We also measured the cytokine production via ELISA, RPA, and semiquantitative competitive RT-PCR. The number of CD4+ cells producing IFN-γ increased as rejection proceeded, in agreement with previous data; we detected no IL-4 production at any time, although relatively low numbers of IL-10-producing cells were identified. In addition, a high percentage of CD8+ cells, which outnumber CD4+ cells at day 6 after transplant, also produce IFN-γ, suggesting that cytotoxic lymphocytes contribute significantly to the local cytokine milieu. This new application of intracellular cytokine staining provides a powerful methodology for studying transplantation immunology. The method may also be easily adapted to the study of other immune-mediated processes. Interplay between T-helper-1 (Th1) and T-helper-2 (Th2) cells is considered important in the development of acute allograft rejection and many other immune-mediated disease processes. Existing methods for evaluating expression of Th1 and Th2 cytokines, including reverse transcriptase polymerase chain reaction (RT-PCR), RNase protection assay (RPA), immunohistochemistry, and enzyme-linked immunosorbent assay (ELISA) all have limitations; alternate techniques to quantify cell populations expressing specific cytokine proteins, generate statistically analyzable data, and allow simultaneous identification of cytokine-secreting cell type are needed. To this end, we adapted a flow cytometric technique for intracellular cytokine immunofluorescence staining for use with cells isolated from solid tissue. To demonstrate the utility of the method, we determined the number of CD4+ and CD8+ cells secreting the prototypical Th1 and Th2 cytokines, interferon (IFN)-γ, and interleukin (IL)-4 in acutely rejecting murine cardiac allografts. We also measured the cytokine production via ELISA, RPA, and semiquantitative competitive RT-PCR. The number of CD4+ cells producing IFN-γ increased as rejection proceeded, in agreement with previous data; we detected no IL-4 production at any time, although relatively low numbers of IL-10-producing cells were identified. In addition, a high percentage of CD8+ cells, which outnumber CD4+ cells at day 6 after transplant, also produce IFN-γ, suggesting that cytotoxic lymphocytes contribute significantly to the local cytokine milieu. This new application of intracellular cytokine staining provides a powerful methodology for studying transplantation immunology. The method may also be easily adapted to the study of other immune-mediated processes. Acute graft rejection is a major cause of morbidity and mortality after solid organ transplantation and is thought to contribute to long-term adverse outcomes, such as the development of occlusive graft arterial disease.1Schoen FJ Libby P Cardiac transplant graft arteriosclerosis.Trends Cardiovasc Med. 1991; 1: 216-223Abstract Full Text PDF PubMed Scopus (47) Google Scholar Helper T lymphocytes are critical mediators of acute rejection; depending on the cytokines they secrete, they may either enhance or modulate rejection severity.2Takeuchi T Lowry RP Konieczny B Heart allografts in murine systems: differential activation of Th2 like effector cells in peripheral tolerance.Transplantation. 1992; 53: 1281-1294Crossref PubMed Scopus (302) Google Scholar, 3Duquesnoy R Demetris A Immunopathology of cardiac transplant rejection.Curr Opin Cardiol. 1995; 10: 193-206Crossref PubMed Scopus (51) Google Scholar Cytotoxic T lymphocytes can also contribute to the local cytokine environment.4Mosmann TR Li L Hengartner H Kagi D Fu W Sad S Differentiation and function of T cell subsets.Ciba Found Symp. 1997; 204: 148-154PubMed Google Scholar Helper T cells can differentiate into two different functional subtypes when exposed to antigen.5Abbas AK Murphy KM Sher A Functional diversity of helper T-lymphocytes.Nature. 1996; 383: 787-793Crossref PubMed Scopus (3833) Google Scholar These subtypes express and secrete different panels of cytokines and therefore are believed to play different roles in immunologically mediated processes. T-helper-1 (Th1) cells are effectors of cell-mediated immunity; they secrete primarily interleukin (IL)-2, which stimulates the growth and activation of T lymphocytes, and interferon (IFN)-γ, which, among other functions, activates macrophages.5Abbas AK Murphy KM Sher A Functional diversity of helper T-lymphocytes.Nature. 