ABSTRACT
BACKGROUND: Failure to recognise acute stroke may result in worse outcomes due to missed opportunity for acute stroke therapies. Our study examines factors associated with stroke misdiagnosis in patients admitted to a large comprehensive stroke centre. METHODS: Retrospective review comparing 156 consecutive stroke patients misdiagnosed in emergency department (ED) with 156 randomly selected stroke controls matched for age, gender, language spoken and stroke subtype for the period 2014-2016. RESULTS: There were 141 ischemic and 15 hemorrhagic misdiagnosed strokes (median age: 77 years, male:female = 1.3: 1). Symptom resolution, altered mental status, nausea/vomiting, dizziness and vertigo favored misdiagnosis (p < 0.05). Hemiparesis and dysarthria favored an accurate diagnosis (p < 0.05). Misdiagnosed patients were more commonly triaged into a lower ED category (62 vs. 42%, p = 0.001), clinically assessed as Face, Arm, Speech and Time (FAST) - negative (78 vs. 22%, p < 0.001) and underwent delayed CT imaging (median 4.1 vs. 1.5 h, p < 0.001). Misdiagnosed patients were more likely to have posterior circulation stroke (PCS; 39 vs. 22%, p = 0.01) and be admitted under non-neurological services (35 vs. 11%, p < 0.001) with worse discharge outcomes including increased mortality. CONCLUSIONS: Patients with stroke misdiagnosis were commonly FAST-negative with nonspecific symptoms including altered mental status, dizziness and nausea/vomiting often associated with PCS. Improved diagnostic accuracy may increase access to acute therapies.
Subject(s)
Brain Ischemia/diagnosis , Ischemic Attack, Transient/diagnosis , Stroke/diagnosis , Adult , Aged , Aged, 80 and over , Case-Control Studies , Diagnostic Errors , Emergency Service, Hospital , Female , Humans , Male , Middle Aged , Retrospective Studies , Risk FactorsABSTRACT
Immune responses to foreign and self-Ags can be controlled by regulatory T cells (Tregs) expressing CD4 and IL-2Rα chain (CD25). Defects in Tregs lead to autoimmunity, whereas induction of Ag-specific CD4+CD25+ Tregs restores tolerance. Ag-specific CD4+CD25+ FOXP3+Tregs activated by the T helper type 2 (Th2) cytokine, IL-4, and specific alloantigen promote allograft tolerance. These Tregs expressed the specific IL-5Rα and in the presence of IL-5 proliferate to specific but not third-party Ag. These findings suggest that recombinant IL-5 (rIL-5) therapy may promote Ag-specific Tregs to mediate tolerance. This study showed normal CD4+CD25+ Tregs cultured with IL-4 and an autoantigen expressed Il-5rα. Treatment of experimental autoimmune neuritis with rIL-5 markedly reduced clinical paralysis, weight loss, demyelination, and infiltration of CD4+ (Th1 and Th17) CD8+ T cells and macrophages in nerves. Clinical improvement was associated with expansion of CD4+CD25+FOXP3+ Tregs that expressed Il-5rα and proliferated only to specific autoantigen that was enhanced by rIL-5. Depletion of CD25+ Tregs or blocking of IL-4 abolished the benefits of rIL-5. Thus, rIL-5 promoted Ag-specific Tregs, activated by autoantigen and IL-4, to control autoimmunity. These findings may explain how Th2 responses, especially to parasitic infestation, induce immune tolerance. rIL-5 therapy may be able to induce Ag-specific tolerance in autoimmunity.
Subject(s)
Autoimmunity/drug effects , CD4 Antigens/metabolism , Interleukin-2 Receptor alpha Subunit/metabolism , Interleukin-5/pharmacology , T-Lymphocytes, Regulatory/drug effects , Animals , Antigens, Differentiation, T-Lymphocyte/metabolism , Autoimmunity/immunology , CHO Cells , Cricetinae , Cricetulus , Down-Regulation/drug effects , Down-Regulation/immunology , Drug Evaluation, Preclinical , Female , Immune Tolerance/drug effects , Rats , Rats, Inbred Lew , Recombinant Proteins/pharmacology , T-Cell Antigen Receptor Specificity/drug effects , T-Lymphocytes, Regulatory/metabolism , T-Lymphocytes, Regulatory/physiologyABSTRACT
OBJECTIVE: Increasing evidence suggests that inflammation and immune dysregulation play an important role in the pathogenesis of bipolar disorder. Because the brain can be affected by various autoimmune processes, it is possible that some psychiatric disorders may have an autoimmune basis. METHOD: This article reviews the literature on peripheral and central immune dysregulation and autoimmunity in bipolar disorder. The mechanisms of the innate and adaptive immune systems in the pathophysiology of bipolar disorder are explored. The clinical features and pathogenesis of neuropsychiatric systemic lupus erythematosus, anti-NMDA encephalitis, and Hashimoto's encephalopathy are summarized. RESULTS: Neuroinflammation and peripheral immune dysregulation may play a role in the pathophysiology of bipolar disorder. This involves a complex interaction between immune cells of the central nervous system and periphery resulting in cellular damage through mechanisms involving excitotoxicity, oxidative stress, and mitochondrial dysfunction. Neuropsychiatric systemic lupus erythematosus, anti-NMDA encephalitis, and Hashimoto's encephalopathy are important differentials for a psychiatrist to consider when suspecting autoimmune encephalopathy. CONCLUSIONS: The link between immune dysregulation, autoimmunity, and bipolar disorder may be closer than previously thought. Psychiatrists should be vigilant for autoimmunity in presentations of bipolar disorder due to its high morbidity and therapeutic implications. Advances in neuroimaging and biomarker identification related to immune dysregulation and neuroinflammation will contribute to our knowledge of the pathophysiology of bipolar disorder.
