key: cord-0816442-x7enb2z6
authors: Vassão, R. C.; Mello, I. G. C.; Pereira, C. A.
title: Role of macrophages, interferon gamma and procoagulant activity in the resistance of genetic heterogeneous mouse populations to mouse hepatitis virus infection
date: 1994
journal: Arch Virol
DOI: 10.1007/bf01309475
sha: fc1c24a1da87324d4bdc3f10334cda3b0f596b6f
doc_id: 816442
cord_uid: x7enb2z6

Genetic heterogeneous mouse populations selected for high (H(III)) and low (L(III)) antibody response were used to study some aspects of mouse hepatitis virus 3 (MHV3) infection, such as the resistance pattern, virus replication in the liver and peritoneal exudate or in cultured peritoneal macrophages, the interferon (IFN) synthesis in the serum and peritoneal exudate and the procoagulant activity (PCA) of the peritoneal exudate (PEC) and spleen cells (SC). The H(III) mice, when compared to their L(III) mice counterparts, were susceptible to MHV3 infection showing higher virus titres in the liver and peritoneal exudate, comparable IFN alpha/beta or IFN gamma titres in the peritoneal exudate or in the serum, and higher levels of PCA of PEC and SC. A higher virus titre was detected in the supernatants of H(III) mouse macrophages infected with MHV3. The activation of H(III) mouse macrophages with LPS, IFN alpha/beta or IFN gamma, in contrast to that of L(III) mouse macrophages, did not induce an antiviral effect with partial restriction of the MHV3 replication. The LPS antiviral activity was shown to be partially exerted by IFN alpha/beta synthesis. The IFN gamma was shown to be more effective in inducing an antiviral state in L(III) macrophages, when compared to IFN alpha/beta. The data obtained are consistent with the notion that the resistance mechanisms to the MHV3 infection involve the PCA and the sensitivity of macrophages to IFN.

The mouse hepatitis virus (MHV) group of coronaviruses is widespread, occurring endemically in mouse colonies, many of which have antibodies against MHV [5, 10, 17, 21, 25] . The MHV3 was isolated by Dick et al. [5] and has been used as a model of viral infection in which resistance is dependent on the genetic background of the mouse strain [1, 6, 8, [13] [14] [15] 24] . Genetic homogeneous A/J mice have been reported to be resistant to MHV3 infection because R.C. Vassgo et al. they develop only a mild disease which disappears 4 to 6 days later. Several other genetic homogeneous mouse strains, such as the BALB/c, have been reported to be susceptible because they develop an acute hepatitis after MHV3 infection and die 3 to 4 days later.

The expression of a monokine with procoagulant activity (PCA), the antiviral state induced by interferon (IFN) and the virus replication in target cells have been implicated in the resistance/susceptibility of these genetic homogeneous mouse strains to the MHV3 infection [1, 7, 12, 18, 24] . We have shown that resistance to MHV3 infection in these genetic homogeneous mouse populations can be a consequence of a T-cell-dependent mechanism, in which the production of IFN gamma and the sensitivity of macrophages to IFN gamma play an essential role 1-13 -15] .

The use of genetic heterogeneous lines of mice, which can be more representative of a natural population since inbreeding was avoided during the selective process, permits a further approach to the study of resistance mechanisms involved in the MHV3 infection. Mice selected for high (H) and low (L) responsiveness, were obtained by bidirectional selective breeding of high or low antibody responder lines of mice and the effect of the polygenic regulation of responsiveness to selection antigens is essentially non-specific. The same group of genes regulates the antibody response to many complex immunogens unrelated with those used during the selective breeding, the antibody response being, as far it has been studied, a consequence of a diversity of immune functions, mainly the regulatory role of the macrophage [2, 3, 11, 22, 23, 26] .

H and L mice of the selection III (H~u and Lm) obtained by several selective breeding experiments based on responsiveness to the flagellar antigen of Salmonellae [22] were used in this study with the aim to investigate aspects of the MHV3 infection that could further elucidate the mechanisms involved in the resistance/susceptibility to this virus infection.

