This web page was produced as an assignment for an undergraduate course at Davidson College*


Michele Ho
BIO 307: Immunology
Dr. A. Malcolm Campbell Please click on ZAP-70 to learn more about this protein

The Role of Immune Mechanisms in Myocarditis

aleffgroup.com/.../tools/ 0-imag-pig-focal-myocarditis.html (permission was granted on 4-15-03)

'Tiger Striping' on the heart of a 4-week old piglet that died due to Focal Myocarditis Disease

Stages of Myocarditis, Nature of Disease, Immune Response, Autoimmune Response, Diagnosis, Treatment, Prevention, References


STAGES OF MYOCARDITIS

     

          Fig. 1 Timeline of myocarditis.  Each stage of the myocarditis has specific key players that are involved in the activation process. 

This figure was borrowed from Medical Progress Review Article Nov 9, 2000.

NATURE OF DISEASE

    Myocarditis is clinically defined as inflammation of the heart muscle.  There is a large variety of infections, systemic diseases, drugs, and toxins that associate with the development of this disease.  Viruses, bacteria, protozoa, and even worms have been implicated as infectious agents (Feldman and McNamara, 2000).  The diagnosis of myocarditis can be made by endomyocardial biopsy and the most frequent cardiotropic viruses detected include Parvo B19, measles, chicken pox, enteroviruses, adenoviruses, cytomegalovirus, Epstein Barr virus, and influenza virus (Maisch et al., 2003).  Viral diseases are more commonly associated with myocarditis in immunocompetent hosts such as human immunodeficiency virus type 1 (HIV-1) and hepatitis C (Hep C).  Recent researches showed that HIV-1 and HIV-1 RNA has been detected in heart tissue from patients with acquired immunodeficiency syndrome (Jacqueline and Blanchfield, 2002).  Dilated cardiomyopathy was evident that in 80 percent of a large group of asymptomatic HIV-postive patients, 83 percent of who had myocarditis and 6 percent of whom had detectable HIV myocardium.  However, there are some studies that fail to show HIV proviral DNA in myocardial samples obtained from HIV-infected children.  Hence it is unclear whether it is HIV itself or the appearance of secondary viruses in an immunocompromised host that accounts for the high incidence of myocarditis in HIV-positive patients.  Another viral disease that is involved in the development of dilated myocardiopathy is Hep C.  It was found that Hep C virus replicate in patients myocardium (Doroshenko, 2002).  The most common myocarditis resulted from bacteria is the Chagasí disease, an inflammatory disease caused by the parasitic protozoan Trypanosoma cruzi.  In addition, drugs can also cause myocardial inflammation by direct toxic effect on the myocyte or through immune-mediated mechanisms.  An example of a drug-induced toxicity is cocaine, which causes cardiac dysfunction by its vasoconstrictor properties.  Patients with drug-induced allergic myocarditis tend to have eosinophilia or an eosinophilic infiltrate in the myocardium (Feldman and McNamara, 2000). 

IMMUNE RESPONSE

                           Fig. 2 Replication of virus followed by the host defense mechanisms.  This figure was borrowed from Viral Infection of the Heart by J.E. Banatvala p. 84

    An example that shows the mechanisms in which the immune system responds can be illustrated by the coxsackievirus B.  The first stage includes RNA viruses are taken into cells by receptor-mediated endocytosis and directly translated inside the cells to produce viral protein.  The virus then replicates in the cytoplasm of the myocytes, but eventually it can also be released into the interstitium, where it undergoes by phagocytosis by macrophages (Feldman and McNamara, 2000).  The second stage of viral infection is characterized by infiltration by inflammatory cells, including natural killer cells and macrophages with the subsequent expression of proinflammatory cytokines.  Recent studies demonstrate that CD1d expression increases early after the infection and that CD1d is essential for pathogenicity of CVB3-induced myocarditis (Huber et al., 2003).  ERK-1/2 is an extracellular signal-regulated kinase which influences the p56(Lck) and in turns activate T cells.  The enhancement of ERK-1/2 activation, therefore, induces myocarditis to those who are susceptible to coxsackievirus (Opavsky et al., 2002).  Moreover, the VP1 protein in Coxsackievirus B group potentially functioned as a predominant antigen inducing detectable IgM antibody following CVB infection (Zhang et al., 2001).  XJEK granules are efficacious in inhibiting CVB3m protecting and curing virus myocarditis.  The granules are also good for inflammation, anti-myocardial ischemia, anti-arrhythmia, and increase the serum IgG level (Wang et al., 2000).  Interestingly, Coxsackievirus group B type 3 (CVB3) have different effects based on genders.  CVB3 induces myocarditis in male but produces little cardiac injury in females.  The males develop cytolytic T lymphocytes (CTL) reactive to heart antigens which primarily cause the inflammation and cardiac injury.  The infected females lack this CTL response because they rapidly produce suppressor cells inhibiting both cellular immunity and cardiac inflammation (Job et al., 1986). 