Subject(s)
Autoimmunity/immunology , Bipolar Disorder/immunology , Brain/immunology , Immune System/physiopathology , Humans , Immune System/immunologyABSTRACT
The quest to understand how allogeneic transplanted tissue is not rejected and how tolerance is induced led to fundamental concepts in immunology. First, we review the research that led to the Clonal Deletion theory in the late 1950s that has since dominated the field of immunology and transplantation. At that time many basic mechanisms of immune response were unknown, including the role of lymphocytes and T cells in rejection. These original observations are reassessed by considering T regulatory cells that are produced by thymus of neonates to prevent autoimmunity. Second, we review "operational tolerance" induced in adult rodents and larger animals such as pigs. This can occur spontaneously especially with liver allografts, but also can develop after short courses of a variety of rejection inhibiting therapies. Over time these animals develop alloantigen specific tolerance to the graft but retain the capacity to reject third-party grafts. These animals have a "split tolerance" as peripheral lymphocytes from these animals respond to donor alloantigen in graft versus host assays and in mixed lymphocyte cultures, indicating there is no clonal deletion. Investigation of this phenomenon excludes many mechanisms, including anti-donor antibody blocking rejection as well as anti-idiotypic responses mediated by antibody or T cells. This split tolerance is transferred to a second immune-depleted host by T cells that retain the capacity to effect rejection of third-party grafts by the same host. Third, we review research on alloantigen specific inhibitory T cells that led to the first identification of the CD4+CD25+T regulatory cell. The key role of T cell derived cytokines, other than IL-2, in promoting survival and expansion of antigen specific T regulatory cells that mediate transplant tolerance is reviewed. The precise methods for inducing and diagnosing operational tolerance remain to be defined, but antigen specific T regulatory cells are key mediators.
Subject(s)
Clonal Deletion , Transplantation Tolerance , Animals , Antigens/pharmacology , Immune Tolerance , Isoantigens , Swine , T-Lymphocytes, RegulatoryABSTRACT
The immune response to an allograft activates lymphocytes with the capacity to cause rejection. Activation of CD4+CD25+Foxp3+T regulatory cells (Treg) can down-regulate allograft rejection and can induce immune tolerance to the allograft. Treg represent <10% of peripheral CD4+T cells and do not markedly increase in tolerant hosts. CD4+CD25+Foxp3+T cells include both resting and activated Treg that can be distinguished by several markers, many of which are also expressed by effector T cells. More detailed characterization of Treg to identify increased activated antigen-specific Treg may allow reduction of non-specific immunosuppression. Natural thymus derived resting Treg (tTreg) are CD4+CD25+Foxp3+T cells and only partially inhibit alloantigen presenting cell activation of effector cells. Cytokines produced by activated effector cells activate these tTreg to more potent alloantigen-activated Treg that may promote a state of operational tolerance. Activated Treg can be distinguished by several molecules they are induced to express, or whose expression they have suppressed. These include CD45RA/RO, cytokine receptors, chemokine receptors that alter pathways of migration and transcription factors, cytokines and suppression mediating molecules. As the total Treg population does not increase in operational tolerance, it is the activated Treg which may be the most informative to monitor. Here we review the methods used to monitor peripheral Treg, the effect of immunosuppressive regimens on Treg, and correlations with clinical outcomes such as graft survival and rejection. Experimental therapies involving ex vivo Treg expansion and administration in renal transplantation are not reviewed.
