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Is the immunohistochemical study of the inflammatory infiltrate helpful in distinguishing villitis of unknown etiology from non-specific infection villitis?
Placenta (2005), 26, 839e841 doi:10.1016/j.placenta.2004.10.012
SHORT COMMUNICATION Is the Immunohistochemical Study of the Inflammatory Infiltrate Helpful in Distinguishing Villitis of Unknown Etiology from Non-specific Infection Villitis? H. Brito, P. Juliano, C. Altemani and A. Altemani* Department of Pathology, Faculty of Medicine, State University of Campinas (UNICAMP), 13084-971 Campinas, Sao Paulo, Brazil Paper accepted 13 October 2004
Keywords: Placenta; Villitis
Villitis is an important placental lesion, which can be caused by specific maternal infections, e.g. rubella, toxoplasmosis, etc, but most of them are of unknown etiology [1]. The clinical significance of villitis depends on the etiology and severity of fetal infection [2] and may lead to intrauterine fetal death, abortion, malformations and fetal growth retardation. The latter has been the major clinical association of villitis of unknown etiology (VUE) [2e4]. Morphologically, infectious villitis may show no specific features and can masquerade as VUE [5]. Both lesions are characterized by chronic inflammatory cells, mainly macrophages and lymphocytes, in the placental villous stroma [2,5]. There are few immunomorphological studies about the immune cells in infectious villitis as well as in VUE [6e9]. Labarrere et al. [6] described that in placentas with VUE the predominant inflammatory cells were activated macrophages expressing class II major histocompatibility complex antigens and helper T cells. Few CD8C T cells and no B cells were detected in the same lesions [6]. These findings contrast with those in villitis caused by maternal infection by Trypanosoma cruzi (Chagas’ disease), in which CD8C cells outnumbered CD4C T cells [7]. The aim of this study was to analyze whether immunohistochemical study of the inflammatory infiltrate in chronic villitis without specific morphological features could distinguish VUE from infectious villitis. All cases of villitis should be reported, particularly in developing countries, where there is a higher incidence of intrauterine infection [10] and the immunohistochemical study could provide objectivity that conventional histological methods lack. * Corresponding author. FCM-UNICAMP, Department of Anatomic Pathology, Caixa Postal 6111, 13084-971 Campinas, Sao Paulo, Brazil. Tel./fax: C55 19 3289 3897. E-mail address: [email protected] (A. Altemani). 0143e4004/$esee front matter
Fifteen placentas with villitis characterized by chronic nongranulomatous inflammatory infiltrate of the villous stroma were examined. Villitis was caused by T. cruzi in 4 cases, Toxoplasma gondii in 3 and of unknown etiology in 8. All placentas were from singleton pregnancies with livebirths but for one stillbirth (toxoplasmosis). Four of the infectious villitis cases were included in a previous report [7]. In all cases of villitis by T. cruzi and T. gondii there was maternal and/or infant evidence of infection and the parasites were identified in the placentas using immunohistochemical methods. In placentas with VUE neither the mother nor the infant had clinical or serological evidence of infection by the usual agents that are investigated during pregnancy (TORCH group, Human
Table 1. Immunohistochemical characterization of the inflammatory cells in placentas with villitis caused by Chagas’ disease, toxoplasmosis and of unknown etiology Monoclonal antibodya UCHL-1 OPD4 CD8 Ratio OPD4:CD8 NK1 L 26 HAM 56 MAC 387 CD15
Chagas [Mean (SD)b]
Toxoplasmosis [Mean (SD)b]
VUE [Mean (SD)C]
91 21 79 28
(G16) (G11) (G11) (G16)
71 26 74 37
(G31) (G14) (G14) (G23)
93 26 74 36
(G11) (G11) (G11) (G20)
03 00 52 8 00
(G05) (G01) (G13) (G16) (G00)
11 00 58 07 10
(G12) (G01) (G08) (G12) (G17)
09 00 48 06 01
(G11) (G00) (G20) (G11) (G02)
a UCHL-1: lymphocyte cells; OPD4: CD4C T lymphocytes; CD8: CD8C T lymphocytes; NK1: natural killer cells; L26: B cells; HAM 56: macrophages; MAC 387: monocytes; CD15: neutrophils. b Mean and standard derivation (SD) of the percentage of positive cells for three high-power fields: at least 100 cells counted for each case.