Fig. 3 CVB3 A schematic representation of a feasible structure for the human coxsackievirus which is depicted that is derived from the crystal structure of the Ig V-like domain.  Image was taken from http://wehih.wehi.edu.au/scop/data/scop.b.html

    The macrophage activation results from the release of viral particles into the interstitium and the release of interferon-y by natural killer cells and other activated white cells.  After activation by interleukin-2, the natural killer cells protect against viral invasion by eliminating virally infected cells, and thus inhibiting virus replication.  Not only natural killer cells release perforin and granzymes, they can exacerbate disease by injuring the cardiomyocytes.  Hence, natural killer cells only interact with virus-infected myocytes, sparing the uninfected cells (Feldman and McNamara, 2000).

    Cell-mediated immunity also has an important role in viral clearing.  Within seven days of infection, antigen-specific T cells infiltrate the mouse myocardium.  Those circulating T cells, which express the alpha and beta chains of the T cell receptor and either CD4 or CD8 coreceptor molecules, include T helper cells and cytotoxic T lymphocytes.  The cytotoxic T cells then recognize the degraded fragments of viral proteins that are presented by the major-histocompatibility-complex class I (MHC I) antigens on the surface of the myocyte membrane.  Cytokines such as interferon-y (IFN-y) induce the up-regulation of the MHC antigens on the surface of the myocytes.  It was found that type I IFNs act as a natural adjuvant for the immune response against myocarditis.  Type I IFN DNA coimmunisation may provide increased efficacy for viral vaccines and subsequently modulate post-viral chronic inflammatory disorders (Feldman and McNamara, 2000). 

    In order to become fully activated, T cells must also receive a second signal from the costimulatory molecules such as B7 on the antigen presenting cells.  When there are appropriate cofactors and antigens, cytotoxic T lymphocytes become activated and are able to lyse virus-infected cardiocytes (Feldman and McNamara, 2000).  Moreover, the cell-to-cell contact allow effective lysis and is mediated by the up-regulation of intercellular adhesion molecules (ICAM-1) on the surface of the infected myocyte, which is induced by tumor necrosis factor alpha (TNF alpha) and INF-y (Cooper, 2003).  These molecules help to further mobilize immunocytes to the injury area to remove the damaged or toxin/virus-contaminated cells.  The ICAM-1 up-regulation can further lead to the coupling of T cells with expressed LFA-1 ligand, leading to killing of the infected host cell by natural killer cells via perforin mechanism (Schultheiss and Schwimmbeck, 1997).  The development of neutralizing antiviral antibodies also helps the clearing of the viral infection.  The following diagram indicates the mechanisms of humoral and cellular immunity:

Fig. 4  Mechanisms of humoral and cellular immunity. This figure was borrowed from Medical Progress Review Article Nov 9, 2000.

AUTOIMMUNE RESPONSE

    

Fig. 5  presents a paradigm for the development of cardiomyopathy. hsc.virginia.edu/.../internal/conf/chiefs/myocarditis.htm by Dr. Scott Robertson ( There was no email address available to request for permission regarding the image.)

 

    The ultimate function of the immune response is to clear virus and allow healing in many instances of myocardial infection.  However, inefficient viral clearing or overaggressive immunologic activation can be observed as well.  For example, in some strains of mice, normal host defense mechanisms are inadequate, and persistent viral replication occurs in the myocytes, resulting in chronic viral disease with cardiac dilation and failure.  The expression of toll-like receptor 4 increases the enteroviral replication in human myocarditis (Satoh et al., 2003).  The persistent activation of infiltrating T cells during viral infection can cause long-term tissue destruction, leading to dilated cardiomyopathy. 

Fig. 6 Infection of neonatal mice with reovirus strain 8B produces myocarditis in neonatal mice due to a direct viral injury of myocytes. The myocardial injury is due to apoptosis. Apoptosis was then verified by TUNEL staining co-localizing to areas of viral antigen, as well as by demonstration of characteristic oligonucleotide laddering of nuclear DNA. The data shows that calpain inhibitors block retrovirus-induced apoptosis in vitro.  Targeting of apoptotic signaling pathways may serve as a novel antiviral strategy.                                    

www.uchsc.edu/.../robertadebiasi/ invivomyocarditis.htm (permission to use image was granted on 4-15-03 by Dr. Roberta DeBiasi)

 

Fig. 7 Cytokine Expressions in the Heart.  AdexCTLA-4IgG suppressed cytokine activation and the onset and progression of experimental autoimmune myocarditis in the rat model.
www.j-circ.or.jp/.../sessions/ reports/66th-ss/ps06-z4.htm
(requested permission on 4-15-03)

   The proinflammatory cytokines also have important roles in the development of chronic inflammatory disease.  Tumor necrosis factor activates endothelial cells, recruits inflammatory cells, enhances the production of inflammatory cytokines, and has significant negative inotropic effects.  Interferons have a critical role in attenuating viral replication when administered endogenously (Feldman and McNamara, 2000).  In addition, cytokines can activate inducible nitric oxide synthase in cardiac myocytes, which can simultaneously give cardiacprotection as well as the development of myocarditis (Ding, 2002).  Nitric oxide is a free radical gas that plays paracrine/autocrine and intracrine roles in maintaining physiological cardiovascular performance.  In the coronary circulation, NO mediates endothelium-dependent vasodilator responses to shear stress and agonist-induced responses to neurohumoral stimulation.  In the heart, NO modulates myocardial relaxation, beta-adrenergic responses, mitochondrial respiration and substrate metabolism and excitation-contraction coupling (Champion and Hare, 2001).  The beneficial and deleterious roles of nitric oxide are still debatable. Moreover, the cellular effector mechanisms such as natural killer cells activity was markedly decreased in the acute state in target cell of patients with perimyocarditis.  In contrast, in myocarditis target cell specific non-major histocompatibility complex (MHC) restricted lysis against living adult allogenic rat myocytes is slightly enhanced (Banatvala, 1993).