Subject(s)
Kidney Transplantation , T-Lymphocytes, Regulatory , Isoantigens , Cytokines/metabolism , Forkhead Transcription Factors/metabolismABSTRACT
CD4(+)CD25(+)Foxp3(+) T cells are regulatory/suppressor cells (Tregs) that include non-antigen (Ag)-specific as well as Ag-specific Tregs. How non-Ag-specific naive CD4(+)CD25(+) Treg develop into specific Tregs is unknown. Here, we generated adaptive Tregs by culture of naive CD4(+)CD25(+)Foxp3(+) T cells with allo-Ag and either interleukin-2 (IL-2) or IL-4. Within days, IL-2 enhanced interferon-gamma receptor (Ifngammar) and Il-5 mRNA and IL-4 induced a reciprocal profile with de novo IL-5Ralpha and increased IFN-gamma mRNA expression. Both IL-2- and IL-4-alloactivated CD4(+)CD25(+) Tregs within 3 to 4 days of culture had enhanced capacity to induce tolerance to specific donor but not to third-party cardiac allografts. These hosts became tolerant as allografts functioned more than 250 days, with a physiologic ratio of less than 10% CD4(+)CD25(+)Foxp3(+) T cells in the CD4(+) population. CD4(+)CD25(+) T cells from tolerant hosts given IL-2-cultured cells had increased Il-5 and Ifngammar mRNA. Those from hosts given IL-4-cultured cells had enhanced IL-5Ralpha mRNA expression and IL-5 enhanced their proliferation to donor but not third-party allo-Ag. Thus, IL-2 and IL-4 activated allo-Ag-specific Tregs with distinct phenotypes that were retained in vivo. These findings suggested that T-helper 1 (Th1) and Th2 responses activate 2 pathways of adaptive Ag-specific Tregs that mediate tolerance. We propose they be known as T-suppressor 1 (Ts1) and Ts2 cells.
Subject(s)
Caspase 1/immunology , Graft Rejection/immunology , Heart Transplantation/immunology , Interleukin-2/immunology , Isoantigens/immunology , Lymphocyte Activation/immunology , Myocardium/immunology , T-Lymphocytes, Regulatory/immunology , Th1 Cells/immunology , Th2 Cells/immunology , Animals , Caspase 1/biosynthesis , Cytokines/biosynthesis , Cytokines/immunology , Graft Rejection/metabolism , Graft Rejection/pathology , Heart Transplantation/pathology , Interleukin-2/biosynthesis , Isoantigens/metabolism , Myocardium/metabolism , Myocardium/pathology , Phenotype , RNA, Messenger/biosynthesis , RNA, Messenger/immunology , Rats , Rats, Inbred Lew , Receptors, Interferon/biosynthesis , Receptors, Interferon/immunology , T-Lymphocytes, Regulatory/metabolism , T-Lymphocytes, Regulatory/pathology , Th1 Cells/metabolism , Th1 Cells/pathology , Th2 Cells/metabolism , Th2 Cells/pathology , Time Factors , Transplantation Tolerance , Interferon gamma ReceptorABSTRACT
Resting and activated subpopulations of CD4+CD25+CD127loT regulatory cells (Treg) and CD4+CD25+CD127+ effector T cells in MS patients and in healthy individuals were compared. Peripheral blood mononuclear cells isolated using Ficoll Hypaque were stained with monoclonal antibodies and analysed by flow cytometer. CD45RA and Foxp3 expression within CD4+ cells and in CD4+CD25+CD127loT cells identified Population I; CD45RA+Foxp3+, Population II; CD45RA-Foxp3hi and Population III; CD45RA-Foxp3+ cells. Effector CD4+CD127+ T cells were subdivided into Population IV; memory /effector CD45RA- CD25-Foxp3- and Population V; effector naïve CD45RA+CD25-Foxp3-CCR7+ and terminally differentiated RA+ (TEMRA) effector memory cells. Chemokine receptor staining identified CXCR3+Th1-like Treg, CCR6+Th17-like Treg and CCR7+ resting Treg. Resting Treg (Population I) were reduced in MS patients, both in untreated and treated MS compared to healthy donors. Activated/memory Treg (Population II) were significantly increased in MS patients compared to healthy donors. Activated effector CD4+ (Population IV) were increased and the naïve/ TEMRA CD4+ (Population V) were decreased in MS compared to HD. Expression of CCR7 was mainly in Population I, whereas expression of CCR6 and CXCR3 was greatest in Populations II and intermediate in Population III. In MS, CCR6+Treg were lower in Population III. This study found MS is associated with significant shifts in CD4+T cells subpopulations. MS patients had lower resting CD4+CD25+CD45RA+CCR7+ Treg than healthy donors while activated CD4+CD25hiCD45RA-Foxp3hiTreg were increased in MS patients even before treatment. Some MS patients had reduced CCR6+Th17-like Treg, which may contribute to the activity of MS.
