JOURNAL OF INTERFERON RESEARCH Volume 3, Number 1, 1983 Mary Ann Lieber!, Inc., Publishers Pp. 45-52
Circulating Interferon in Cytomegalovirus Infected Bone-Marrow-Transplant Recipients and in Infants with Congenital Cytomegalovirus Disease A.
RHODES-FEUILLETTE,1 M. CANIVET,1 H. CHAMPSAUR,2 E. GLUCKMAN,3 M.C. MAZERON,4 and J. PERIES1
ABSTRACT a study concerning five CMV-infected bone-marrow-transplant recipients, five congenital CMV diseases and appropriate controls, presence of high levels of circulating interferon (IFN) was demonstrated exclusively during the course of CMV disease. This interferon was predominantly "immune" or gamma interferon (7-IFN). These results suggest that during CMV disease the interferon compartment of the immune response is
In
modified.
INTRODUCTION
CMV
infection
is
common
among adults and
newborns, but varies from asymptomatic infection
to disseminated disease with immune deficiency and symptoms which may lead to death. CMV disease and the immune status have been well studied in man as in mice experimentally in-
fected with mouse cytomegalovirus (MCMV). Yet, some discrepancy appears concerning the production of interferon (IFN) during CMV infection. Some investigators report a detection of IFN production in several in vitro systems.'1"4' Others report the contrary.'5 6) In view of several recent publications dealing with the in vitro effects of various inductors of leukocyte interferons during CMV disease in humans, as well as in mice, the hypothesis of a different status for a- and 7-IFN production can be considered.'79' For instance, an abnormal state of CMV infected mononuclear cells could result in a different effect on each species of IFN: a deficiency in a-IFN'7' and a modification in 7-IFN.'8'9' In order to shed light on the IFN mechanisms involved in vivo during CMV disease, and since until our present work only one case report had stated the presence of serum-IFN in a CMV infection,'101 we decided to study circulating IFN in CMV-infected patients.
MATERIALS AND METHODS Serum-IFN-like activity was investigated in 10 CMV-infected patients, among which five were adult medullary-graft-recipients and the five others were infants with congenital CMV disease.
'Experimental Oncology Department, U. 107 INSERM, LOI CNRS, Institut de Recherches sur les Maladies du Sang, Hôpital Saint-Louis, 75475 Paris Cedex 10; laboratory of Virology, Hôpital du Kremlin Bicêtre, 94 Kremlin Bicêtre; 3Bone Marrow Transplant Unit. Centre Hayem, Hôpital Saint-Louis, 75475 Paris Cedex 10; "Central Laboratory of Bacteriology and Virology, Hôpital Saint-Louis, 75475 Paris Cedex 10. 91
RHODES-FEUILLETTE ET AL. In all cases, CMV infection was confirmed by viral isolations, anti-CMV antibodies and typical clinical disorders. Bone-marrow recipients had received high dose cyclophosphamide and total body irradiation with lung protection. Circulating IFN-levels were monitored at least bimonthly during seven months postgraft while patients were receiving methotrexate and additional immunosuppressive medication such as methyl-prednisolone and antithymocyte globulin (ALS) for graft-versus-host disease (GVH). CMV disease occurred in five of the patients, with CMV viremia, anti-CMV antibodies, and typical symptoms. This study group (Cases 1-5) was completed by two graft-recipients, considered as controls, who developed no viral disease. Included in this study are five congenital CMV diseases with CMV viruria, anti-CMV IgM antibodies, and typical symptoms (e.g., microcephaly, hepatosplenomegaly). IFN-like activity was tested in sera positive for IgG antibodies towards CMV. In Cases 6-9, samples taken before IgGs to CMV had appeared were also tested. This study group (Cases 6-10) was completed by six healthy infants for which two samples of serum were assayed. Titration of IFN-like activity in sera was done by a modification of the micromethod employed by Havell and Vil&k,'11' using amniotic cell line AV3, with vesicular stomatitis virus (VSV) as challenge virus. Briefly, 30,000 AV3 cells in 0.1 ml McCoy + 10% FBS were seeded in each well of microtiter plates (Falcon 3040, Microtest II tissue culture plates). Cells were cultured 24 h at 37°C in a humidified atmosphere of 5% C02 in air. Then four replicates of serial two-fold dilutions of sera were added to the wells. Twenty-four hours later, sera were washed out before addition of VSV. The IFN-like titer was expressed as the reciprocal of the highest dilution of assayed sera which protected at least 50% of the cell monolayer from the cytopathic effect (CPE) induced by 1000 CPED50 VSV. To minimize subjective interpretation of the degree of protection against VSV-CPE, titrations including reference leucocyte IFN (Laboratory of Professor Kishida, Kyoto Prefectorial University, Kyoto, Japan) and sera from healthy blood donors, were blindly read by two investigators. Results expressed in u/ml were standardized to reference human leukocyte IFN. In study-sera, anti-VSV activity was further characterized as IFN by the following standard tests:
(1) Trypsin digestion, 2 h at 37°C, followed by assay of treated and nontreated sera on AV3 cells. (2) Protection of AV3 cells from the CPE of a virus other than VSV: EMC (encephalomyocarditis virus, kindly provided by C. Chany) (3) Simultaneous titration on heterologous (mouse) and homologous (human) cells. In
an
effort
to
determine the
IFN-species,
those
sera
available
were
submitted to the
following
tests:
(1) Dialysis at pH 2, during 48 h, followed by assay of treated and nontreated sera on AV3 cells as
well
bovine MDBK cells.'121 Seroneutralization. (2) Samples mixed 1:1 with a 1/60 dilution of specific antibody were incubated 1 h at 37°C and 2 h at +4°C. Specific antibodies were provided, respectively, for a-IFN by C. Chany (Hôpital Saint-Vincent-de-Paul, Paris) and for 7-IFN by S. Baron (University of Texas Medical Branch, Galveston, TX) and by E. Falcoff (Hôpital Curie, Paris). Similarly treated controls included the previously described human reference leukocyte a-IFN and a semipurified human 7-IFN provided by D. Fradelizi (Laboratory of J. Dausset, Hôpital Saint-Louis, Paris). as on
RESULTS
Circulating IFN in medullary-graft recipients: Cases
1-5
Whereas no IFN-like activity was detected in sera from 40 healthy adult controls, in all seven patients followed post-graft, including those two who developed no viral disease, this type of activity was found in several sera. High levels of IFN-like activity were found only in the course of CMV disease (Cases 1-5). Other-
92
SERUM-IFN IN CMV DISEASE
wise transitory and low levels (
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SERUM-IFN IN CMV DISEASE CMV disease, whereas only transitory and low levels of serum-IFN
graft.
otherwise detected post-
cases studied, pH 2-sensitivity was found among several of the all with sera Moreover, pH 2-sensitive IFN were shown to offer little on no antiviral acbovine MDBK cells (
Circulating Interferon in Cytomegalovirus Infected Bone-Marrow-Transplant Recipients and in Infants with Congenital Cytomegalovirus Disease A.
RHODES-FEUILLETTE,1 M. CANIVET,1 H. CHAMPSAUR,2 E. GLUCKMAN,3 M.C. MAZERON,4 and J. PERIES1
ABSTRACT a study concerning five CMV-infected bone-marrow-transplant recipients, five congenital CMV diseases and appropriate controls, presence of high levels of circulating interferon (IFN) was demonstrated exclusively during the course of CMV disease. This interferon was predominantly "immune" or gamma interferon (7-IFN). These results suggest that during CMV disease the interferon compartment of the immune response is
In
modified.
INTRODUCTION
CMV
infection
is
common
among adults and
newborns, but varies from asymptomatic infection
to disseminated disease with immune deficiency and symptoms which may lead to death. CMV disease and the immune status have been well studied in man as in mice experimentally in-
fected with mouse cytomegalovirus (MCMV). Yet, some discrepancy appears concerning the production of interferon (IFN) during CMV infection. Some investigators report a detection of IFN production in several in vitro systems.'1"4' Others report the contrary.'5 6) In view of several recent publications dealing with the in vitro effects of various inductors of leukocyte interferons during CMV disease in humans, as well as in mice, the hypothesis of a different status for a- and 7-IFN production can be considered.'79' For instance, an abnormal state of CMV infected mononuclear cells could result in a different effect on each species of IFN: a deficiency in a-IFN'7' and a modification in 7-IFN.'8'9' In order to shed light on the IFN mechanisms involved in vivo during CMV disease, and since until our present work only one case report had stated the presence of serum-IFN in a CMV infection,'101 we decided to study circulating IFN in CMV-infected patients.
MATERIALS AND METHODS Serum-IFN-like activity was investigated in 10 CMV-infected patients, among which five were adult medullary-graft-recipients and the five others were infants with congenital CMV disease.