Ó 2005 Published by Elsevier Ltd.
840
Placenta (2005), Vol. 26
Figure 1. Villitis of unknown etiology. (A) CD8C T lymphocytes. (B) Same area as A showing CD4C T cells. Note that the CD8C T lymphocytes outnumber the CD4C T cells (Envision System Technique, DAKO, SA, Denmark). Bar Z 40 mm.
immunodeficiency virus and hepatitis B virus). Paraffin sections of all placentas were stained with haematoxylin and eosin and by immunohistochemistry according to the Envision System Technique (DAKO, SA, Denmark) [11] using monoclonal antibodies (DAKO, SA, Denmark) specific for leukocytes CD45Rb (LCA clone PD7/26/16 C 2B11), T cells e CD45RO (clone UCHL-1), B cells e CD20 (clone L26), T helper lymphocytes CD4C (clone OPD4), T lymphocytes CD8C (clone C8/144B), natural killer cells (clone NK1), neutrophils CD15C (clone C3De1), monocytes (clone MAC 387) and macrophages (clone HAM 56). Any CD45ROpositive cell was used as the criterion for villitis. The positive inflammatory cells in affected villi were then counted in three different high-power fields. The relative numbers of leukocytes were considered in relation to CD45Rb-positive cells, except for CD4/OPD4C and CD8C T lymphocytes, the percentage of which was calculated using the number of CD45ROC cells as denominator. In all cases of villitis (infectious and of unknown etiology), the inflammatory infiltrate was composed mainly of HAM 56C macrophages, T lymphocytes, and a few natural killer cells (Table 1). Among T cells, CD8C cells outnumbered CD4C cells in all placentas. CD4C:CD8C ratios ranged from 0.04 to 0.38 in villitis by T. cruzi, from 0.11 to 0.56 in T. gondii infection and from 0.13 to 0.67 in VUE (Figure 1). B cells were absent. Monocytes were present in small numbers in some cases of villitis of unknown as well as of infectious etiology (Table 1).
These cells were usually adhered to inflamed villi with trophoblastic necrosis. Few CD15C neutrophils were detected in some villitis by T. gondii. Placental villitis is a histological finding that should be mentioned in a pathological report since the clinical implications vary according to the etiologic agent if known. Our findings showed that immunohistochemical study of the inflammatory infiltrate was not helpful in distinguishing VUE from non-specific infectious villitis by known agents. In both cases lesions were constituted mainly by HAM 56C macrophages and CD8C T cells. In relation to VUE two hypotheses have been considered for its etiology: an unidentified infection and an immunologic reaction due to maternal rejection of paternal antigens exposed within villi [4,12,13]. Among the infections, virus has been considered a possible cause of VUE because the agent is one of the most difficult for diagnosis and may cause chronic villitis without illness in the neonate [5,14,15]. Although cases of viral villitis were not used for comparison in our study, the similarity of the cellular composition of the inflammatory infiltrate in the two studied groups (VUE and infectious villitis) supports the hypothesis that VUE could result from an infectious cause. In addition we also could conclude that HAM 56C macrophages and CD8C T cells probably predominate in all forms of villitis, independent of its etiology, since villitis by unusual agents (such as T. cruzi and T. gondii) as well as VUE present this kind of cellular composition.
ACKNOWLEDGMENTS Supported by FAPESP (process number 02/00755-7).
REFERENCES [1] Knox WF, Fox H. Villitis of unknown aetiology: its incidence and significance in placentae from a British population. Placenta 1984;5: 395e402.
[2] Fox H. Infections and inflammatory lesions of the placenta. In: Fox H, editor. Pathology of the placenta, 2nd ed. London: Saunders; 1997, p. 294e343. [3] Altshuler G, Russell P. The human placental villitides: a review of chronic intrauterine infection. Curr Top Pathol 1975;60:63e112.