    There are studies that show humoral immunity is also essential in the development of postinfectious myocarditis.  The experiments showed that a T-cell-dependent myocarditis could be demonstrated in mice after immunization with cardiac C protein or streptococcal M protein peptide, adoptive transfer of myosin-reactive cells or plenocytes after myocardial infarction, or transplantation of a normal heart into a virus-treated host after documentation of viral clearance.  The researchers suggested that there is an autoimmune myocarditis with the evidence of cross-reactive epitopes between cardiac myosin and infectious agents (Kishimoto et al., 2001).  Upon secondary exposure of enteroviruses, T cell-mediated immune responses to a conserved antigenic epitope (Kishimoto et al., 2001).  Not only there is a relation between virus and the subsequent development of cardiomyopathy, there are other factors such as physical-activitylevel, sex, age, and genetic background which determine the virulence of the infection.

    Patients with myocarditis normally have an imbalance between helper and cytotoxic T cells; an inappropriate expression of the MHC on cardiac tissues; and circulating organ-specific autoantibodies in the serum.  The cytotoxic activity against healthy cardiomyocytes was myocyte-specific, induced by CD8 lymphocytes and MHC restricted.  Cytotoxic T lymphocytes are activated following myocardial infarction and can recognize and kill healthy myocytes in vitro (Varda-Bloom et al., 2000).  In patients with diated cardiomyopathy, autoantibodies have been identified that react with heart mitochondria, the adenine nucleotide translocator, the muscarinic receptor, myosin heavy chain, or laminin. 

              Fig. 8 Mechanisms that lead to autoimmune response. This figure was borrowed from Viral Infections of The Heart by J.E. Banatvala, p. 85

DIAGNOSIS

    Patients with myocarditis disease normally have flulike syndrome accompanied by fever, arthralgias, and malaise.  Clinicians can also perform laboratory tests to find out if patients have an elevated sedimentation rate, eosinophilia, an elevation in the cardiac fraction of creatine kinase, cardiac troponin T or troponin I.  Other diagnostic tests include autoimmune serum markers or the induction of the MHC and intercellular adhesion molecules on cardiac myocytes to identify patients with autoimmune myocarditis.   To further confirm the diagnosis, usually the physician would obtain a cardiac catherization (Feldman and McNamara, 2000). 

    Researchers believe that complement plays a critical role in the development of autoimmune myocarditis and that it acts through complement receptor type 1 (CR1) and type 2 (CR2).  There is a subset of CD44 and CD62L T cells that expresses CR1 and CR2, which suggest that both receptors are involved in the B and T cell activation, T cell proliferation, and cytokine production.   Since the activated complement is the key product of the innate immune response, it modulates the induction of an autoimmune disease (Kaya et al., 2001). 

    Activation of dendritic cells is another important component of myocarditis.  Interleukin 1 receptor 1  (IL-1R1) triggering is required for efficient activation of dendritic cells, which is in turn a prerequisite for induction of autoreactive CD4+ T cells and autoimmunity (Eriksson et al., 2003).  CD4+ T cells induce local eosinotaxis, mediated by IL-5, and participate in myocardiocyte injury via Eo induction (Hirawasa et al., 2003).  IL-6 is required for the expansion of autoimmune CD4+ T cells and the pathogenesis of autoimmune myocarditis by upregulate complement C3 (Eriksson, et al., 2003).  CD83+ dendritic cells (CDs) are involved in the inflammatory process and triggered by an imbalance of DCs and their failure to confer tolerance to self-antigen (Schoppet et al., 2003).  Cardiac antigen-specific CD8+ T cells are involved in the autoimmune component of human myocarditis and that IL-12 is required for the differentiation of pathogenic CD8+ T cell effectors (Grabie et al., 2003). 

TREATMENT

    Treatments of myocarditis include antibiotics to fight of the infection if the cause is bacterial infection.  However, if the case is severe, the only option that allows complete recovery is heart transplantation (Long and Blanchfield, 2002).  Drugs that reduce the basal levels of cytokines are amlodipine, pentoxifylline, and beta-blockers.  There are also drugs that help reduce endotoxin-induced cytokine gene expression: ouabain, amiodarone, adenosine, angiotensin converting enzyme inhibitors (e.g. captopril, enalapril, and lisinopril), angiotensin II-receptor blockers (Godsel et al., 2003).  Direct blockade of the deleterious actions of elevated plasma levels of cytokines recently became possible through intravenous infusion of a soluble TNF-alpha receptor fusion protein, which resulted in an increase in exercise tolerance and left ventricle performance (Paulus, 2000).  Immunosuppressive therapy is not recommended in patients with infectious or postinfectious myocarditis.  However, immunosuppression is essential for patients with cardiac dysfunction due to a systemic autoimmune disease and it can be achieved by the help of biventricular mechanical assist (BVS 5000) (Marelli et al., 2003).  Successful treatment of enterovirus-induced myocarditis with interferon-alpha was proven to be a reliable therapy (Daliento et al., 2003).  Embryonic stem cells significantly increase the survival of viral myocarditis mice and also decrease the necrosis and infiltration of inflammatory cells (Wang et al., 2002).  In addition, the suppressor of cytokine signaling-1 (SOCS1) is a novel therapeutic target for enterovirus-induced cardiac injury by inhibiting the signaling of Janus Kinase (JAK) and signal transducers and activators of transcription (STAT) (Yasukawa et al., 2003).  An antiviral agent can reduce the number of infected cells and virus identified in human myocardial fibroblasts.  An alternative approach to the treatment of viral myocarditis has been the development of virus-specific vaccines.  Attenuated vaccines have successfully prevented the development of myocarditis after viral challenge in mice, pigs, and elephants.  The usefulness of vaccines in humans remains unclear (Feldman and McNamara, 2000).

