key: cord-0007824-cxo9npjj
authors: Schindler, L.; Klimetzek, V.; Kirchner, H.
title: Bone-marrow derived macrophages as targets for the replication of mouse hepatitis virus type 3
date: 2002-11-12
journal: Immunol Lett
DOI: 10.1016/0165-2478(83)90042-1
sha: 8b5ceed1959d72acf8c21d0ef6a0ef7c07221bf4
doc_id: 7824
cord_uid: cxo9npjj

Bone-marrow (BM) derived macrophages are sensitive target cells for replication of mouse hepatitis virus type 3 (MHV3). These cells can be grown in large numbers and the percentage of defined macrophages increased until day 10 when 100% of the cells represented macrophages. MHV3 replicated within these cells to high titers and caused the formation of multi-nucleated giant cells. This effect was seen with very low virus inocula in BM macrophages of C57BL/6 mice that are highly susceptible to in vivo infection with MHV3 whereas macrophages from resistant A/J mice did not show a cytopathic effect at these virus doses. 1000-fold higher virus doses, however, caused the cytopathic effect in macrophages of both C57BL/6 and A/J mice.

(Received 29 July 1983) (Accepted 12 August 1983)

Bone-marrow (BM) derived macrophages are sensitive target cells for replication of mouse hepatitis virus type 3 (MHV3). These cells can be grown in large numbers and the percentage of defined macrophages increased until day l0 when 100% of the cells represented macrophages. MHV3 replicated within these cells to high titers and caused the formation of multi-nucleated giant cells. This effect was seen with very low virus inocula in BM macrophages of C57BL/6 mice that are highly susceptible to in vivo infection with MHV3 whereas macrophages from resistant A/J mice did not show a cytopathic effect at these virus doses. 1000-fold higher virus doses, however, caused the cytopathic effect in macrophages of both C57BL/6 and A/J mice.

Mouse hepatitis virus type 3 (MHV3), a member of the genus coronavirus [1] produces a characteristic cytophatic effect (CPE) in mouse peritoneal exudate cells (PEC) by fusing the macrophages into multi-nucleated giant cells [2] . The appearance of this CPE closely parallels viral replication and thus provides a useful tool for in vitro studies of virus macrophage interactions [3, 4] . However, mouse PEC represent a heterogenous cell population [5] Key words: bone marrow macrophages mouse hepatitis virus type 3 -genetics of resistance and divergent data have been reported on genetic differences between the capacity of PEC from different inbred mouse strains [6 8] . We have therefore used in vitro cultivated bone marrow (BM) macrophages for replication and titration of MHV3. These can be readily grown in large numbers and with a high degree of purity. MHV3 replicated to high titers in these cultures. The purpose of this report is two-fold: (a) we wish to report the usefulness of this tissue culture system for the replication of MHV3, and (b) we have reexamined the differences of in vitro MHV3 replication in macrophages derived from different inbred mouse strains.

MHV3 originally a gift of R. J. Huebner (Baltimore, MD, USA) to Dr. G. Streissle (Wuppertal, F.R.G.) was passaged in 3-wk-old CF-I mice (Winkelmann, Bochem, F.R.G.) by intraperitoneal (i.p.) infection. Livers from dead mice were pooled, and a homogenate was prepared in buffer and frozen at -70 ° C. The LDs0 of this material for 8-wk-old C57BL/6 mice was 102 MID, and the LDs0 for 8wk-old A/J mice was 105 MID (see section 3.5 for explanation of the term MID).

Male A/J mice were obtained from Jackson Laboratories (Bar Harbor, ME, U.S.A.) and male C57BL/6 mice from the Gesellschaft ftir Versuch-stierkunde (Hannover, F.R.G.). All mice were obtained at the age of 8 wk and used in the experiments within 3 to 6 wk of purchase. Upon arrival, they were tested for serum antibodies against MHV3 by indirect immunofluorescence (courtesy of Dr. W. Nicklas, German Cancer Research Center, Heidelberg, F.R.G.). The mice used in this study were consistently negative.