Brito et al.: Immunohistochemical Study of the Inflammatory Infiltrate [4] Labarrere C, Althabe O, Telenta M. Chronic villitis of unknown aetiology in placentae of idiopathic small for gestational age infants. Placenta 1982;3:309e18. [5] Benirschke K, Kaufmann P. Infectious diseases. In: Bernirschke K, Kaufmann P, editors. Pathology of the human placenta, 2nd ed. New York: Springer; 2000, p. 591e684. [6] Labarrere CA, McIntyre JA, Faulk WP. Immunohistologic evidence that villitis in human normal term placentas is an immunologic lesion. Am J Obstet Gynecol 1990;162:515e22. [7] Altemani A, Bittencourt AL, Lana AM. Immunohistochemical characterization of the inflammatory infiltrate in placental Chagas’ disease: a qualitative and quantitative analysis. Am J Trop Med Hyg 2000;62(3): 319e24. [8] Schwartz DA, Khan R, Stoll B. Characterization of the fetal inflammatory response to cytomegalovirus placentitis. Arch Pathol Lab Med 1992;116:21e7. [9] Labarrere CA, Faulk WP. MHC class II reactivity of human villous trophoblast in chronic inflammation of unestablished etiology. Transplantation 1990;50(5):812e6.
841 [10] Naeye RL, Blanc WA. Relation of poverty and race to antenatal infection. N Engl J Med 1970;283:555e60. [11] Sabattini E, Bisgaard K, Ascani S, Poggi S, Piccioli M, Ceccarelli C, et al. The EnVisionÔC system: a new immunohistochemical method for diagnostics and research. Critical comparison with the APAAP, ChemMateÔ, CSA, LABC, and SABC techniques. J Clin Pathol 1998; 51(7):506e11. [12] Labarrere CA, Carson SD, Faulk SP. Tissue factor in chronic villitis of unestablished etiology. J Reprod Immunol 1991;19:225e35. [13] Redline RW, Patterson P. Villitis of unknown etiology is associated with major infiltration of fetal tissues by maternal inflammatory cells. Am J Pathol 1993;143:473e9. [14] Garcia AGP, Basso NG, Fonseca MEF, Outanni HN. Congenital ECHO virus infection e morphological and virological study of fetal and placental tissue. J Pathol 1990;160:123e7. [15] Garcia AGP, Basso NG, Fonseca MEF, Zuardi JAT, Outanni HN. Enteroviruses associated placental morphology: a light, virological, electron microscopic and immunologic study. Placenta 1991;12: 533e47.
SHORT COMMUNICATION Is the Immunohistochemical Study of the Inflammatory Infiltrate Helpful in Distinguishing Villitis of Unknown Etiology from Non-specific Infection Villitis? H. Brito, P. Juliano, C. Altemani and A. Altemani* Department of Pathology, Faculty of Medicine, State University of Campinas (UNICAMP), 13084-971 Campinas, Sao Paulo, Brazil Paper accepted 13 October 2004
Keywords: Placenta; Villitis
Villitis is an important placental lesion, which can be caused by specific maternal infections, e.g. rubella, toxoplasmosis, etc, but most of them are of unknown etiology [1]. The clinical significance of villitis depends on the etiology and severity of fetal infection [2] and may lead to intrauterine fetal death, abortion, malformations and fetal growth retardation. The latter has been the major clinical association of villitis of unknown etiology (VUE) [2e4]. Morphologically, infectious villitis may show no specific features and can masquerade as VUE [5]. Both lesions are characterized by chronic inflammatory cells, mainly macrophages and lymphocytes, in the placental villous stroma [2,5]. There are few immunomorphological studies about the immune cells in infectious villitis as well as in VUE [6e9]. Labarrere et al. [6] described that in placentas with VUE the predominant inflammatory cells were activated macrophages expressing class II major histocompatibility complex antigens and helper T cells. Few CD8C T cells and no B cells were detected in the same lesions [6]. These findings contrast with those in villitis caused by maternal infection by Trypanosoma cruzi (Chagas’ disease), in which CD8C cells outnumbered CD4C T cells [7]. The aim of this study was to analyze whether immunohistochemical study of the inflammatory infiltrate in chronic villitis without specific morphological features could distinguish VUE from infectious villitis. All cases of villitis should be reported, particularly in developing countries, where there is a higher incidence of intrauterine infection [10] and the immunohistochemical study could provide objectivity that conventional histological methods lack. * Corresponding author. FCM-UNICAMP, Department of Anatomic Pathology, Caixa Postal 6111, 13084-971 Campinas, Sao Paulo, Brazil. Tel./fax: C55 19 3289 3897. E-mail address: [email protected] (A. Altemani). 0143e4004/$esee front matter