PREVENTION

      Even though myocarditis is an unpredictable disease, the following steps can help prevent its onset:

*take extra measures to avoid infections, and obtain appropriate treatment for infections.

*limit alcohol consumption to no more than one or two drinks a day, if any.

*maintain current immunizations against diphtheria, tetanus, measles, rubella, and polio.

*avoid anything that may cause the abnormal heart to work too hard, including salt and vigorous exercise (Long and Blanchfield, 2002).

REFERENCES

Banatvala, J.E.  1993.  Viral Infections of The Heart.  Boston, MA: Hodder and Stoughton, pp.84-161.

Champion, H.C., and J.M. Hare.  2001.  Emerging therapeutic targets in nitric oxide-dependent cardiac disease.  Expert Opin Ther Targets 5(5):547-556.

Cooper, L.T.  2003.  Myocarditis From Bench to Bedside.  Totowa, NJ: Humana Press, pp.101-2.

Cull, V.S., S. Broomfield, E.J. Bartlett, N.L. Brekalo, and C.M. James.  2002.  Coimmunisation with type I IFN genes enhances protective immunity against cytomegalovirus and myocarditis in gB DNA-vaccinated mice.  Gene Ther 9(20):1369-78.

Daliento, L., F. Calabresse, F. Tona, A.L. Caforio, G. Tarsia, A. Angelini, and G. Thiene.  2003.  Successful treatment of enterovirus-induced myocarditis with interferon-alpha.  J Heart Lung Transplant 22(2):214-7.

Ding, G.F.  2002.  Involvement of immune system in the pathogenesis of viral myocarditis.  Sheng Li Ke Xue Jin Zhan 33(1):30-7.

Doroshenko, B.H.  2002.  Hemostasis status in patients with acute viral myocarditis.  Lik Sprava 5-6:22-3.

Eriksson, U., M.O. Kurrer, N. Schmitz, S.C. Marsch, A. Fontana, H.P. Eugster, and M. Kopf.  2003.  Interleukin-6-deficient mice resist development of autoimmune myocarditis associated with impaired upregulation of complement C3.  Circulation 107(2):320-5.

Eriksson, U., M.O. Kurrer, I. Sonderegger, G. Iezzi, A. Tafuri, L. Hunziker, S. Suzuki, K. Bachmaier, R.M. Bingisser, J.M. Penninger, and M. Kopf.  2003.  Activation of dendritic cells through the interleukin 1 receptor is critical for the induction of autoimmune myocarditis.  J Exp Med 197(3):323-31.

Feldman, A.M., and D. McNamara.  2000.  Myocarditis.  The New England Journal of Med 343(19):1388-98.

Godsel, L.M., J.S. Leon, and D.M. Engman.  2003.  Angiotensin Converting Enzyme Inhibitors and Angiotensin II Receptor Antagonists in Experimental Myocarditis.  Curr Pharm Des 9(9):723-35.

Grabie, N., M.W. Delfs, J.R. Westrich, V.A. Love, G. Stavrakis, F. Ahmad, C.E. Seidman, J.G. Seidman, and A.H. Lichtman.  2003.  IL-12 is required for differentiation of pathogenic CD8+ T cell effectors that cause myocarditis.  J Clin Invest 111(5):671-80.

Hirasawa, M., H. Deguchi, A. Ukimura, and Y. Kitaura.  2003.  Immunologic interaction between infiltrating eosinophils and T lymphocytes in murine spontaneous eosinophilic myocarditis.  Int Arch Allergy Immunol 130)1):73-81.

Huber, S., D. Sartini, and M. Exley.  2003.  Role of CD1d in coxsackievirus b3-induced myocarditis.  J Immunol 170(6):3147-53.

Job, L.P., D.C. Lyden, and S.A. Huber.  1986.  Demonstration of suppressor cells in coxsackievirus group B, type 3 infected female Balb/c mice which prevent myocarditis.  Cell Immunol 89)1):104-13.

Kaya, Z., M. Afanasyeva, Y. Wang, K.M. Dohmen, J. Schlichting, T. Tretter, D. Fairweather, V.M. Holers, and N.R. Rose. 2001.  Contribution of the innate immune system to autoimmune myocarditis: a role for complement.  Nat Immunol 2(8):739-45.

Kishimoto, C., Y. Hiraoka, and H. Takada.  2001.  T cell-mediated immune response enhances the severity of myocarditis in secondary cardiotropic virus infection in mice.  Basic Res Cardiol 96(5):439-45.