1996; 383: 787-793Crossref PubMed Scopus (3833) Google Scholar Th1 cytokines likely stimulate the development of acute rejection, and their transcript levels have been correlated with acute graft rejection severity.6Cunningham DA Dunn MJ Yacoub MH Rose ML Local production of cytokines in the human cardiac allograft: a sequential study.Transplantation. 1995; 57: 1333-1337Crossref Scopus (75) Google Scholar, 7Piccotti JR Chan SY Goodman RE Magram J Eichwald EJ Bishop DK IL-12 antagonism induces T helper 2 responses, yet exacerbates cardiac allograft rejection.J Immunol. 1996; 157: 1951-1957PubMed Google Scholar, 8Chan SY DeBruyne LA Goodman RE Eichwald EJ Bishop DK In vivo depletion of CD8+ T cells results in Th2 cytokine production and alternate mechanisms of allograft rejection.Transplantation. 1995; 59: 1155-1161Crossref PubMed Scopus (166) Google Scholar Conversely, T-helper-2 (Th2) cells express IL-4, IL-5, and IL-10 and appear to antagonize the effects of Th1 cells in vivo.5Abbas AK Murphy KM Sher A Functional diversity of helper T-lymphocytes.Nature. 1996; 383: 787-793Crossref PubMed Scopus (3833) Google Scholar For example, Th2 cells may modulate acute rejection and the development of allograft tolerance.2Takeuchi T Lowry RP Konieczny B Heart allografts in murine systems: differential activation of Th2 like effector cells in peripheral tolerance.Transplantation. 1992; 53: 1281-1294Crossref PubMed Scopus (302) Google Scholar Th2 cytokine transcripts correlate with milder rejection episodes.6Cunningham DA Dunn MJ Yacoub MH Rose ML Local production of cytokines in the human cardiac allograft: a sequential study.Transplantation. 1995; 57: 1333-1337Crossref Scopus (75) Google Scholar, 9Salom R Maguire J Esmore D Hanock W Endothelial cell activation and cytokine expression during acute graft rejection.Transplant Proc. 1995; 27: 2164-2165PubMed Google Scholar Similarly, cytotoxic T lymphocytes may differentiate into T cytotoxic (Tc)1 and Tc2 populations with cytokine profiles analogous to those seen in T helper subpopulations.4Mosmann TR Li L Hengartner H Kagi D Fu W Sad S Differentiation and function of T cell subsets.Ciba Found Symp. 1997; 204: 148-154PubMed Google Scholar The degree to which strict Th1/2 and Tc1/2 differentiation takes place in humans, and the precise roles that these cells play in rejection processes, still remains unclear. Experimental methods used to examine the presence and contribution of discrete T cell subpopulations in acute graft rejection include reverse transcriptase polymerase chain reaction (RT-PCR), RNase protection assay (RPA), in situ hybridization, immunohistochemistry, radioimmunoassay (RIA), and ELISA. Each method has important limitations. For example, levels of cytokine transcript detected by RT-PCR may not always correlate with cytokine protein levels; many important cytokine transcripts contain AU sequences in the 3′ untranslated region that confer mRNA instability.10Shaw G Kamen R Conserved AU sequence from a 3′-untranslated region of GM-CSF mRNA mediates selective mRNA degradation.Cell. 1986; 46: 659-667Abstract Full Text PDF PubMed Scopus (3103) Google Scholar In addition, RT-PCR is at best semiquantitative and is usually performed on whole-tissue homogenates, giving no information about the source of the detected transcript. Likewise, RPA cannot distinguish the source of particular cytokines. Although in situ hybridization can be used to identify secreting cell types, it also detects RNA levels, not protein. It is technically difficult, and levels of cytokine transcript present in vivo may be insufficient for detection. Immunohistochemistry also allows identification of cytokine-secreting cell type and detects cytokine protein directly; however, it gives variable results and lacks a high degree of sensitivity, and the process of generating statistically meaningful data are tedious.Ex vivo methods such as RIA, ELISA, and