Subject(s)
Antigens, CD/immunology , Forkhead Transcription Factors/immunology , Multiple Sclerosis/immunology , T-Lymphocytes, Regulatory/immunology , Adult , Aged , CD4-Positive T-Lymphocytes/immunology , Case-Control Studies , Female , Humans , Immunophenotyping , Male , Middle Aged , Multiple Sclerosis/blood , Young AdultABSTRACT
CD4+CD25+Foxp3+T cell population is heterogenous and contains three major sub-groups. First, thymus derived T regulatory cells (tTreg) that are naïve/resting. Second, activated/memory Treg that are produced by activation of tTreg by antigen and cytokines. Third, effector lineage CD4+CD25+T cells generated from CD4+CD25- T cells' activation by antigen to transiently express CD25 and Foxp3. We have shown that freshly isolated CD4+CD25+T cells are activated by specific alloantigen and IL-4, not IL-2, to Ts2 cells that express the IL-5 receptor alpha. Ts2 cells are more potent than naïve/resting tTreg in suppressing specific alloimmunity. Here, we showed rIL-5 promoted further activation of Ts2 cells to Th2-like Treg, that expressed foxp3, irf4, gata3 and il5. In vivo, we studied the effects of rIL-5 treatment on Lewis heart allograft survival in F344 rats. Host CD4+CD25+T cells were assessed by FACS, in mixed lymphocyte culture and by RT-PCR to examine mRNA of Ts2 or Th2-like Treg markers. rIL-5 treatment given 7 days after transplantation reduced the severity of rejection and all grafts survived ≥60d whereas sham treated rats fully rejected by day 31 (p<0.01). Treatment with anti-CD25 or anti-IL-4 monoclonal antibody abolished the benefits of treatment with rIL-5 and accelerated rejection. After 10d treatment with rIL-5, hosts' CD4+CD25+ cells expressed more Il5ra and responded to specific donor Lewis but not self. Enriched CD4+CD25+ cells from rIL-5 treated rats with allografts surviving >60 days proliferated to specific donor only when rIL-5 was present and did not proliferate to self or third party. These cells had more mRNA for molecules expressed by Th2-like Treg including Irf4, gata3 and Il5. These findings were consistent with IL-5 treatment preventing rejection by activation of Ts2 cells and Th2-like Treg.
Subject(s)
Graft Rejection/immunology , Interleukin-5/pharmacology , Lymphocyte Activation/immunology , T-Lymphocyte Subsets/immunology , T-Lymphocytes, Regulatory/immunology , Allografts , Animals , Heart Transplantation/adverse effects , Rats , Rats, Inbred F344 , Rats, Inbred Lew , Receptors, Interleukin-5/immunologyABSTRACT
BACKGROUND: It is important to establish the validity of diagnostic coding in administrative datasets used in stroke and transient ischemic attack (TIA) research. This study examines the accuracy of emergency department (ED) TIA diagnosis and final diagnostic coding after hospital admission. METHODS: Using administrative datasets, we identified all patients with an ED TIA diagnosis (435.9; ICD-9) admitted to Liverpool Hospital from January 2003 to December 2007. ED and hospital admission records were matched and final diagnosis codes (ICD-10-AM) recorded. All records were expertly reviewed to determine coding validity. RESULTS: 570 patients were admitted with an ED TIA diagnosis. According to ICD-10-AM coding, 46% had TIA, 29% stroke and 25% TIA mimic diagnoses. Expert review determined final diagnoses of TIA in 51.4%, stroke in 26.1% and TIA mimic in 22.5% of the patients. The positive predictive value of a final TIA diagnosis (ICD-10-AM) was 88.2% when subjected to expert review. TIA mimic disorders diagnosed after admission included serious conditions. CONCLUSIONS: Half of the emergency diagnoses retained a TIA diagnosis after hospital admission. In the setting of neurological admission there were small percentage differences between coded final diagnosis for TIA, stroke and mimic and diagnoses at expert review. Admission of ED TIA cases permitted identification of TIA mimics with serious conditions requiring non-TIA management.
Subject(s)
Ischemic Attack, Transient/diagnosis , Stroke/diagnosis , Databases, Factual , Diagnosis, Differential , Emergency Service, Hospital , Female , Humans , International Classification of Diseases , Male , New South WalesABSTRACT
Experimental autoimmune neuritis (EAN) induced by peripheral nerve myelin (PNM) is self-limiting and re-immunization with PNM does not re-activate disease. This study showed inhibition of EAN by CD4+CD25+T cells both from sensitized hosts or from naïve hosts after ex-vivo activation by PNM and rIL-2. Transfer of naïve CD4+CD25+T cells has no effect on EAN, nor did naïve CD4+CD25+T cells activated with rIL-2 and renal tubular antigen. Culture of naive CD4+CD25+Treg with rIL-2 and PNM induced mRNA for the IFN-gamma receptor. We showed naïve CD4+CD25+T cells activated by specific auto-antigen and rIL-2 produced more potent antigen-specific Treg that may have therapeutic potential.