Long, J.L., and D.S. Blanchfield.  2002.  The Gale Encyclopedia of Medicine.  Farmington Hills, MI: Gale Group, pp. 2288-91.

Maisch, B., A.D. Ristic, I. Portig, and S. Pankuweit.  2003.  Human viral cardiomyopathy.  Front Biosci 8:S39-67.

Marellin, D., R. Kermani, J. Bresson, M.C. Fishbein, M. Hamilton, J. Moriguchi, G.C. Fonarow, B. Cohen, J. Kobashigawa, and H. Laks.  2003.  Tex Heart Inst J 30(1)50-6.

Opavsky, M.A., T. Martino, M. Rabinovitch, J. Penninger, C. Richardson, M. Petric, C. Trinidad, L. Butcher, J. Chan, and P.P. Liu.  2002.  Enhanced ERK-1/2 activation in mice susceptible to coxsackievirus-induced myocarditis.  J Clin Invest 109(12):1561-9.

Paulus, W.J.  2000.  Cytokines and heart failure.  Heart Fail Monit 1(2):50-6.

Satoh, M., M. Nakamura, T. Akatsu, J. Iwasaka, Y. Shimoda, I. Segawa, and K. Hiramori.  2003.  The expression of toll-like receptor 4 associated with enteroviral replication in human Myocarditis.  Clin Sci (Lond) [epub ahead of print].

Schoppet, M., S. Pankuweit, and B. Maisch.  2003.  CD83+ dendritic cells in inflammatory infiltrates of Churg-Strauss myocarditis.  Arch Pathol Lab Med 127(1):98-101.

Schultheiss, H.P., and P. Schwimmbeck.  1997.  The Role of Immune Mechanisms in Cardiovascular Disease.  Berlin, Germany: Springer, pp. 47-48.

Varda-Bloom, N., J. Leor, D.G. Ohad, Y. Hasin, M. Amar, R. Fixler, A. Battler, M. Eldar, and D. Hasin.  2000.  Cytotoxic T lymphocytes are activated following myocardial infarction and can recognize and kill healthy myocytes in vitro.  J Mol Cell Cardiol 32(12):2141-9.

Wang, J.F., Y. Yang, G. Wang, J. Min, M.F. Sullivan, P. Ping, Y.F. Xiao, and J.P. Morgan.  2002.  Cell Transplant 11(8):753-8.

Yasukawa, H., T. Yajima, H. Duplain, M. Iwatate, M. Kido, M. Hoshijima, M.D. Weitzman, T. Nakamura, S. Woodard, D. Xiong, A. Yoshimura, K.R. Chien, and K.U. Knowlton.  2003.  The suppressor of cytokine signaling-1 (SOCS1) is a novel therapeutic target for enterovirus-induced cardiac injury.  J Clin Invest 111(4):469-78.

  Wang, Q.M., G.L. Chen, Y.J. Wang, H.S. Wang, M.H. Gao, and Y.Z. Gong.  2000.  An experimental study on inhibitor effect of xinjierkang granules on virus myocarditis.  Zhongguo Zhong Yao Za Zhi 25(5):293-6.

  Zhang, T., G. Ma, and L. Ma.  2001.  Study on specificity of IgM antibody response in patients with coxsackie virus B infection.  Zhonghua Shi Yan he Lin Chuang Bing Du Xue Za Zhi 15(1):66-8.

____________________________________________________________________________

 

 

Are you intrigued by the effects of Zeta-Associated Protein 70?

 

ZAP-70

 

Human's Syk tyrosine kinase is part of the SH2 domain family which includes ZAP-70.

Image was taken from http://wehih.wehi.edu.au/scop/data/scop.b.html

 


 

Structure and Function

Mutations of ZAP-70 and Related Disorders

Other related defects that cause SCID  

ZAP-70 Related Treatments for Immunodeficiency Disorders

Alterations of ZAP-70 Signaling

References

 


 

    Zeta-associated protein or ZAP-70 is one of the principal targets of Lck in T cells, which is essential in propagating the signal onward.  It is a non-src family protein kinase which associates with phosphorylated CD3 zeta chain, and plays an important role in TCR-CD3 complex signaling (Delves and Roitt 1998).  ZAP-70 binds to the phosphorylated zeta chain ITAMs and is phosphorylated and activated by Lck when the coreceptor binds to the MHC ligand (Janeway et al., 2001).   The expression of ZAP-70 in developing T cells promotes the development of single positive from double positive thymocytes.  Current studies show that ZAP-70 tyrosine kinase is required for the up-regulation of Fas ligand in activation-induced T cell apoptosis (Eischen et al., 1997).  An interesting research discovered that T cells from a substantial proportion of elderly humans exhibit significant reductions in the catalytic activity, but not expression of ZAP-70 when stimulated by ligation of the TCR/CD3 with cross-linked anti-CD3 monoclonal antibody OKT3.  Hence, age-related impairments of ZAP-70 activation in anti-CD3 stimulated T cells are associated with reduced tyrosine phosphorylations of zeta chains and autophosphorylations of the PTKs p56lck/p59fyn (Whisler et al., 1999).