bioassay detect the net secretions of heterogeneous, captive, and therefore artificial, culture populations that have often spent days outside the allograft environment; these methods also provide no information about secreting cell type. The need clearly exists for techniques that can quantify cytokine protein expression, generate statistically analyzable data, and allow simultaneous identification of the source of cytokine protein. Intracellular cytokine staining (ICCS) with flow cytometric analysis was recently described as a technique for examining cytokine expression in cloned T cell lines.11Sander B Andersson J Andersson U Assessment of cytokines by immunofluorescence and the paraformaldehyde-saponin procedure.Immunol Rev. 1991; 119: 65-93Crossref PubMed Scopus (410) Google Scholar, 12Jung T Schauer U Heusser C Neumann C Reiger C Detection of intracellular cytokines by flow cytometry.J Immunol Methods. 1993; 159: 197-207Crossref PubMed Scopus (894) Google Scholar, 13Carter LL Swain SL Single cell analyses of cytokine production.Curr Opin Immunol. 1997; 9: 177-182Crossref PubMed Scopus (90) Google Scholar Briefly, intracellular cytokine staining involves incubating cells with an anti-cytokine antibody in the presence of a mild detergent that permeabilizes the cell surface and allows the antibody to pass through cytoplasmic and organelle membranes and bind to intracellular cytokine protein. Cells can then be resealed and stained with monoclonal antibodies against cell-surface markers, such as CD4 or CD8, so that cytokine expression can be correlated with cell type using multicolor flow cytometry. When applied to the study of acute graft rejection, this method offers many advantages. First, cell-surface staining may be used to identify a specific subpopulation (eg, CD4+ or CD8+) of graft-infiltrating lymphocytes from within a heterogeneous mixture, without the need for physical isolation of the cells. Using intracellular staining techniques and multicolor flow cytometry, one may define the particular cytokines produced by a specific cell type and the number of cells producing a given cytokine. Flow cytometry permits analysis of a large number of cells and the generation of statistically significant data. To evaluate the applicability of this technique to the study of solid tissue processes, we developed a method for isolating and stimulating cells from acutely rejecting allografts and optimized intracellular staining conditions for the isolated lymphocyte populations. We defined the CD4+ and CD8+ lymphocyte populations within rejecting allografts by surface staining and demonstrated an increase in both populations as acute rejection proceeded. Finally, we used ICCS to determine the relative numbers of CD4+ and CD8+ cells within rejecting allografts producing IFN-γ or IL-4 at each time point. Our results demonstrate increasing Th1-type and Tc1-type infiltrates as acute rejection proceeds, without any detectable IL-4. Moreover, we find that, at the time of graft failure, CD8+ T lymphocytes predominate as a source of IFN-γ. C57/BL6 (B6, H-2b) and BALB/c wild-type mice were obtained from Taconic Farms (Germantown, NY) or the Jackson Laboratories (Bar Harbor, ME). All mice were maintained in the pathogen-free Harvard Medical School facility on acidified water; all experiments conformed with approved animal care protocols. The Riboquant multiprobe RNase protection assay (RPA) kit, in vitro transcription kit and mCK-1 template set, biotinylated anti-IFN-γ (rat IgG1clone XMG1.2), biotinylated anti-IL-4 (rat IgG1, clone BVD6–24G2), biotinylated anti-IL-10 (rat IgG2b, clone JES5–16E3), biotinylated control antibody (anti-IgE rat IgG1 clone R35–92 or IgG1 isotype-matched control, clone R3–34), fluorescein isothiocyanate (FITC)- conjugated anti-CD4 (clone RM4–5), Cy-chrome (CyC)- conjugated anti-CD4 (clone RM4–5), phycoerythrin (PE)-conjugated anti-CD8 (clone 53-6.7), Cy-C-conjugated anti-CD8 (clone 53-6.7), PE-conjugated streptavidin, unlabeled anti-IFN-γ (rat IgG1, clone R4–6A2), unlabeled anti-IL-4 (rat IgG1, clone 11B11), and unlabeled anti-CD16/CD32 (clone 2.4G2, Fc block) were obtained