CoChat
Sign Up
Cited by 124Viral Infections and Immunology ResearchCardiac Fibrosis and RemodelingOcular Diseases and Behçet’s Syndrome

Quantitative Analysis of Myocardial Inflammation by Flow Cytometry in Murine Autoimmune Myocarditis

Open access
Afanasyeva, Marina, Georgakopoulos, Dimitrios, Belardi, Diego F., Ramsundar, Amrish C., Barin, Jobert G., Kass, David A., Rose, Noel R.·2004·The American Journal of Pathology
DOI 10.1016/s0002-9440(10)63169-0PMC PMC1613271
Open this paper in CoChat — free accountOpen PDF

Abstract

Inflammation has been increasingly recognized as an important pathological component of heart failure. Existing methods of assessing myocardial infiltrate are labor-intensive and provide data that are difficult to quantify and not representative of the whole heart. As a result, little effort has been made to systematically assess the components of myocardial inflammation. We established an alternative method of quantitative assessment of myocardial inflammation by flow cytometry after enzymatic digestion of hearts to characterize the infiltrate and study the association between inflammation and cardiac function in murine experimental autoimmune myocarditis. The severity of acute myocarditis uniquely correlated with the proportion of neutrophils, but not T cells, B cells, or macrophages. Both acute and chronic phases were characterized by the presence of CD44high (activated) T cells in the heart, whereas T cells trafficking through normal hearts exhibited CD44low phenotype. During the chronic phase, the proportion of CD4+ T cells was associated with increased left-ventricular volumes and deterioration of systolic function, the hallmarks of dilated cardiomyopathy. We conclude that flow cytometry on uniformly digested mouse hearts provides sensitive and reproducible assessment of myocardial infiltrate and can be used to dissect out the specific role of individual immune components from the overall inflammatory response in the heart.

The claims and statistics of this paper haven’t been checked yet.

Run the check — free, no account needed. It reads the open-access text and checks the sources it cites.

A few free checks a day · papers already verified open instantly · results are public

Integrity signals

Registry

Crossref · 10.1016/s0002-9440(10)63169-0

Cites

Statistics

Claims the paper makes

The claims and statistics of this paper haven’t been checked yet.

Run the check — free, no account needed. It reads the open-access text and checks the sources it cites.

A few free checks a day · papers already verified open instantly · results are public

Claims other papers make about this one

Each is checked against this paper’s own text: does it say what the citing paper says it does?

  • A model of EAM at different phases, including normal control (day 0), acute inflammatory (day 14), subacute inflammatory (day 21), and myopathy (day 60), was established.

    “A model of EAM at different phases, including the normal control phase (day 0), acute inflammatory phase (day 14), subacute inflammatory phase (day 21), and myopathy phase (day 60), was established. 11”

    From Single-Cell RNA Sequencing to Dissect the Immunological Network of Autoimmune Myocarditis. (2020) · cites this paper as [11]

    The Flow Cytometric Assessment section compares unimmunized day-0 controls with mice examined on day 15 and days 22–24, and describes approximately day 21 as peak inflammation. The Association between Inflammation and Cardiac Dysfunction section identifies days 58–61 as the chronic phase, with impaired cardiac function. The paper does not establish the claimed four-phase classification: it does not label day 21 subacute or day 60 a myopathy phase, and its early measurement is day 15 rather than day 14. Page numbers were not available in our copy. (LLM, conf=0.88)

    Experimental — accuracy not yet measured

  • Previous studies on immune cells in myocarditis clustered immune cells into major cell types based on flow cytometry data.

    “Previous studies on immune cells in myocarditis clustered immune cells into major cell types based on flow cytometry data. 11”

    From Single-Cell RNA Sequencing to Dissect the Immunological Network of Autoimmune Myocarditis. (2020) · cites this paper as [11]

Self-check

Percentages, sums and reported differences re-computed from the paper’s own numbers.

The self-check needs the paper’s full text.