Subject(s)
Autoantigens/immunology , Immunotherapy, Adoptive , Interleukin-2/pharmacology , Neuritis, Autoimmune, Experimental/immunology , T-Lymphocytes, Regulatory/immunology , Animals , CD4 Antigens/analysis , Cells, Cultured , Convalescence , Female , Interleukin-2 Receptor alpha Subunit/analysis , Lymphocyte Activation/drug effects , Myelin Sheath/immunology , Neuritis, Autoimmune, Experimental/prevention & control , Rats , Rats, Inbred Lew , Recombinant Proteins/pharmacology , Recurrence , T-Cell Antigen Receptor Specificity , T-Lymphocytes, Regulatory/transplantationABSTRACT
This study was designed (1) to compare the prevalence of emergency department (ED) presentations in Western Zone Sydney South West Area Health Service (WZS) between 1998-2002 and 2003-2007 for epilepsy (including status epilepticus (SE) and convulsions), hospital admission rates, and proportion of first seizure presentations; and (2) to compare these data with those for New South Wales (NSW) and Australia-wide figures. Using health department data sets, we found 19,834 presentations to WZS EDs between 1998 and 2007 (24.85/10,000 population/year). When the periods 2003-2007 and 1998-2002 in WZS are compared, ED presentations fell by 3% (P=0.03) and hospital admissions fell by 6% (P=0.001). The prevalence of ED presentations for seizures in NSW did not change (P=0.92), but hospital admissions fell by 3% (P<0.0001). When 1999/2000-2002/2003 was compared with 2003/2004-2006/2007, the prevalence of hospital admissions in Australia fell by 1% (P=0.0002). Rates of presentation for epilepsy in WZS have fallen over the last decade. Most presentations were first seizures rather than recurrences. The reason for this is speculative, but may reflect improved levels of education and health care delivery.
Subject(s)
Emergency Service, Hospital/statistics & numerical data , Patient Admission/statistics & numerical data , Seizures/epidemiology , Adolescent , Adult , Australia/epidemiology , Child , Child, Preschool , Female , Hospital Information Systems/statistics & numerical data , Humans , Infant , Infant, Newborn , Male , Middle Aged , Prevalence , Retrospective Studies , Seizures/classification , Time Factors , Young AdultABSTRACT
Therapy with alloantigen-specific CD4+CD25+ T regulatory cells (Treg) for induction of transplant tolerance is desirable, as naïve thymic Treg (tTreg) are not alloantigen-specific and are weak suppressor cells. Naïve tTreg from DA rats cultured with fully allogeneic PVG stimulator cells in the presence of rIL-2 express IFN-gamma receptor (IFNGR) and IL-12 receptor beta2 (IL-12Rß2) and are more potent alloantigen-specific regulators that we call Ts1 cells. This study examined additional markers that could identify the activated alloantigen-specific Treg as a subpopulation within the CD4+CD25+Foxp3+Treg. After culture of naïve DA CD4+CD8-CD25+T cells with rIL-2 and PVG alloantigen, or rIL-2 without alloantigen, CD8α was expressed on 10-20% and CD8ß on <5% of these cells. These cells expressed ifngr and Il12rb2. CD8α+ cells had increased Ifngr that characterizes Ts1 cells as well was Irf4, a transcription factor induced by TCR activation. Proliferation induced by re-culture with rIL-12 and alloantigen was greater with CD4+CD8α+CD25+Treg consistent with the CD8α+ cells expressing IL-12R. In MLC, the CD8α+ fraction suppressed responses against allogeneic stimulators more than the mixed Ts1 population, whereas the CD4+CD8-CD25+T cells were less potent. In an adoptive transfer assay, rIL-2 and alloantigen activated Treg suppress rejection at a ratio of 1:10 with naïve effector cells, whereas alloantigen and rIL-2 activated tTreg depleted of the CD8α+ cells were much less effective. This study demonstrated that expression of CD8α by rIL-2 and alloantigen activation of CD4+CD8-CD25+Foxp3+T cells was a marker of activated and potent Treg that included alloantigen-specific Treg.
Subject(s)
CD8 Antigens/immunology , Gene Expression Regulation/immunology , Isoantigens/immunology , Lymphocyte Activation , T-Lymphocytes, Regulatory/immunology , Transplantation Tolerance , Animals , Gene Expression Regulation/drug effects , Interleukin-2/pharmacology , Rats , Rats, Inbred LewABSTRACT
BACKGROUND: Naïve CD4+CD25+T cells suppress immune responses in a non-antigen specific manner. The effects of naïve CD4+CD25+T cells in suppressing alloimmune responses as assayed in the mixed lymphocyte culture (MLC) is poorly understood. METHOD: The alloreactivity of naïve CD4+CD25+, CD4+CD25(-) and unfractionated CD4+T cells from DA rats was compared in MLC with MHC incompatible stimulator cells. The response of Lewis and PVG cells to semi-allogeneic (LewisxPVG)F1 cells and fully allogeneic stimulators were compared. Potential mechanisms of suppression were examined by blocking T cell cytokines, produced by activated CD4+CD25+T cells. RESULTS: Proliferation of CD4+CD25(-)T cells was significantly greater than unfractionated CD4+T cells to both allogeneic and syngeneic stimulator cells. CD4+CD25+T cells had no response to syngeneic stimulators and very low proliferative responses to alloantigen due to the Foxp3(-) cells. Admixing CD4+CD25+T cells with CD4+CD25(-)T cells at a ratio of 1:10 reduced the proliferation to that of unfractionated CD4+ T cells. At a ratio of 1:1 proliferation was nearly totally suppressed, IL-2, IL-4 and IL-5 mRNA induction was reduced but IFN-gamma, IL-10, TGF-beta and inducible nitric oxide (iNOS) mRNA induction was spared. The inhibition by CD4+CD25+ T cells was not due to their consumption of IL-2 nor to anti-CD25mAb that had been used to enrich the cells being releases and blocking the IL-2 receptor on CD4+CD25(-)T cells that had been activated by alloantigen and induced to express CD25. Blocking IFN-gamma, IL-10, TGF-beta, IL-5 or iNOS did not prevent CD4+CD25+T cell's inhibition of CD4+CD25(-)T cell proliferation. Blocking IFN-gamma or iNOS enhanced CD4+CD25(-)T cell proliferation only in the absence of CD4+CD25+T cells. Depletion of CD4+CD25+T cells enhanced responses to syngeneic stimulator cells, but this anti-self suppression did not regulate the response to alloantigen on semi-allogeneic stimulators. CONCLUSIONS: Two independent mechanisms that control proliferation of CD4+CD25(-)T cells in MLC were identified that naive CD4+CD25+T cells mediated by cell to cell contact and not release of cytokines produced in the cultures, and that CD4+CD25(-)T cells producing IFN-gamma to induce iNOS.