from PharMingen (San Diego, CA). Horseradish peroxidase (HRP)-conjugated ultra-avidin was from Leinco Technologies, Ballwin, MO. Ionomycin, phorbol myristate acetate (PMA), brefeldin A (BFA), collagenase (C1030, type I), saponin, bovine serum albumin (BSA), 2,2′-azino-bis(3-ethylbenz-thiazoline-6-sulfonic acid), and paraformaldehyde were obtained from Sigma Chemical Co. (St. Louis, MO). IL-2 from the supernatant of X63 cell cultures was the generous gift of Dr. Andrew Lichtman, Brigham and Women's Hospital, Boston, MA. RPMI 1640 media was provided by BioWhittaker (Walkersville, MD), nonessential amino acids, l-glutamine, HEPES buffer, minimal essential medium sodium pyruvate, penicillin/streptomycin, 2-mercaptoethanol, heat-inactivated fetal calf serum, Superscript reverse transcriptase, Taq polymerase, 50 mmol/L MgCl solution, TRIzol, and 10X PCR buffer were from Gibco BRL/Life Technologies (Grand Island, NY). Ficoll lymphocyte separation medium was from Organon Teknika (Durham, NC). IFN-γ and IL-4 primers for PCR were as published by Murray et al,14Murray LJ Lee R Martens C In vivo cytokine gene expression in T cell subsets of the autoimmune MRL/Mp-lpr/lpr mouse.Eur J Immunol. 1990; 20: 163-170Crossref PubMed Scopus (218) Google Scholar and dNTPs were from Pharmacia (Piscataway, NJ). The competitive fragment for quantitation of cytokine mRNA was the generous gift of Dr. Jim Lederer, Brigham and Women's Hospital. Vascularized cardiac allografts were generated using the abdominal heterotopic murine cardiac transplantation model originally described by Corry et al15Corry RJ Winn HJ Russell P Primary vascularized allografts of hearts in mice.Transplantation. 1973; 16: 343-350Crossref PubMed Scopus (777) Google Scholar and detailed by Nagano et al.16Nagano H Mitchell RN Taylor MK Hasegawa S Tilney NL Libby PL Interferon-γ deficiency prevents coronary arteriosclerosis but not myocardial rejection in transplanted mouse hearts.J Clin Invest. 1997; 100: 550-557Crossref PubMed Scopus (229) Google Scholar Total surgical time was roughly 60 minutes, and heart ischemic time was approximately 20 minutes. At the time of graft harvest, the host was sacrificed by exsanguination under methoxyflurane anesthesia, and the graft was recovered. BALB/c (H2d) donor hearts were transplanted into nonimmunosuppressed C57BL/6 (H2b) recipients; using this total allogeneic mismatch strain combination, acute graft rejection is evident histologically by 4 to 6 days, and graft failure (cessation of palpable contractions) due to rejection occurs by 7 ± 1 days.17Nagano H Libby P Taylor MK Hasegawa S Stinn JL Becker G Tilney NL Mitchell RN Coronary arteriosclerosis following T cell-mediated injury in murine cardiac allografts: role of interferon-γ.Am J Pathol. 1998; 152: 1187-1197PubMed Google Scholar Animals were sacrificed 3 hours after surgery to establish the baseline of cells that immediately infiltrated the graft or were already resident. Additional animals were sacrificed on days 4, 6, and 8 after transplantation. Transplanted hearts were harvested at day 7 for RNA extraction for both competitive RT-PCR and RPAs. In most experiments, recovered cardiac tissue was minced with a sterile razor blade and placed in 10 ml of borate-buffered saline with 2% BSA and 2 mg/ml collagenase. This mixture was rocked at 37°C for 2 to 3 hours and then strained through a 70-μm nylon cell strainer (Becton Dickinson, Franklin Lakes, NJ). Dead lymphocytes and red blood cells were removed by centrifugation through Ficoll for 10 minutes at 2000 rpm; resulting interface lymphocytes were washed in RPMI and resuspended in C/10 media (RPMI 1640 supplemented with 1% nonessential amino acids, 1% l-glutamine, 1% HEPES buffer, 1% minimal essential medium sodium pyruvate, 1% penicillin/streptomycin, 0.1% 2-mercaptoethanol, and 10% heat-inactivated fetal calf serum). Mechanical dissociation of heart tissue through a cytoscreen, without collagenase treatment, was also used to extract heart cells from some samples. Spleens from transplant recipient animals were passed through a cytoscreen into 7 ml of C/10, and cells and residue were pelleted at 1200 rpm