The citation lists haven’t been fetched yet

References 48 · Cited by 124

Research works printed but not linked: 44

  1. Wolfgram LJ, Beisel KW, Herskowitz A, Rose NR: Variations in the susceptibility to coxsackievirus B3-induced myocarditis among dif- ferent strains of mice. J Immunol 1986, 136:1846 -1852
  2. Hill SL, Afanasyeva M, Rose NR: Autoimmune myocarditis. The Mo- lecular Pathology of Autoimmune Diseases. Edited by CA Bona, AN Theophilopoulos. Ann Arbor, Taylor & Francis, 2002, pp 951-964
  3. Gauntt C, Huber S: Coxsackievirus experimental heart diseases. Front Biosci 2003, 8:E23-E35
  4. Kawai C: From myocarditis to cardiomyopathy: mechanisms of in- flammation and cell death: learning from the past for the future. Circulation 1999, 99:1091-1100
  5. Neu N, Rose NR, Beisel KW, Herskowitz A, Gurri-Glass G, Craig SW: Cardiac myosin induces myocarditis in genetically predisposed mice. J Immunol 1987, 139:3630 -3636
  6. Wang Y, Afanasyeva M, Hill SL, Rose NR: Characterization of murine autoimmune myocarditis induced by self and foreign cardiac myosin. Autoimmunity 1999, 31:151-162
  7. Penninger JM, Neu N, Timms E, Wallace VA, Koh DR, Kishihara K, Pummerer C, Mak TW: The induction of experimental autoimmune myocarditis in mice lacking CD4 or CD8 molecules. J Exp Med 1993, 178:1837-1842
  8. Hirasawa M, Deguchi H, Ukimura A, Kitaura Y: Immunologic interac- tion between infiltrating eosinophils and T lymphocytes in murine spontaneous eosinophilic myocarditis. Int Arch Allergy Immunol 2003, 130:73-81
  9. Sonnenblick EH: Correlation of myocardial ultrastructure and func- tion. Circulation 1968, 38:29 -44
  10. Weber KT: Cardiac interstitium in health and disease: the fibrillar collagen network. J Am Coll Cardiol 1989, 13:1637-1652
  11. Wasowska B, Baldwin WM, Howell DN, Sanfilippo F: The effects of donor-specific blood transfusion enhancement of rat renal allografts on cytotoxic activity and phenotypes of peripheral blood lympho- cytes, splenocytes, and graft-infiltrating cells. Transplantation 1991, 51:451-459
  12. Gavett SH, Chen X, Finkelman F, Wills-Karp M: Depletion of murine CD4ϩ T lymphocytes prevents antigen-induced airway hyperreactiv- ity and pulmonary eosinophilia. Am J Respir Cell Mol Biol 1994, 10:587-593
  13. Zhou YY, Wang SQ, Zhu WZ, Chruscinski A, Kobilka BK, Ziman B, Wang S, Lakatta EG, Cheng H, Xiao RP: Culture and adenoviral infection of adult mouse cardiac myocytes: methods for cellular ge- netic physiology. Am J Physiol 2000, 279:H429 -H436
  14. Fiset C, Clark RB, Larsen TS, Giles WR: A rapidly activating sustained Kϩ current modulates repolarization and excitation-contraction cou- pling in adult mouse ventricle. J Physiol 1997, 504:557-563
  15. Georgakopoulos D, Mitzner WA, Chen CH, Byrne BJ, Millar HD, Hare JM, Kass DA: In vivo murine left ventricular pressure-volume relations by miniaturized conductance micromanometry. Am J Physiol 1998, 274:H1416 -H1422
  16. Georgakopoulos D, Kass D: Minimal force-frequency modulation of inotropy and relaxation of in situ murine heart. J Physiol 2001, 534: 535-545
  17. Shiverick KL, Thomas LL, Alpert NR: Purification of cardiac myosin. Application to hypertrophied myocardium. Biochim Biophys Acta 1975, 393:124 -133
  18. Donermeyer DL, Beisel KW, Allen PM, Smith SC: Myocarditis-induc- ing epitope of myosin binds constitutively and stably to I-Ak on antigen-presenting cells in the heart. J Exp Med 1995, 182:1291- 1300
  19. Glower DD, Spratt JA, Snow ND, Kabas JS, Davis JW, Olsen CO, Tyson GS, Sabiston Jr DC, Rankin JS: Linearity of the Frank-Starling relationship in the intact heart: the concept of preload recruitable stroke work. Circulation 1985, 71:994 -1009
  20. Kass DA, Beyar R: Evaluation of contractile state by maximal ventric- ular power divided by the square of end-diastolic volume. Circulation 1991, 84:1698 -1708
  21. Gaasch WH, Battle WE, Oboler AA, Banas Jr JS, Levine HJ: Left ventricular stress and compliance in man. With special reference to normalized ventricular function curves. Circulation 1972, 45:746 -762
  22. Raff GL, Glantz SA: Volume loading slows left ventricular isovolumic relaxation rate. Evidence of load-dependent relaxation in the intact dog heart. Circ Res 1981, 48:813-824