Fifteen placentas with villitis characterized by chronic nongranulomatous inflammatory infiltrate of the villous stroma were examined. Villitis was caused by T. cruzi in 4 cases, Toxoplasma gondii in 3 and of unknown etiology in 8. All placentas were from singleton pregnancies with livebirths but for one stillbirth (toxoplasmosis). Four of the infectious villitis cases were included in a previous report [7]. In all cases of villitis by T. cruzi and T. gondii there was maternal and/or infant evidence of infection and the parasites were identified in the placentas using immunohistochemical methods. In placentas with VUE neither the mother nor the infant had clinical or serological evidence of infection by the usual agents that are investigated during pregnancy (TORCH group, Human
Table 1. Immunohistochemical characterization of the inflammatory cells in placentas with villitis caused by Chagas’ disease, toxoplasmosis and of unknown etiology Monoclonal antibodya UCHL-1 OPD4 CD8 Ratio OPD4:CD8 NK1 L 26 HAM 56 MAC 387 CD15
Chagas [Mean (SD)b]
Toxoplasmosis [Mean (SD)b]
VUE [Mean (SD)C]
91 21 79 28
(G16) (G11) (G11) (G16)
71 26 74 37
(G31) (G14) (G14) (G23)
93 26 74 36
(G11) (G11) (G11) (G20)
03 00 52 8 00
(G05) (G01) (G13) (G16) (G00)
11 00 58 07 10
(G12) (G01) (G08) (G12) (G17)
09 00 48 06 01
(G11) (G00) (G20) (G11) (G02)
a UCHL-1: lymphocyte cells; OPD4: CD4C T lymphocytes; CD8: CD8C T lymphocytes; NK1: natural killer cells; L26: B cells; HAM 56: macrophages; MAC 387: monocytes; CD15: neutrophils. b Mean and standard derivation (SD) of the percentage of positive cells for three high-power fields: at least 100 cells counted for each case.
Ó 2005 Published by Elsevier Ltd.
840
Placenta (2005), Vol. 26
Figure 1. Villitis of unknown etiology. (A) CD8C T lymphocytes. (B) Same area as A showing CD4C T cells. Note that the CD8C T lymphocytes outnumber the CD4C T cells (Envision System Technique, DAKO, SA, Denmark). Bar Z 40 mm.
immunodeficiency virus and hepatitis B virus). Paraffin sections of all placentas were stained with haematoxylin and eosin and by immunohistochemistry according to the Envision System Technique (DAKO, SA, Denmark) [11] using monoclonal antibodies (DAKO, SA, Denmark) specific for leukocytes CD45Rb (LCA clone PD7/26/16 C 2B11), T cells e CD45RO (clone UCHL-1), B cells e CD20 (clone L26), T helper lymphocytes CD4C (clone OPD4), T lymphocytes CD8C (clone C8/144B), natural killer cells (clone NK1), neutrophils CD15C (clone C3De1), monocytes (clone MAC 387) and macrophages (clone HAM 56). Any CD45ROpositive cell was used as the criterion for villitis. The positive inflammatory cells in affected villi were then counted in three different high-power fields. The relative numbers of leukocytes were considered in relation to CD45Rb-positive cells, except for CD4/OPD4C and CD8C T lymphocytes, the percentage of which was calculated using the number of CD45ROC cells as denominator. In all cases of villitis (infectious and of unknown etiology), the inflammatory infiltrate was composed mainly of HAM 56C macrophages, T lymphocytes, and a few natural killer cells (Table 1). Among T cells, CD8C cells outnumbered CD4C cells in all placentas. CD4C:CD8C ratios ranged from 0.04 to 0.38 in villitis by T. cruzi, from 0.11 to 0.56 in T. gondii infection and from 0.13 to 0.67 in VUE (Figure 1). B cells were absent. Monocytes were present in small numbers in some cases of villitis of unknown as well as of infectious etiology (Table 1).