 

Structure and Function

    ZAP-70 has two SH2 domains in its amino terminal halves and a carboxy-terminal kinase domain.  The crystal structure of the tandem SH2 domains of human ZAP-70 in complex with a peptide derived from the zeta-subunit of the T cell receptor reveals an unanticipated interaction between the two domains.  A coiled coil of alpha-helices connects the two SH2 domains, producing an interface that constitutes one of the tow critical phosphotyrosine binding sites (Hatada et al., 1995).  As each SH2 domain binds to one phosphotyrosine, ZAP-70 preferentially binds to motifs with two phosphotyrosines spaced a precise distance apart.  ZAP-70 is thus recruited to the receptor complex upon full phosphorylation of the ITAMs.  Binding to the ITAM peptide induces large movements between the two SH2 domains and the actual binding sites.  The conformation of the ITAM-free protein is partly governed by a hydrophobic cluster between the linker region and the C-terminal SH2 domain (Folmer et al., 2002).  The activation of ZAP-70 is mainly contributed by Lck, which in turn phosphorylates LAT (Linker of Activation in T cells) and SLP-76 (Second Linker Protein) (Pelosi et al., 1999).  SLP-76 and LAT are each critical for the expansion and differentiation of double-negative thymocytes and that SLP-76 is essential for allelic exclusion at the TCR beta locus (Pivniouk and Geha, 2000).  Tec kinases then activate PLC-y and guanine-nucleotide exchange factors activate Ras which propagates the signal from the cell membrane into the nucleus to begin gene transcription.  Another function of ZAP-70 is that in the immune synapse, ZAP-70 controls T cell polarization and recruitment of signaling proteins but not formation of the synaptic pattern (Blanchard et al., 2002). 

Fig. 1  Activated ZAP-70 phosphorylated SLP-76 and LAT initiating ras/MAPK and PLCgl signaling cascades.  Tyrosine phosphorylated LAT which is constitutively located in glycolipid enriched membrane microdomains additionally recruits PLCgl1to the membrane, placing PLCg1 in close proximity with its substrate, PIP2.  PIP2 is hydrolyzed IP3 and DAG which leads to increases of cytosolic free calcium and activation of protein kinase C.  Tyrosine phosphorylated LAT binds to the Grb2 SH2 domain which recruits Sos to the plasma membrane thus innitiating Ras/ERK activation.  Since Gads constitutively associates with SLP-76, theSLP-&6/Gads complex may be recruited to LAT leading to ras/MAPK and PLCg1 activation.  Tyrosine phosphorylated SLP-76/Vav/Nck/Pak complex which may be important for the regulation of cytoskeletal rearrangements.  SLP-76 additionally binds to the SH2 domain of Itk which subsequently phosphorylates and further activates PLCg1.  (Requested permission from Dr. Gary Koretzky, M.D., Ph.D. on 3/13/03)< http://www.med.upenn.edu/immun/koretzky.html>

To view Figure 2, please click on the following website: http://www.hhmi.org/lectures/biointeractive/animations/tcell/tcell_frames.htm

Fig. 2  The animation summerizes the importance of ITAM and ZAP-70 in propagating the signal onward.  In order to be activated, the molecules contributing to the transduction cascade need to be present together in the correct part of the cell. The inactivated molecule ZAP-70 (zeta-associated protein-70), a key player in the cascade, may randomly bump into the ITAM subunit of the T-cell receptor. However, nothing will come of these contacts unless ITAM is in the activated state. The binding of the APC to the T-cell receptor triggers a series of events leading to ITAM activation.  (There was no email contact listed to request for permission to use the animation.)

Mutations of ZAP-70 and Related Disorders

    Patients who make a defective form of the cytosolic protein tyrosine kinase ZAP-70, which transmit signals from the T cell receptor, their CD4 T cells emerge from the thymus in normal numbers, whereas CD8 T cells are absent.  However, the CD4 T cells that mature fail to respond to stimuli that normally activate via the T cell receptor; hence, cause immunodeficiency (Janeway 2001).Thymocytes expressing both CD4 and CD8 (double positive) are present while CD4- CD8+ thymocytes are absent, indicating that ZAP-70 is indispensable for the development of CD8 single positive cells.  Lack of ZAP-70 may be partly compensated by the presence of syk, allowing the development of CD4+ T cells but not CD8+ T cells (Noraz et al., 2000).  However, CD4+ T cells fail to respond to anti-CD3, mitogens or allogenic cells in vitro, indicating a defective signal transduction and the activity of natural killer (NK) cell is normal.  Researches show that mice with defective ZAP-70 lack both CD4+ and CD8+ T-cells in peripheral lymphoid organs, which suggest that the requirement of ZAP-70 and syk for T-cell development is different between mice and human ( Hivroz and Fischer, 1994).  In addition, patients with chronic lymphocytic leukemia (CLL) have low-level expression of CD38 and do not express a detectable amount of ZAP-70 protein.  Leukemia cells from identical twins with CLL were found discordant for expression of ZAP-70, suggesting that B-cell expression of ZAP-70 is not genetically predetermined (Chen et al., 2002).