'Experimental Oncology Department, U. 107 INSERM, LOI CNRS, Institut de Recherches sur les Maladies du Sang, Hôpital Saint-Louis, 75475 Paris Cedex 10; laboratory of Virology, Hôpital du Kremlin Bicêtre, 94 Kremlin Bicêtre; 3Bone Marrow Transplant Unit. Centre Hayem, Hôpital Saint-Louis, 75475 Paris Cedex 10; "Central Laboratory of Bacteriology and Virology, Hôpital Saint-Louis, 75475 Paris Cedex 10. 91
RHODES-FEUILLETTE ET AL. In all cases, CMV infection was confirmed by viral isolations, anti-CMV antibodies and typical clinical disorders. Bone-marrow recipients had received high dose cyclophosphamide and total body irradiation with lung protection. Circulating IFN-levels were monitored at least bimonthly during seven months postgraft while patients were receiving methotrexate and additional immunosuppressive medication such as methyl-prednisolone and antithymocyte globulin (ALS) for graft-versus-host disease (GVH). CMV disease occurred in five of the patients, with CMV viremia, anti-CMV antibodies, and typical symptoms. This study group (Cases 1-5) was completed by two graft-recipients, considered as controls, who developed no viral disease. Included in this study are five congenital CMV diseases with CMV viruria, anti-CMV IgM antibodies, and typical symptoms (e.g., microcephaly, hepatosplenomegaly). IFN-like activity was tested in sera positive for IgG antibodies towards CMV. In Cases 6-9, samples taken before IgGs to CMV had appeared were also tested. This study group (Cases 6-10) was completed by six healthy infants for which two samples of serum were assayed. Titration of IFN-like activity in sera was done by a modification of the micromethod employed by Havell and Vil&k,'11' using amniotic cell line AV3, with vesicular stomatitis virus (VSV) as challenge virus. Briefly, 30,000 AV3 cells in 0.1 ml McCoy + 10% FBS were seeded in each well of microtiter plates (Falcon 3040, Microtest II tissue culture plates). Cells were cultured 24 h at 37°C in a humidified atmosphere of 5% C02 in air. Then four replicates of serial two-fold dilutions of sera were added to the wells. Twenty-four hours later, sera were washed out before addition of VSV. The IFN-like titer was expressed as the reciprocal of the highest dilution of assayed sera which protected at least 50% of the cell monolayer from the cytopathic effect (CPE) induced by 1000 CPED50 VSV. To minimize subjective interpretation of the degree of protection against VSV-CPE, titrations including reference leucocyte IFN (Laboratory of Professor Kishida, Kyoto Prefectorial University, Kyoto, Japan) and sera from healthy blood donors, were blindly read by two investigators. Results expressed in u/ml were standardized to reference human leukocyte IFN. In study-sera, anti-VSV activity was further characterized as IFN by the following standard tests:
(1) Trypsin digestion, 2 h at 37°C, followed by assay of treated and nontreated sera on AV3 cells. (2) Protection of AV3 cells from the CPE of a virus other than VSV: EMC (encephalomyocarditis virus, kindly provided by C. Chany) (3) Simultaneous titration on heterologous (mouse) and homologous (human) cells. In
an
effort
to
determine the
IFN-species,
those
sera
available
were
submitted to the
following
tests:
(1) Dialysis at pH 2, during 48 h, followed by assay of treated and nontreated sera on AV3 cells as
well
bovine MDBK cells.'121 Seroneutralization. (2) Samples mixed 1:1 with a 1/60 dilution of specific antibody were incubated 1 h at 37°C and 2 h at +4°C. Specific antibodies were provided, respectively, for a-IFN by C. Chany (Hôpital Saint-Vincent-de-Paul, Paris) and for 7-IFN by S. Baron (University of Texas Medical Branch, Galveston, TX) and by E. Falcoff (Hôpital Curie, Paris). Similarly treated controls included the previously described human reference leukocyte a-IFN and a semipurified human 7-IFN provided by D. Fradelizi (Laboratory of J. Dausset, Hôpital Saint-Louis, Paris). as on
RESULTS
Circulating IFN in medullary-graft recipients: Cases
1-5
Whereas no IFN-like activity was detected in sera from 40 healthy adult controls, in all seven patients followed post-graft, including those two who developed no viral disease, this type of activity was found in several sera. High levels of IFN-like activity were found only in the course of CMV disease (Cases 1-5). Other-
92
SERUM-IFN IN CMV DISEASE
wise transitory and low levels (
O O N
N
o N
_«
14
14
14
14
Q, O. u-
e c C u o o q 14 14 c e C
DC X
14
14
B C O O O C
c c e
í~ + ?-1
Q
zz
2
14
14
14
14
14
14
CCCCC O O O O O ccccc
+ + 8 a
0 ?-'
cl>
0û z z
14
14
14
14
14
c
c
c c c
14
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o o o o o o c c
c c
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+ + û û
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a a
Z Z
;-
£ in
oo
o o *t oo
•s
u
îl s g
CTn
O
a
96
8 Z to
Q Z
SERUM-IFN IN CMV DISEASE CMV disease, whereas only transitory and low levels of serum-IFN
graft.
otherwise detected post-
cases studied, pH 2-sensitivity was found among several of the all with sera Moreover, pH 2-sensitive IFN were shown to offer little on no antiviral acbovine MDBK cells (