  23. Afanasyeva M, Wang Y, Kaya Z, Park S, Zilliox MJ, Schofield BH, Hill SL, Rose NR: Experimental autoimmune myocarditis in A/J mice is an interleukin-4-dependent disease with a Th2 phenotype. Am J Pathol 2001, 159:193-203
  24. Afanasyeva M, Wang Y, Kaya Z, Stafford EA, Dohmen KM, Sadighi Akha AA, Rose NR: Interleukin-12 receptor/STAT4 signaling is re- quired for the development of autoimmune myocarditis in mice by an interferon-gamma-independent pathway. Circulation 2001, 104: 3145-3151
  25. Afanasyeva M, Rose NR: Immune mediators in inflammatory heart disease: insights from a mouse model. Eur Heart J Supple 2002, 4:I31-I36
  26. Austyn JM, Hankins DF, Larsen CP, Morris PJ, Rao AS, Roake JA: Isolation and characterization of dendritic cells from mouse heart and kidney. J Immunol 1994, 152:2401-2410
  27. Stinn JL, Taylor MK, Becker G, Nagano H, Hasegawa S, Furakawa Y, Shimizu K, Libby P, Mitchell RN: Interferon-gamma-secreting T-cell populations in rejecting murine cardiac allografts: assessment by flow cytometry. Am J Pathol 1998, 153:1383-1392
  28. Furukawa Y, Mandelbrot DA, Libby P, Sharpe AH, Mitchell RN: As- sociation of B7-1 co-stimulation with the development of graft arterial disease. Studies using mice lacking B7-1, B7-2, or B7-1/B7-2. Am J Pathol 2000, 157:473-484
  29. Huber SA, Mortensen A, Moulton G: Modulation of cytokine expres- sion by CD4ϩ T cells during coxsackievirus B3 infections of BALB/c mice initiated by cells expressing the gamma delta ϩ T-cell receptor. J Virol 1996, 70:3039 -3044
  30. Pummerer C, Berger P, Fruhwirth M, Ofner C, Neu N: Cellular infiltrate, major histocompatibility antigen expression and immunopathogenic mechanisms in cardiac myosin-induced myocarditis. Lab Invest 1991, 65:538 -547
  31. Poon BY, Ward CA, Giles WR, Kubes P: Emigrated neutrophils reg- ulate ventricular contractility via alpha4 integrin. Circ Res 1999, 84: 1245-1251
  32. Poon BY, Ward CA, Cooper CB, Giles WR, Burns AR, Kubes P: Alpha(4)-integrin mediates neutrophil-induced free radical injury to cardiac myocytes. J Cell Biol 2001, 152:857-866
  33. Tepper RI, Coffman RL, Leder P: An eosinophil-dependent mecha- nism for the antitumor effect of interleukin-4. Science 1992, 257:548 - 551
  34. Patella V, de Crescenzo G, Marino I, Genovese A, Adt M, Gleich GJ, Marone G: Eosinophil granule proteins are selective activators of human heart mast cells. J Immunol 1996, 157:1219 -1225
  35. Ramalingam T, Rajan B, Lee J, Rajan TV: Kinetics of cellular re- sponses to intraperitoneal Brugia pahangi infections in normal and immunodeficient mice. Infect Immun 2003, 71:4361-4367
  36. Shapiro HM: Practical Flow Cytometry. Hoboken, John Wiley & Sons, 2003, p 486
  37. Cauley LS, Cauley KA, Shub F, Huston G, Swain SL: Transferable anergy: superantigen treatment induces CD4ϩ T cell tolerance that is reversible and requires CD4ϪCD8Ϫ cells and interferon gamma. J Exp Med 1997, 186:71-81
  38. Azuma T, Takahashi T, Kunisato A, Kitamura T, Hirai H: Human CD4ϩ CD25ϩ regulatory T cells suppress NKT cell functions. Cancer Res 2003, 63:4516 -4520
  39. Eck M, Greiner A, Kandolf R, Schmausser B, Marx A, Muller-Her- melink HK: Active fulminant myocarditis characterized by T-lympho- cytes expressing the gamma-delta T-cell receptor: a new disease entity? Am J Surg Pathol 1997, 21:1109 -1112
  40. Huber SA: Coxsackievirus-induced myocarditis is dependent on dis- tinct immunopathogenic responses in different strains of mice. Lab Invest 1997, 76:691-701
  41. Huber SA, Sartini D, Exley M: Vgamma4(ϩ) T cells promote autoim- mune CD8(ϩ) cytolytic T-lymphocyte activation in coxsackievirus B3-induced myocarditis in mice: role for CD4(ϩ) Th1 cells. J Virol 2002, 76:10785-10790
  42. Smith SC, Allen PM: Myosin-induced acute myocarditis is a T cell- mediated disease. J Immunol 1991, 147:2141-2147
  43. Neu N, Pummerer C, Rieker T, Berger P: T cells in cardiac myosin- induced myocarditis. Clin Immunol Immunopathol 1993, 68:107-110
  44. Grabie N, Delfs MW, Westrich JR, Love VA, Stavrakis G, Ahmad F, Seidman CE, Seidman JG, Lichtman AH: IL-12 is required for differ- entiation of pathogenic CD8ϩ T cell effectors that cause myocarditis. J Clin Invest 2003, 111:671-680