Subject(s)
Immune Tolerance/immunology , Interleukin-2 Receptor alpha Subunit/metabolism , T-Lymphocyte Subsets/immunology , T-Lymphocytes, Regulatory/immunology , Animals , Cell Differentiation/immunology , Cell Proliferation , Cell Separation , Coculture Techniques , Lymphocyte Culture Test, Mixed , Rats , Rats, Inbred Lew , T-Lymphocyte Subsets/metabolism , T-Lymphocytes, Regulatory/metabolismABSTRACT
BACKGROUND: The mechanisms by which CD4+T cells, especially CD4+ CD25+T cells, transfer allograft specific tolerance are poorly defined. The role of cytokines and the effect on antigen-presenting cells is not resolved. METHODS: Anti-CD3 monoclonal antibody (mAb) therapy induced tolerance to PVG heterotopic cardiac transplantation in DA rats. Peripheral CD4+T cells or CD4+ CD25+ and CD4+ CD25-T cell subsets were adoptively transferred to irradiated DA hosts grafted with PVG heart grafts. For specificity studies, tolerant CD4+T cells were transferred to hosts with Lewis or (PVGxLewis)F1 heart grafts. Cytokine mRNA induction and the requirement for interleukin (IL)-4 and transforming growth factor (TGF)-beta in the transfer of tolerance was assessed. RESULTS: CD4+T cells transferred specific tolerance and suppressed naïve CD4+T cells capacity to effect rejection of PVG but not Lewis grafts. (PVGxLewis)F1 grafts had a major rejection episode but recovered. Later these hosts accepted PVG but not Lewis skin grafts. Adoptive hosts restored with tolerant or naïve cells had similar levels of mRNA expression for all Th1 and Th2 cytokines and effector molecules assayed. Transfer of tolerance by CD4+T cells was not blocked by mAb to IL-4 or TGF-beta. CD4+ CD25-T cells from either naïve or tolerant hosts effected rejection. In contrast neither tolerant nor naïve CD4+ CD25+T cells restored rejection. CONCLUSIONS: Specific tolerance transfer required CD4+ containing CD4+ CD25+T cells. An inflammatory response with induction of mRNA for Th1 and Th2 cytokines plus cytotoxic effector molecules occurred, but IL-4 and TGF-beta were not essential. Inhibition of antigen presenting cells was not the sole mechanism as there was no linked tolerance.
Subject(s)
Antigens/immunology , CD4-Positive T-Lymphocytes/immunology , Immune Tolerance/immunology , Aging/immunology , Animals , Animals, Newborn , Heart Transplantation/immunology , Immune Tolerance/genetics , Interleukin-4/genetics , Interleukin-4/immunology , Mice , RNA, Messenger/genetics , Rats , Skin Transplantation/immunology , Survival Rate , T-Lymphocyte Subsets/immunology , Transforming Growth Factor beta/genetics , Transforming Growth Factor beta/immunology , Transplantation, Homologous/immunologyABSTRACT
BACKGROUND: Specific transplant tolerance is mediated by CD4 T cells that die unless supported by T-cell derived cytokines and donor antigen. This study examined the role of Th1 and Th2 cytokines in the maintenance of tolerance. METHODS: Tolerance to fully allogeneic PVG cardiac allografts in DA rats was induced by short-term anti-CD3 monoclonal antibody therapy. Responses of tolerant cells to donor and third party antigen were assessed in vivo by examination of the infiltrate in the heart and application of skin grafts, and in vitro in mixed lymphocyte culture. Cell subsets were stained, induction of cytokine mRNA assayed by reverse-transcriptase polymerase chain reaction and the role of cytokines determined by treating with blocking monoclonal antibody to cytokines or cytokine administration. RESULTS: Tolerated grafts had a T cell and macrophage infiltrate with increased mRNA for Th1 cytokines, interleukin (IL)-2, and interferon (IFN)-gamma but not Th2 cytokines. Peripheral lymphocytes proliferated in mixed lymphocyte culture and expressed Th1 cytokine mRNA. Tolerant hosts accepted PVG and rejected Lewis skin allografts and the lymph nodes draining both these grafts had similar induction of Th1 and Th2 cytokine mRNA. Treatment of tolerant rats with Th1 cytokines IL-2, IFN-gamma, and IL-12p70 or monoclonal antibody that blocked IL-4, IL-5, and transforming growth factor-beta did not prevent acceptance of PVG skin grafts. CONCLUSIONS: These studies in a model of tolerance regulated by CD4CD25 T cells demonstrated there was no defect in Th1 responses. Tolerance was due to regulation that was not solely dependent on IL-4, IL-5, or transforming growth factor-beta and was not inactivated or overwhelmed by administration of Th1 cytokines, IL-2, IFN-gamma or IL-12p70.