These cells were usually adhered to inflamed villi with trophoblastic necrosis. Few CD15C neutrophils were detected in some villitis by T. gondii. Placental villitis is a histological finding that should be mentioned in a pathological report since the clinical implications vary according to the etiologic agent if known. Our findings showed that immunohistochemical study of the inflammatory infiltrate was not helpful in distinguishing VUE from non-specific infectious villitis by known agents. In both cases lesions were constituted mainly by HAM 56C macrophages and CD8C T cells. In relation to VUE two hypotheses have been considered for its etiology: an unidentified infection and an immunologic reaction due to maternal rejection of paternal antigens exposed within villi [4,12,13]. Among the infections, virus has been considered a possible cause of VUE because the agent is one of the most difficult for diagnosis and may cause chronic villitis without illness in the neonate [5,14,15]. Although cases of viral villitis were not used for comparison in our study, the similarity of the cellular composition of the inflammatory infiltrate in the two studied groups (VUE and infectious villitis) supports the hypothesis that VUE could result from an infectious cause. In addition we also could conclude that HAM 56C macrophages and CD8C T cells probably predominate in all forms of villitis, independent of its etiology, since villitis by unusual agents (such as T. cruzi and T. gondii) as well as VUE present this kind of cellular composition.
ACKNOWLEDGMENTS Supported by FAPESP (process number 02/00755-7).
REFERENCES [1] Knox WF, Fox H. Villitis of unknown aetiology: its incidence and significance in placentae from a British population. Placenta 1984;5: 395e402.
[2] Fox H. Infections and inflammatory lesions of the placenta. In: Fox H, editor. Pathology of the placenta, 2nd ed. London: Saunders; 1997, p. 294e343. [3] Altshuler G, Russell P. The human placental villitides: a review of chronic intrauterine infection. Curr Top Pathol 1975;60:63e112.
Brito et al.: Immunohistochemical Study of the Inflammatory Infiltrate [4] Labarrere C, Althabe O, Telenta M. Chronic villitis of unknown aetiology in placentae of idiopathic small for gestational age infants. Placenta 1982;3:309e18. [5] Benirschke K, Kaufmann P. Infectious diseases. In: Bernirschke K, Kaufmann P, editors. Pathology of the human placenta, 2nd ed. New York: Springer; 2000, p. 591e684. [6] Labarrere CA, McIntyre JA, Faulk WP. Immunohistologic evidence that villitis in human normal term placentas is an immunologic lesion. Am J Obstet Gynecol 1990;162:515e22. [7] Altemani A, Bittencourt AL, Lana AM. Immunohistochemical characterization of the inflammatory infiltrate in placental Chagas’ disease: a qualitative and quantitative analysis. Am J Trop Med Hyg 2000;62(3): 319e24. [8] Schwartz DA, Khan R, Stoll B. Characterization of the fetal inflammatory response to cytomegalovirus placentitis. Arch Pathol Lab Med 1992;116:21e7. [9] Labarrere CA, Faulk WP. MHC class II reactivity of human villous trophoblast in chronic inflammation of unestablished etiology. Transplantation 1990;50(5):812e6.
841 [10] Naeye RL, Blanc WA. Relation of poverty and race to antenatal infection. N Engl J Med 1970;283:555e60. [11] Sabattini E, Bisgaard K, Ascani S, Poggi S, Piccioli M, Ceccarelli C, et al. The EnVisionÔC system: a new immunohistochemical method for diagnostics and research. Critical comparison with the APAAP, ChemMateÔ, CSA, LABC, and SABC techniques. J Clin Pathol 1998; 51(7):506e11. [12] Labarrere CA, Carson SD, Faulk SP. Tissue factor in chronic villitis of unestablished etiology. J Reprod Immunol 1991;19:225e35. [13] Redline RW, Patterson P. Villitis of unknown etiology is associated with major infiltration of fetal tissues by maternal inflammatory cells. Am J Pathol 1993;143:473e9. [14] Garcia AGP, Basso NG, Fonseca MEF, Outanni HN. Congenital ECHO virus infection e morphological and virological study of fetal and placental tissue. J Pathol 1990;160:123e7. [15] Garcia AGP, Basso NG, Fonseca MEF, Zuardi JAT, Outanni HN. Enteroviruses associated placental morphology: a light, virological, electron microscopic and immunologic study. Placenta 1991;12: 533e47.