for 10 minutes and resuspended in 5 ml of ammonium chloride buffer (5 mmol/L Tris, 0.83% NH4Cl, pH 7.2) at 37°C for 5 minutes to lyse red blood cells. Splenocytes were washed twice with RPMI medium and resuspended in C/10 for subsequent stimulation. Extracted cells were stimulated with 25 μmol/L ionomycin and 10 ng/ml PMA for 4 hours at 37°C under a 5% CO2 humidified atmosphere, and 10 μg/ml of the fungal antimetabolite BFA was added for the duration of the culture to block cytokine secretion and thereby improve cytokine detection. After stimulation, cells were centrifuged at 1200 rpm for 5 minutes and washed with 10 μg/ml BFA in cold phosphate-buffered saline (PBS). Cells were fixed at room temperature with 4% paraformaldehyde in PBS for 15 minutes and then washed twice with PBS. Before the initial staining, a maximum of 1 × 107 cells/tube were washed with saponin/PBS buffer (0.5% saponin, 1% bovine serum albumin, 0.1% NaN3 in PBS) to permeabilize the plasma and intracellular membranes. Saponin permeabilization is reversible so that saponin needs to be present throughout the intracellular staining procedure.11Sander B Andersson J Andersson U Assessment of cytokines by immunofluorescence and the paraformaldehyde-saponin procedure.Immunol Rev. 1991; 119: 65-93Crossref PubMed Scopus (410) Google Scholar To reduce background staining, Fc block (0.25 μg) was applied 5 minutes before the first staining per the manufacturer's instructions. Cells in 100 μl of saponin/PBS buffer were labeled with 10 μg/ml of a primary biotinylated anti-cytokine antibody or isotype-matched, irrelevant control antibody for 30 minutes at room temperature, followed by two washes with saponin/PBS. The cells were incubated with PE-conjugated streptavidin (2.0 μg/ml) for 30 minutes at room temperature. After two washes with saponin/PBS, the cells were washed with PBS to seal the membranes and then stained with FITC- and/or CyC-conjugated surface-marker antibodies (2.5 μg/ml) for 20 minutes at room temperature. Cells were washed twice with PBS. Flow cytometric analysis was usually performed immediately; alternatively, cells could be stored at 4°C for up to 24 hours before analysis after the addition of 200 μl of 1% paraformaldehyde in PBS. Flow cytometry was performed on a FACScan flow cytometer (Becton Dickinson, Mountain View, CA), equipped with a 15-mW argon laser and filter settings for FITC (530 nm), CyC (650 nm), and PE (585 nm), using CellQuest software (Macintosh). Lymphocytes were distinguished from other cardiac-infiltrating cells on the basis of light-scatter characteristics. Scatter regions for lymphocytes were established before each collection using stimulated splenocytes. Collection of cytokine staining data from allografts was restricted to this gated region (to exclude nonlymphocyte cells). Data were collected on 2,000 to 10,000 cells within the lymphocyte scatter region. TRIzol was used to perform mRNA isolation according to the manufacturer's protocol. The RNA pellet was dissolved in 50 μl of diethylpyrocarbonate (DEPC)-treated water and stored at −20°C. For RPAs, the RNA was quantitated by optical densitometry, and approximately 20 μg of RNA from fresh tissue or 5 μg of RNA from extracted and stimulated lymphocytes was assayed using the Riboquant protocol (PharMingen). Specifically, a panel of radiolabeled probes of staggered sizes, complementary to cytokine mRNAs of interest, are hybridized to sample mRNA. Any remaining single-strand probe or RNA is enzymatically digested; resulting mRNA protected probes are electrophoresed on a sequencing gel where each cytokine migrates a specific distance based on the size of its probe. The gel is then exposed to radiographic film or a phospor plate for detection and quantitation. Semiquantitative competitive RT-PCR was performed as described by Platzer et al.18Platzer C Richter G Uberla K Muller W Blocker H Diamantstein T Blankenstein T Analysis of cytokine mRNA levels in interleukin-4 transgenic mice by quantitative polymerase chain reaction.Eur J Immunol. 