    A mutation in ZAP-70 can result in severe combined immunodeficiency (SCID), which is characterized by the absence of both cellular and humoral immunity.  SCID mutation lacks appropriate rearrangements of TRC and Ig which results in absence of mature T and B cells, and abnormal sensitivity to ionization radiation which causes DNA double-strand to break.  Intensive researches demonstrate that ZAP-70 is essential for human T cell function and suggest that CD4+ and CD8+ T cells depend on different intracellular signaling pathways to support their development or survival (Elder et al., 1994).  One clinical case showed that T cells in ZAP-70 deficient patients are assumed to have no helper functions for B-cell immunoglobulin synthesis.  The particular patient had immunoglobulin E (IgE) antibodies specific to food allergens and the scientists investigated the mechanisms of switching to IgE.  It was found that the peripheral blood mononuclear cells from the patient did not proliferate upon stimulation with the antigens but produced distinct levels of IL-4.  Cell sorting analysis indicated that the cells that produced IL-4 in response to the antigens were enriched in CD4+ T cells.   Purified CD4+ T cells from that patient expressed CD40L upon stimulation with anti-CD3 and induced mature epsilon transcript on naÔve B cells.  The results demonstrated that there was sufficient T cell receptor signaling remained to exert antigen-specific IgE switching on B cells (Toyabe et al., 2001).

Other related defects that cause SCID include:

1.      X-linked SCID-defects in common y chain for interleukin 2 (IL-2), IL-4, IL-7,

IL-9 and IL-15 receptors.

2.      Adenosine deaminase (ADA) deficiency- defect in the enzyme of interconversion pathways of purine metabolism which leads to the accumulation of deoxyadenosine.

3.      Purine nucleotide phosphorylase (PNP) deficiency- different mutations in PNP gene on chromosome 14 causes central nervous system of hypotonia, spasticity, neutropenia or megaloblastic anemia.

4.      JAK3 kinase deficiency- mutation of JAK3 kinase in T-cell signal transduction pathway is a rare cause of SCID.

5.      Reticular dysgenesis- the most severe type of SCID due to an inherited defect in a pluripotential bone marrow stem cell.

6.      RAG defects- prevents VDJ rearrangement of immunoglobulin and T cell receptor genes.

7.      MHC class II deficiency- caused by genetic defects in transcription factors and fails to express MHC class II on lymphocytes and macrophages.

8.      Omennís syndrome- characterized by thickened eczematous skin, massive lymphadenopathy and hepathosplenomegaly due to infiltration of polyclonal lymphocytes, histiocytes, and eosinophils.

9.      Cellular immune defects and dwarfism- characterized by short limb dwarfism, thin hair and variable T-cell deficiency with severe hypoplastic anemia.

10. Defects in NF-AT transcription factor- defects of NF-AT do not have the capability to control the transcription of many cytokines (Delves and Roitt, 1998).

Related Treatments

    Even though the disease is extremely rare, nearly all patients with ZAP-70 defects presented with typical features of SCID in early life: severe pulmonary infection often sustained by opportunistic pathogen (Pneumocystis carinii), chronic diarrhea, failure to thrive, and persistent candidiasis.  ZAP-70 deficiency is ultimately fatal unless patients undergo bone marrow transplantation (BMT) (Barata et al., 2001).  Patients with SCID can be treated with intravenous immunoglobulin infusions at regular intervals and prophylaxis for pneumocystic carinii.  Bacterial or fungal infections are treated with intravenous infusions of antibiotics or antifungal agents.  Epstein-Barrvirus, herpes virus and cytomegalovirus infection may result in systemic and lethal disorders, hence it can be treated with antiviral reagents.  As for cytomegalovirus patients, a prophylactic infusion of immunoglobulin is another treatment option.  Bone marrow transplantation (BMT) is the most favorable treatment to reconstitute the impaired immune system in SCID (Taylor et al., 1996).  Chemotherapy, total body radiation, and antilymphocyte globulin can be used to improve engraftment (Otsu et al., 2002).  HLA-haploidentical marrow from a parent is one of the primary sources for BMT.  Adenosine deaminase (ADA) deficiency patients have two possibilities: enzyme replacement with polyethylene glycol-modified ADA (PEG-ADA) or gene therapy inserted in a retroviral vector (Delves and Roitt, 1998).

Alterations of ZAP-70 signaling

    No drugs are currently known to affect the ZAP-70 protein.  However, drug targeting of single SH2 domain within ZAP-70 and Syk are comparable in their abilities to mediate hematopoietic antigen receptor function (Kong et al., 1995).  An experiment reported that Herpesvirus saimiri, which does not code for a ZAP-70 homologue, can replace this tyrosine kinase because H. saimiri is an oncogenic virus that has the ability to transform human T cells to stable growth based on mutual CD2 and CD3-mediated activation.   In the ZAP-70 deficient patientís cell lines, CD2 and CD3 activation were restored in terms of (Ca++), MAPK activation, cytokine production, and proliferation.  The transformed cells expressed a high level of ZAP-70 related kinase Syk; therefore, it was concluded that wild type H. saimiri can restore CD2 and CD3-mediated activation in signaling deficient human T cells (Meinl et al., 2001).

References

Barata, L.T., R. Henriques, C. Hivroz, E. Jouanguy, A. Paiva, A.M. Freitas, H.B.Coimbra, A. Fischer, and H.C. da Mota.  2001.  Primary

immunodeficiency secondary to ZAP-70 deficiency.  Acta Med Port 14(4):413-7.

 

Blanchard, N., V. Di Bartolo, and C. Hivroz.  2002.  In the immune synapse, ZAP-70 controls T cell polarization and recruitment of signaling    

proteins but not formation of the synaptic pattern.  Immunity 17(4):389-99.