Subject(s)
Cytokines/pharmacology , Interleukin-4/immunology , Interleukin-5/immunology , Th1 Cells/immunology , Transforming Growth Factors/immunology , Transplantation Tolerance/immunology , Adjuvants, Immunologic/pharmacology , Animals , Antibodies, Monoclonal/pharmacology , Heart Transplantation/immunology , Interferon-gamma/pharmacology , Interleukin-12/pharmacology , Interleukin-2/pharmacology , Interleukin-4/metabolism , Interleukin-5/metabolism , Mice , Mice, Inbred BALB C , RNA, Messenger/metabolism , Rats , Rats, Inbred Lew , Rats, Inbred Strains , Skin Transplantation/immunology , Th1 Cells/metabolism , Transforming Growth Factors/metabolism , Transplantation Tolerance/drug effectsABSTRACT
Th2 cytokines, especially IL-4 and IL-10, may facilitate transplant tolerance induction but the role of IL-13, another Th2 cytokine, is not known. This study examined the effects of rat recombinant IL-13 (rIL-13) on alloimmune responses. In vitro effects of rIL-13 were compared in mixed lymphocyte cultures (MLC) on rat lymphocytes cultured with PVG stimulator cells. DA rats grafted with fully allogeneic PVG neonatal heart grafts were treated with 40,000 units of rIL-13 for 10 days and graft survival monitored by ECG. Cytokine mRNA expression in the graft and lymphoid tissues was studied by RT-PCR and alloantibody levels assayed. rIL-13 had no effect on MLC, unlike rIL-4 which enhanced proliferation and induced Th2 and inhibited Th1 cytokines in MLC. rIL-13 inhibited IL-12p35, IL-12p40 and TNF-alpha mRNA induction in dendritic cell cultures. Treatment with rIL-13 prolonged fully allogeneic PVG neonatal heart graft survival to 18-21 (13-27) days (median (range)); compared to 12 (9-15) days in untreated normal rejection (p<0.05) and 14 (10-24) days in sham treated controls (p<0.05). RT-PCR studies on graft tissue identified reduced mRNA expression for the dendritic cell/macrophage molecules iNOS, TNF-alpha and IL-12 compared to normal rejection. rIL-13 treatment did not increase Th2 cytokines as compared to normal rejection, or the Th2 dependent IgG1 alloantibody response, while IL-4 did. These studies demonstrated that rIL-13 can prolong allograft survival associated with inhibition of IL-12, TNF-alpha and iNOS mRNA induction, and suggest IL-13 could modify graft rejection by inhibition of dendritic cell and/or macrophage function.
Subject(s)
Cytokines/biosynthesis , Graft Survival/immunology , Interleukin-13/immunology , Macrophage Activation/immunology , Macrophages/immunology , Animals , Autoantibodies/blood , CHO Cells , Cricetinae , Cricetulus , Dendritic Cells/immunology , Graft Rejection/prevention & control , Heart Transplantation , Interleukin-13/metabolism , Lymphocyte Culture Test, Mixed , Macrophages/metabolism , RNA, Messenger/analysis , Rats , Recombinant Proteins/biosynthesis , Reverse Transcriptase Polymerase Chain Reaction , Transplantation, HomologousABSTRACT
CD4+T cells mediate antigen-specific allograft tolerance, but die in culture without activated lymphocyte derived cytokines. Supplementation of the media with cytokine rich supernatant, from ConA activated spleen cells, preserves the capacity of tolerant cells to transfer tolerance and suppress rejection. rIL-2 or rIL-4 alone are insufficient to maintain these cells, however. We observed that activation of naïve CD4+CD25+FOXP3+Treg with alloantigen and the Th2 cytokine rIL-4 induces them to express interleukin-5 specific receptor alpha (IL-5Rα) suggesting that IL-5, a Th2 cytokine that is produced later in the immune response may promote tolerance mediating Treg. This study examined if recombinant IL-5(rIL-5) promoted survival of tolerant CD4+, especially CD4+CD25+T cells. CD4+T cells, from DA rats tolerant to fully allogeneic PVG heart allografts surviving over 100days without on-going immunosuppression, were cultured with PVG alloantigen and rIL-5. The ability of these cells to adoptively transfer tolerance to specific-donor allograft and suppress normal CD4+T cell mediated rejection in adoptive DA hosts was examined. Tolerant CD4+CD25+T cells' response to rIL-5 and expression of IL-5Rα was also assessed. rIL-5 was sufficient to promote transplant tolerance mediating CD4+T cells' survival in culture with specific-donor alloantigen. Tolerant CD4+T cells cultured with rIL-5 retained the capacity to transfer alloantigen-specific tolerance and inhibited naïve CD4+T cells' capacity to effect specific-donor graft rejection. rIL-5 promoted tolerant CD4+CD25+T cells' proliferation in vitro when stimulated with specific-donor but not third-party stimulator cells. Tolerant CD4+CD25+T cells expressed IL-5Rα. This study demonstrated that IL-5 promoted the survival of alloantigen-specific CD4+CD25+T cells that mediate transplant tolerance.