1992; 22: 1179-1184Crossref PubMed Scopus (131) Google Scholar Briefly, cDNA was created from the mRNA samples using specific cytokine primers according to the Superscript reverse transcriptase protocol. Serial dilutions of known concentration of a competitive fragment (designed to produce products slightly larger than the native cytokine mRNA were added to equal quantities of cDNA, and PCR was performed with specific primers to cytokines of interest. When the intensity of the authentic cytokine band is equal to the intensity of the competitive fragment band, the concentrations of the two are approximately equal. To validate both our RPA and ICCS techniques, we generated Th2-like (IL-4-secreting) and Th1-like (IFN-γ-secreting) control cells in vitro. Th2 cells were generated by incubating splenocytes from C57BL/6 mice with equal numbers of irradiated BALB/c splenocytes and exogenously added IL-4 (2000 U/ml) and anti-IFN-γ (40 μg/ml) for 5 days. C57BL/6 splenocytes tend to differentiate into Th1 cells without additional cytokine stimulation, so our IFN-γ-secreting cells were generated by incubating splenocytes from C57BL/6 mice with equal numbers of irradiated BALB/c splenocytes for 5 days without additional cytokines. After washing and resting overnight in medium (with 200 U/ml IL-4 for the Th2 cells), the cells were treated like the graft-infiltrating cells, and either they were re-stimulated with PMA/ionomycin for 4 hours in the presence of BFA for subsequent ICCS or their RNA was extracted for RPA Leukocyte infiltrates in rejecting allografts were quantitated by immunohistochemical staining against leukocyte common antigen (LCA) or CD45, followed by counting positively stained cells. Sections of allografts were frozen in OCT compound (Ames, Division of Miles Laboratories, Elkhart, IN) and stored at −80°C. Frozen, 4- to 5-μm-thick sections of heart were fixed in acetone for 10 minutes and incubated with rat anti-CD45 by standard techniques. The sections were then incubated with rabbit biotinylated anti-rat IgG, developed by the avidin-biotin complex method, and counterstained with hematoxylin. Positively stained cells were counted and averaged from 10 high-power fields (×100) per allograft; two allografts were analyzed at each time point (days 0, 4, 6, and 8 after transplant). Initial reports of the technique of ICCS used stable in vitro lymphocyte cell lines as a source of cytokine-producing cells.19O'Garra A Murphy K Role of cytokines in determining T cell function (mouse).in: Weir DM Handbook of Experimental Immunology. Blackwell Science Cambridge, UK1996: 221-226Google Scholar, 20Openshaw P Murphy EE Hosken NA Maino V Davis K Murphy K O'Garra A Heterogeneity of intracellular cytokine synthesis at the single cell level in polarized T helper 1 and T helper 2 populations.J Exp Med. 1995; 182: 1357-1367Crossref PubMed Scopus (514) Google Scholar These cells represent a homogeneous population of selected, transformed lymphocytes that produce high concentrations of cytokine protein. Primary cells are more vulnerable to cell death and may produce cytokines with lesser frequency and at lower concentrations than cultured cells. Therefore, it was necessary to re-optimize isolation and stimulation conditions for maximal lymphocyte survival and cytokine detection in cells extracted directly from cardiac allografts. Lymphocytes were obtained by digesting minced heart tissue with collagenase and then pushing the fragments through a cell strainer and removing dead lymphocytes and red blood cells by centrifugation through Ficoll. Estimates based on the number of mononuclear cells in standard tissue histology sections and the number of sections per mouse heart indicate there are 5 × 106 to 40 × 106 graft-infiltrating mononuclear cells per acutely rejecting heart. Lymphocyte extraction by mechanical grinding, without collagenase treatment, yielded approximately 25,000 to 50,000 cells within the lymphocyte scatter region, or approximately 0.5% to 5% of the anticipated total. Collagenase digestion of the heart tissue increased the yield of mononuclear cells to greater than 106 per acutely rejecting heart (data not shown). Some existing methods for studying rejection use a separate enrichment step, such as cell