 

Chen, L., G. Widhopf, L. Huynh, L. Rassenti, K.R. Rai, A. Weiss, and T.J. Kipps.  2002.  Expression of ZAP-70 is associated with increased B

cells receptor signaling in chronic lymphocytic leukemia.  Blood 100(13):4609-14.

 

Cloning an Army of T Cells for Immune Defense. < http://www.hhmi.org/lectures/biointeractive/animations/tcell/tcell_frames.htm> Accessed 2003 17 Feb.

 

Elder, M.E., D. Lin, J. Clever, A.C. Chan, T.J. Hope, A. Weiss, and T.G. Parslow.  1994.  Human severe combined immunodeficiency due to a

defect in ZAP-70, a T cell tyrosine kinase.  Science 264(5165):1596-9.

 

Eischen, C.M., B.L. Williams, W. Zhang, L.E. Samelson, D.H. Lynch, R.T. Abraham, and P.J. Leibson.  1997.  ZAP-70 tyrosine kinase is required for the up-regulation of Fas ligand in activation-induced T cell apoptosis.  Journal of Immunology 159(3):1135-9.

 

Folmer, R.H., S. Geschwindner, and Y. Xue.  2002.  Crystal structure and NMR studies of the apo SH2 domains of ZAP-70: two bikes rather  than a tandem.  Biochemistry 41(48):14176-84.

 

Gelfand, E.W., K. Weinberg, B.D. Mazxer, T.A. Kadlecek, and A. Weiss.  1995.  Absence of ZAP-70 prevents signaling through the antigen

receptor on peripheral blood T cells but not on thymocytes.  Journal of Exp Med 182(4):1057-65.

 

Hatada, M.H., X. Lu, E.R. Laird, J. Green, J.P. Morgenstern, M. Lou, C.S. Marr, T.B. Phillips, M.K. Ram, and K. Theriault.  1995.  Molecular

basis for interaction of the protein tyrosine kinase ZAP-70 with the T cell receptor.  Nature 376(6544):17-8.

 

Janeway, A.C., P. Travers, M. Walport, J.M. Shlomchik.  2001.  Immunobiology: The Immune System in Health and Disease.  New York, NY: Elsevier Science Ltd./ Garland Publishing, pp. 467, 1275, 1279, 2173, 2324.

 

Kong, G.H., J.Y. Bu, T. Kurosaki, A.S. Shaw, and A.C. Chan.  1995.  Reconstitution of Syk function by the ZAP-70 protein tyrosine kinase. 

Immunity 2:485-92.

 

Koretzky, G.  Molecular mechanisms of lymphocyte activation.  <http://www.med.upenn.edu/immun/koretzky.html>  Accessed 2003 13 Mar.

 

Meinl, E., T. Derfuss, R. Pirzer, N. Blank, D. Lengenfelder, A. Blancher, F. Le Deist, B. Fleckenstein, and C. Hivroz.  2001.  Herpesvirus saimiri replaces ZAP-70 for CD3 and CD2-mediated T cell activation.  Journal of Biol Chem 276(40):36902-8.

 

Noraz, N., K. Schwarz, M. Steinberg, V.  Dardalhon, C. Rebouissou, R. Hipskind, W. Friedrich, H. Yssel, K. Bacon, and N. Taylor.  2000.  

Alternative antigen receptor (TCR) signaling in T cells derived from ZAP-70-deficient patients expressing high levels of Syk.  Journal of       

Biol Chem 275(21):15832-8.

 

Otsu, M., M, Steinberg, C. Ferrand, P. Merida, C. Rebouissou, P. Tigerghien, N. Taylor, F. Candotti, and N. Noraz.  2002.  Reconstitution of

lymphoid development and function in ZAP-70-deficient mice following gene transfer into bone marrow cells.  Blood 100(4):1248-56.

 

Pelosi, M. V. Bartolo, V. Mounier, D. Mege, J.M. Pascussi, E. Dufour, A. Blondel, and O. Acuto.  1999.  Tyrosine 319 in the interdomain B of ZAP-70 is a binding site for the Src homology 2 domain of Lck.  Journal of Biol Chem 274:14229-37.

 

Pivniouk, V., and R.S. Geha.  2000.  The role of SLP-76 and LAT in lymphocyte development.  Current Opinion in Immunology 12:173-178.

 

Taylor, N., et al.  Immunomodulation et immunotherapie.  <http://www.igm.cnrs-mop.fr/pagedesequipes/webNTL.htm>  Accessed 2003 17 Feb.

 

Toyabe, S., A. Watanabe, W. Harada, T. Karasawa, and M. Uchiyama.  2001.  Specific immunoglobulin E responses in ZAP-70-deficient 

 patients are mediated by Syk-dependent T cell receptor signaling.  Immunology 103(2):164-71.

 

Whisler, R., M. Chen, B. Liu, and Y. Newhouse.  1999.  Age-related impairments in TCR/CD3 activation of ZAP-70 are associated with reduced

 tyrosine phosphorylations of zeta chains and  p56lck/p59fyn in human T cells.  Mechanisms of Ageing and Development 111:1:49-66.

 


Davidson Immunology Home Page

 

Davidson College Home Page

 

Questions or Comments, please email Michele Ho