High and low antibody responder mice, 8 to 12-weeks old, from the selection III (Hn~ and Lm), obtained by selective breeding based on responsiveness to the flagellar antigen of Salmonellae [22] , from the Laboratorio de Imunogenetica, Instituto Butantan, were used in the experiments. Certain animals were periodically sacrificed, and the peritoneal exudate and liver tissue obtained. These were ground, resuspended in 2ml of RPMI 1640 medium containing 10% fetal calf serum (FCS) (Gibco Ltd., Paisley, Scotland), penicillin (100 U/ml) plus streptomycin (100 gg/ml) and tested for presence of MHV in L929 cells. The peritoneal exudates were collected by peritoneal lavage with 5 mt of medium, centrifuged at 750 g for 10 min and the presence of MHV in the supernatants tested on L929 cells. No animal was found to have MHV in the liver or peritoneal exudate. Nevertheless, all the animals tested were found to have antibodies against MHV3 in comparable titres, as measured by ELISA of blood from retroorbital sinus. Virus MHV3, originally obtained from Dr. J. L. Virelizier, Institut Pasteur, Paris, France, was cloned by limiting dilution. One plaque was selected and amplified on L929 cells to serve as the inoculum for future stocks [16] to limit spontaneous mutations. The stocks were always titrated by plaque assay on L929 cells as previously described [19] . Aliquots containing 2 x 105 plaque forming units per milliliter (PFU/ml) were stored at -80 °C and used in the experiments. The MHV3 titres in the peritoneal exudate or liver of infected animals or in supernatants of macrophage cultures were expressed as PFU/ml of peritoneal exudate or cell supernatant and as PFU/g of liver.

Peritoneal exudate cells were collected by peritoneal tavage with RPMI 1640 containing 10~o of FCS and cultured on 96-well plates (Limbro Chemical, Hamden, U.S.A.) at a concentration of 2 x 105 cells per well. The cells were incubated for 2 h at 37 °C in 5~o CO 2 and washed three times with medium after vigorous shaking to remove nonadherent cells. Ninety per cent of the cells were macrophages as determined by their ability to take up zymosan particles.

Peritoneal macrophages were treated with 10 gg/ml of lipopolyssacharide (LPS) from Escherichia coil O111 :B4 (Difco Laboratories, Detroit, U.S.A.)or 100 U/ml of murine IFN alpha/beta (laboratory standard purified to 107 units of protein per mg from supernatants of L929 cells infected with Sendai virus devoid of IFN gamma activity) or of murine recombinant IFN gamma (Holland Biotechnology, Leiden, Holland). Twenty-four hours later, activated or nonactivated cultures were infected with MHV3 at a multiplicity of infection (moi) of 0.01, in order to study the inhibition of MHV3 replication. The supernatants of cell cultures, collected at various intervals after infection, were tested for the virus titre by plaque assay [19] .

A cytopathic effect reduction test technique, described in detail elsewhere [19, 20] was used for the IFN titre determinations. Briefly, monolayers of L929 cells in microtitre plates were incubated for 18 h with different dilutions of samples. Supernatants were removed, and the monolayers were infected with 200 tissue culture infective doses 50~o (TCIDso) of encephalomyocarditis virus. Unadsorbed virus was removed 2h later by washing the monolayers, and fresh minimum essential medium (MEM) supplemented with 10~o FCS was added. Microtitre plates were incubated for 24 h, and the IFN titre, in units/ml (U/ml) was expressed as the reciprocal dilution of the supernatant able to inhibit 50~o of virus replication. For characterization of IFN alpha/beta or IFN gamma in the samples, polyclonal antibodies to mouse IFN alpha/beta and monoclonal antibodies to mouse recombinant IFN gamma (Holland Biotechnology, Leiden, Holland) with activity of 2 x 103 and 2 x t05 neutralizing units per mg, respectively, were always used. The antibodies showed no cross-reactivity, and controls for IFN characterization included internal and well-known preparations of IFN alpha/beta, IFN gamma and a mixture of both to assure the assay specificity. 

The PCA was determined by a technique already described [9] . Briefly, phosphate buffer saline (PBS), pH 7.2-suspended peritoneal exudate (PEC) and spleen cells (SC) from mice noninfected or infected with 103 PFU of MHV3, obtained 2 days after infection, were freeze-thawed, sonicated and assayed for the capacity to shorten the clotting time of rabbit citrated platelet-poor plasma. For the determination of standard milliunits of PCA (mU PCA), a rabbit brain thromboplastin standard (Roche Laboratories, Rio de Janeiro, Brasil) was used to construct a standard curve of PCA, where 100~ of thromboplastin activity corresponded to 10000mU of PCA/106 cells. The data were linear with rabbit citrated platelet-poor plasma and expressed as the mean ± standard deviation of assays from 5 mice in 3 different experiments.

As shown in Tables 1 and 2, the Hill mice were fully susceptible and the LIH mice were fully resistant to the MHV3 infection, regardless the dose of virus used to the experimental infection and whether they were inoculated intraperitoneally, intravenously or subcutaneously. The mean survival time (mst) of Hil I mice inoculated ip with 10 ~ to 10 3 PFU of MHV3 was of 4.0_+ 0.3 to 4.2 _+ 0.4, with no correlation with the MHV3 dose used for the experimental infection (Table t) . The mst of Hm mice inoculated sc with 103 PFU of MHV3 was longer (7.0 _++ 1.6) than that observed for the Hll I mice inoculated ip or iv (4.0 +_ 0.4 and 4.2 _+ 0.5, respectively) with the same dose of virus (Table 2) .