sorting, to isolate lymphocytes from rejecting tissue. This additional step is likely to reduce cell yield and might alter cytokine expression. We therefore took advantage of the distinctive pattern of light scatter by lymphocytes in flow cytometry and selected a lymphocyte-enriched population from a heterogeneous group of cells on the basis of forward-scatter and side-scatter characteristics alone (Figure 2). Ficoll fractionation of collagenase-digested explants, as routinely used, resulted in decreased numbers of nonlymphocytic cells (eg, myocytes; not shown). Cells obtained after Ficoll centrifugation or after dissociation were directly cultured. Figure 3 shows control stains with stimulated primary lymphocytes to demonstrate our ability to stain for both intracellular IL-4 (Figure 3A) and IFN-γ (Figure 3B). The staining intensity of the isotype-matched control antibody (solid dark line in both panels) was used to set the threshold for the anti-cytokine-stained samples. The threshold for positive cytokine signal was set where fewer than 5% of cells showed autofluorescence or background staining with control antibody; background signal was subtracted from cytokine-positive signal. To increase cytokine production, we stimulated cells with PMA and ionomycin for 3 to 5 hours before fixation and staining. Without this stimulation step, we could not detect any cytokine signals (Figure 4, A and B). Cell survival was judged by number, viability, and surface staining characteristics as detected by flow cytometry. In initial experiments, optimal cell survival was obtained with shorter culture times (ie, 4 to 6 hours versus12 to 48 hours). PMA/ionomycin stimulation has been reported to reduce CD4 expression;21Anderson SJ Coleclough C Regulation of CD4 and CD8 expression on mouse T cells.J Immunol. 1993; 151: 5123-5134PubMed Google Scholar however, we found, in our protocol, that the PMA/ionomycin treatment did not affect the percentage of cells staining for CD4 or CD8, although it occasionally and variably decreased the signal intensity (Figure 4C). The loss of signal intensity was most pronounced with CD4 staining of highly stimulated lymphocytes (eg, from acutely rejecting hearts shortly before they stopped beating), with less effect seen on Thy1 or CD8 staining. We also verified that the stimulation protocol did not alter the cytokine profile of the graft-infiltrating cells. This was demonstrated by using an RPA with RNA extracted from day 7 acutely rejecting hearts immediately after explantation and with RNA from extracted and PMA/ionomycin-stimulated lymphocytes (Figure 5A). IL-2, IL-10, and IFN-γ transcripts are detected in both unstimulated (lane 2) and stimulated (lane 3) extracted cells. No new cytokine message was induced by the stimulation protocol. The loss of IL-6 and IL-15 signals in the RNA from the stimulated cells is presumably due to decreased recovery of macrophages after the 4-hour culture time required for PMA/ionomycin stimulation. The ratio of IFN-γ to GAPDH housekeeping signal in the stimulated cells was 10 times greater than that seen in nonstimulated lymphocytes recovered directly from homogenized tissue. Notably, there is no IL-4 signal either before or after stimulation of lymphocytes from acutely rejecting hearts although IL-4 is detected in the control RNA (lane 1). To increase the intracellular concentration of cytokine protein within primary ex vivo cells, stimulation was performed in the presence of BFA. BFA is a fungal metabolite that blocks cytokine secretion but that does not interfere with activation and synthesis.22Picker LJ Singh MK Zdraveski Z Treer JR Waldrop SL Bergstressa PR Maino VC Direct demonstration of cytokine heterogeneity among human memory/effector cells by flow cytometry.Blood. 1995; : 1408-1419PubMed Google Scholar Exposure to BFA thus causes the accumulation of recently synthesized cytokine protein within the cell before a" @default.
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• W2104976130 title "Interferon-γ-Secreting T-Cell Populations in Rejecting Murine Cardiac Allografts" @default.
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