Authors
Afanasyeva, M., Georgakopoulos, D., Belardi, D. F., Ramsundar, A. C., Barin, J. G., Kass, D. A., & Rose, N. R.
Affiliations
  • Johns Hopkins University (US)
  • Johns Hopkins Medicine (US)
Corresponding author
Rose, Noel R.
ORCID
Venue
The American Journal of Pathology
Publisher
Elsevier BV
Published
2004-03
Volume
164
Issue
3
Pages
807-815
ISSN
0002-9440 (ISSN-L)
DOI
10.1016/s0002-9440(10)63169-0
PMC
PMC1613271
OpenAlex
W2160485534
Cited by
124
References
48
Citations by year
2026: 1 · 2025: 8 · 2024: 7 · 2023: 2 · 2022: 1 · 2021: 2 · 2020: 3 · 2019: 6 · 2018: 8 · 2017: 5 · 2016: 8 · 2015: 4 · 2014: 9 · 2013: 7 · 2012: 4
FWCI
3.93
Type
journal article
Language
en
Open access
Licence
www.elsevier.com/tdm/userlicense/1.0/ (tdm)
Locations
  • American Journal Of Pathology (publishedVersion)
  • PubMed Central (publishedVersion)
  • PubMed (publishedVersion)
Funders
  • National Institutes of Health (HL70729)
  • National Heart, Lung, and Blood Institute (HL 67290, R01HL067290, HL 70729, R01HL070729)
  • National Institute of Allergy and Infectious Diseases (AI 51835, R21AI051835)
Topics
  • Health Sciences › Medicine › Cardiology and Cardiovascular Medicine › Viral Infections and Immunology Research
  • Health Sciences › Medicine › Cardiology and Cardiovascular Medicine › Cardiac Fibrosis and Remodeling
  • Health Sciences › Medicine › Ophthalmology › Ocular Diseases and Behçet’s Syndrome
Keywords
myocarditis, autoimmune myocarditis, neutrophils, CD4+ T cells, dilated cardiomyopathy, heart failure, cardiac function, flow cytometry quantification
MeSH terms
Animals, Autoimmune Diseases, B-Lymphocytes, Disease Models, Animal, Female, Flow Cytometry, Lymphocyte Activation, Macrophages, Male, Myocarditis, Neutrophils, Sensitivity and Specificity, T-Lymphocytes, Reproducibility of Results, Hyaluronan Receptors, Mice

About these checks

Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.

Statistics and arithmetic are recomputed, not judged.

Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.

What counts as a claim ›How we measure confidence ›How we check this ›

Are you an author, or do you see a problem with this page? Request a review