Hm and LII 1 mice Table 3 shows that the virus replication in the liver and peritoneum of ip MHV3 infected Hln and Liil mice correlated with their susceptibility. In both tissues, Figure 1 shows the kinetics of IFN alpha/beta and IFN gamma synthesis in serum and peritoneal exudate of mice infected ip with MHV3. In both mouse lines, comparable levels of IFN alpha/beta or IFN gamma synthesis were found in peritoneal exudate or in serum. No IFN alpha/beta was found in their sera. Except in this case, a peak of IFN alpha/beta or IFN gamma in both mouse lines was observed 2 to 3 days after the MHV3 infection, and higher levels of IFN gamma synthesis were always detected in both Hn~ and Lm mice.

As shown in Fig. 2 , the maximal MHV3 titers in macrophage cultures of Hm or Lm mice were observed 2 days after infection, with a higher virus titre in cell cultures from the susceptible HxH mice. Typical syncytia of MHV3 replication could be found in both cell cultures (data not shown). The results shown in Table 4 indicate that the macrophages from resistant Lm mice were sensitive to the induction of an anti-MHV3 state by LPS, IFN alpha/beta or IFN gamma. However, at the same conditions, no anti-MHV3 effect could be induced in macrophages from H.~ mice. Macrophages from Lm mice were shown to be more sensitive to IFN gamma than to IFN alpha/beta. The HII 1 and LII I mouse macrophage treatment with LPS, led to the synthesis of comparable titres of IFN alpha/beta (data not shown), which was shown to be partially responsible for the antiviral state induced by LPS in Lm mouse macrophages (Table 5) . The "in vitro" treatment with LPS, IFN alpha/beta or with IFN gamma, which induce an anti-MHV3 effect only in Lm mouse macrophages, correlated with the "in vivo" resistance observed in Lin mice after MHV3 infection.

The data obtained of PCA of PEC and SC from MHV3 infected or noninfected Hm and Lm mice ( Tables 1 and 2 .

The mouse hepatitis virus strains of coronavirus are viruses responsible for well known epizootics of enteritis which occurs endemically in most mouse colonies. Most of the animals in these colonies were found to have antibodies against different types of MHV, such as MHV3, which have been isolated from a variety of mouse strains under diverse conditions [1, 5, 10, 15, 21, 25] . This study was undertaken in an attempt to investigate the resistance or susceptibility of genetic heterogeneous mouse populations to our strain of MHV3. The Hll I and Lnt lines of mice obtained by several selective breeding based on antibody responsiveness to an unrelated antigen [22] , are representative of a natural state of a mouse population concerning genetical and immunological aspects, since inbreeding was avoided during the entire selective process. Furthermore, we have observed a reconstitution of the normal distribution of the original F0 genetically heterogeneous population among an F2 segregant population [2, 3, 11] . In addition, these mice are very useful for the studies of resistance/susceptibility to infectious agents due to their characteristic macrophage activity [-41 .

Both lines of mice, although showing no MHV in their tissues, had in their sera comparable levels of antibodies against MHV3. These antibodies have been shown to exert an "in vitro" neutralizing activity, but their involvement in resistance mechanisms against MHV3 have not been clearly demonstrated. Hnl mice were shown to be fully susceptible and LIH mice fully resistant to the experimental infection with MHV3, regardless the route of inoculation or the dose of virus used for the infection (Tables 1 and 2 ). The MHV3 replication in their tissues (Table 3 ) correlated with the resistance/susceptibility pattern (Tables 1 and 2 ) and the high levels of IFN gamma detected in sera and mainly in the peritoneal exudates (Fig. 1) , can be explained by the immunological primed state of the animals. In this case, the experimental virus infection provided a secondary stimulus for the immune system to synthesize the early and high levels of IFN gamma.

As it has already been shown in genetic homogeneous mouse populations [7, 13] , the two proposed mechanisms of resistance to MHV3 infection, or, the activation of macrophages by IFN gamma and the PCA, were shown to be correlated with resistance/susceptibility in these genetic heterogeneous mouse populations (Tables 4 and 6 ). Only macrophages originated from resistant Lni mice were able to be activated by IFN gamma in order to restrict the MHV3 replication (Table 4 ) and only PEC and SC from susceptible Hin mice were able to show an elevation of the PCA (Table 6) .