Subject(s)
Graft Rejection/prevention & control , Heart Transplantation , Interleukin-5/pharmacology , Isoantigens/immunology , T-Lymphocytes, Regulatory/immunology , Transplantation Tolerance/drug effects , Allografts , Animals , Cell Survival/drug effects , Cell Survival/immunology , Graft Rejection/immunology , Graft Rejection/pathology , Interleukin-5/immunology , Rats , Rats, Inbred Lew , T-Lymphocytes, Regulatory/pathologyABSTRACT
Transplant tolerance induced in adult animals is mediated by alloantigen-specific CD4+CD25+ T cells, yet in many models, proliferation of CD4+ T cells from hosts tolerant to specific-alloantigen in vitro is not impaired. To identify changes that may diagnose tolerance, changes in the patterns of proliferation of CD4+, CD4+CD25+, and CD4+CD25- T cells from DA rats tolerant to Piebald Virol Glaxo rat strain (PVG) cardiac allografts and from naïve DA rats were examined. Proliferation of CD4+ T cells from both naïve and tolerant hosts was similar to both PVG and Lewis stimulator cells. In mixed lymphocyte culture to PVG, proliferation of naïve CD4+CD25- T cells was greater than naïve CD4+ T cells. In contrast, proliferation of CD4+CD25- T cells from tolerant hosts to specific-donor PVG was not greater than CD4+ T cells, whereas their response to Lewis and self-DA was greater than CD4+ T cells. Paradoxically, CD4+CD25+ T cells from tolerant hosts did not proliferate to PVG, but did to Lewis, whereas naïve CD4+CD25+ T cells proliferate to both PVG and Lewis but not to self-DA. CD4+CD25+ T cells from tolerant, but not naïve hosts, expressed receptors for interferon (IFN)-γ and IL-5 and these cytokines promoted their proliferation to specific-alloantigen PVG but not to Lewis or self-DA. We identified several differences in the patterns of proliferation to specific-donor alloantigen between cells from tolerant and naïve hosts. Most relevant is that CD4+CD25+ T cells from tolerant hosts failed to proliferate or suppress to specific donor in the absence of either IFN-γ or IL-5. The proliferation to third-party and self of each cell population from tolerant and naïve hosts was similar and not affected by IFN-γ or IL-5. Our findings suggest CD4+CD25+ T cells that mediate transplant tolerance depend on IFN-γ or IL-5 from alloactivated Th1 and Th2 cells.
ABSTRACT
CD4+T cells that transfer alloantigen-specific transplant tolerance are short lived in culture unless stimulated with specific-donor alloantigen and lymphocyte derived cytokines. Here, we examined if IFN-γ maintained survival of tolerance transferring CD4+T cells. Alloantigen-specific transplant tolerance was induced in DA rats with heterotopic adult PVG heart allografts by a short course of immunosuppression and these grafts functioned for >100days with no further immunosuppression. In previous studies, we found the CD4+T cells from tolerant rats that transfer tolerance to an irradiated DA host grafted with a PVG heart, lose their tolerance transferring ability after 3days of culture, either with or without donor alloantigen, and effect rejection of specific-donor grafts. If cultures with specific-donor alloantigen are supplemented by supernatant from ConA activated lymphocytes the tolerance transferring cells survive, suggesting these cells depend on cytokines for their survival. In this study, we found addition of rIFN-γ to MLC with specific-donor alloantigen maintained the capacity of tolerant CD4+T cells to transfer alloantigen-specific tolerance and their ability to suppress PVG allograft rejection mediated by co-administered naïve CD4+T cells. IFN-γ suppressed the in vitro proliferation of tolerant CD4+T cells. Tolerant CD4+CD25+T cells did not proliferate in MLC to PVG stimulator cells with no cytokine added, but did when IFN-γ was present. IFN-γ did not alter proliferation of tolerant CD4+CD25+T cells to third-party Lewis. Tolerant CD4+CD25+T cells' expression of IFN-γ receptor (IFNGR) was maintained in culture when IFN-γ was present. This study suggested that IFN-γ maintained tolerance mediating alloantigen-specific CD4+CD25+T cells.