During the experimental infection with MHV3, the high levels of IFN gamma synthesis that are observed in already MHV-primed mice are able to efficiently restrict the MHV3 replication only in resistant mice, which have IFN gamma-sensitive macrophages, allowing time for the local and systemic responses to clear the infective particles. On the other hand, and acting synergistically, the higher PCA in susceptible mice, which demonstrates prothrombin cleaving activity [6] , was shown to induce abnormalities of the microcirculation, such as granular blood flow and sinusoidal microthrombi, that may disturb the virus clearence and precedes the higher viral replication in susceptible animals [6, 7] , which are unable to restrict the virus replication by an impaired macrophage activity. As a consequence, focal avascular lesions appear and progress to confluent necrosis (data not shown) that results in mortality.

The IFN alpha/beta seems therefore to play a secondary role in the resistance of L,I mice to MHV3, since it is synthesized in lower amounts during the MHV3 infection (Fig. 1) , and because L.~ mouse macrophages are less sensitive to IFN alpha/beta than to IFN gamma ( Table 4 ). The same reasoning could be applied to the LPS, which exert its function partially through the IFN alpha/beta synthesis (Table 5 ).

In conclusion, these data from the study of MHV3 infection using genetic heterogeneous mouse populations, which better represents a natural population, confirm those obtained in studies with genetic homogeneous mouse strains [7, 13] , representing individuals in a given population in the sense that the two main mechanisms involved in the resistance/susceptibility of mice to MHV3 infection are: 1. The synthesis of IFN gamma and the sensitivity of macrophages to IFN gamma, and 2. The PCA levels exhibited by peritoneal exudate and spleen cells.

Adult mouse hepatocytes in primary monolayer culture express genetic resistance to mouse hepatitis virus type 3

Genetics of immunoresponsiveness to natural antigens in the mouse

Genetic selections for relevant immunological functions

A major role of the macrophage in quantitative genetic regulation of immunoresponsiveness and anti-infectious immunity

A virus related to that causing hepatitis in mice (MHV)

The immune response to mouse hepatitis virus: expression of monocyte procoagutant activity and plasminogen activator during infection in vivo

Susceptibility/resistance in mouse hepatitis virus strain 3 and macrophage procoagulant activity are linked and controlled by two non-H-2 linked genes

Genetically determined resistance to mouse hepatitis virus type 3 is expressed in hematopoietic donor cells in radiation chimeras

Macrophage procoagulant activity as a measure of cell-mediated immunity in the mouse

New strain of mouse hepatitis virus as the cause of lethal enteritis in infant mice

Poligenic control of quantitative antibody responsiveness: restriction of the multispecific effect related to the selection antigen

Immunopathology of mouse hepatitis virus type 3 infection. I. Role of humoral and cell-mediated immunity in resistance mechanisms

A major role of macrophage activation by interferon gamma during mouse hepatitis virus type 3 infection. I. Genetically dependent resistance

A major role of macrophage activation by interferon gamma during mouse hepatitis virus type 3 infection. II. Age dependent resistance

Acquired immunity of A/J mice to mouse hepatitis virus 3 infection: dependence on interferon gamma synthesis and macrophage sensitivity to interferon gamma

Temperature sensitive mutants of mouse hepatitis virus type 3 (MHV3): isolation, biochemical and genetic characterization

Coronaviruses. In: Fields BN (ed) Virology. Raven Press

Interaction between mouse hepatitis viruses and primary cultures of Kupffer and endothelial liver cells from resistant and susceptible inbred mouse strains

Induction of natural killer cells and interferon during mouse hepatitis virus infection of resistant and susceptible inbred mouse strains

Inhibition of mouse hepatitis virus type 3 multiplication in activated Kupffer cells

MHV3 infection in genetic heterogeneous mice

Mouse hepatitis virus infection as a highly contagious, prevalent, enteric infection in mice

Selective breeding of mice for antibody responsiveness to flagellar and somatic antigens of Salmonellae

Non-specific regulation of antibody responsiveness in the mouse

Role of interferon in the pathogenesis of viral diseases of mice as demonstrated by the use of anti-interferon serum. V. Protective role in mouse hepatitis virus type 3 infection of susceptible and resistant strains 0f mice

Isolation of a murine hepatitis virus from Swiss mice treated with antilymphocyte serum

Differences in antigen handling by peritoneal macrophages from the Biozzi high and tow responder lines of mice

This work was supported by grants from the Funda~fio de Amparo fi Pesquisa do Estado 

Authors' address: Dr. C.A. Pereira, Laboratorio de Imunologia Viral, Instituto Butantan, Av. Vital BrasiI 1500, 05503-900 Silo Pauto SP, Brasil.Received January 25, 1994