In vitro cultures of BM macrophages were set up according to a protocol described previously [9] . In brief, BM cells were obtained by flushing femurs and tibias with Balanced Salt Solution (BSS). A single cell suspension was prepared by passage through a 25 gauge needle. The cells were plated at densities of 200,000/ml in 24-well plates (Code 76-003-05, Linbro, Flow Labs, Bonn, F.R.G.) and cultured in Dulbecco's Minimal Essential Medium (D-MEM, Seromed, Mtinchen, F.R.G.) supplemented with 15% heat inactivated fetal bovine serum (FBS), 5% heat inactivated horse serum (Seromed, Mianchen, F.R.G.), 1% essential and non-essential amino acids, 200 mM L-glutamine, 50/~g/ml gentamycin (Gibco, NY, U.S.A.) and 20% L-cell conditioned medium (the conditioned L-cell medium was prepared in our own laboratory). BM cells were refed daily starting on day 5 and were cultured for a total of 8 10 days.

PEC were obtained by rinsing the peritoneal cavity and cultured at a concentration of 3 × 106/ml in 0.2 ml of medium RPMI 1640 supplemented with 5% FBS, 100 IU/ml penicillin, 100/~g/ml streptomycin, and 2 mM glutamine (Gibco, NY, U.S.A.) in the wells of flat-bottom 96 well plates (Code 3049, Falcon Plastics, Oxnard, CA, U.S.A.). These were incubated for 45 rain at 37 ° C and rinsed three times with medium. The adherent cell population was further cultured for 2 days.

For titration of the virus, BM cells were grown in 24-well plates for 8-10 days. After this time the cells were infected with MHV3 and incubated for an additional 48 h. MHV3 caused the formation of giant cells in these cultures, subsequently referred to as CPE. Virus titers are expressed as the reciprocal 108 value of the virus dilution that still caused this effect (also designated MID = macrophage infecting dose).

BM macrophages can be grown easily in large numbers and their yield is considerably higher than the yield of peritoneal macrophages from the same number of mice. In BM cultures grown in L cellconditioned medium the number of morphologically defined macrophages increased linearly until days 8-10 and then remained constant. From day 10 on 100% of the cells were macrophages, according to the criteria of phagocytosis, adherence to plastic surfaces and to non-specific esterase staining. These data (that are not extensively documented here) are in full accordance with previous data of one of us [10] and Falk et al. [11] . 

A/J and C57BL/6 mice BM macrophages from mice of different strains at a comparable cell density were investigated for the appearance of multi-nucleated giant cells at different times after infection with graded dilutions of MHV3. Cultures derived from resistant A/J mice failed to produce giant cells except in cultures infected with high inocula of 104 MID or greater. In cultures in C57BL/6 macrophages, however, a CPE was observed after 48 h even when doses of 10 MID were inoculated (Fig. 1 ).

In further experiments BM macrophages of A/J, 

In order to further document the applicability of the BM tissue culture system for viral titration, we have tested the virus titers in the peritoneal wash-out fluid of mice injected with MHV3. A/J or C57BL/6 mice were injected i.p. with 103 MID and virus titers were determined at various times thereafter. Table 3 shows that there were marked differences in the virus titers between the two mouse strains. These were in the magnitude of 3 logl0 both 52 h and 72 h after infection. In further experiments (data not documented) we have also shown that virus titers in the blood and in homogenates of liver or spleen could be titrated readily in BM macrophages of C57BL/6 mice.

In the murine model of infection with MHV3 resistance is genetically controlled. As established by Bang and Warwick [6] and confirmed by Virelizier and Allison [3] C57BL/6 mice are highly susceptible to MHV3 and die of hepatitis 34 d after infection.

A/J mice, in contrast are resistant. Resistance is expressed on the level of the macrophages in that macrophages of resistant mice are unable to replicate MHV3. More recently, Macnaughton and Matterson [7] and Taguchi et al. [8] have studied the replication of MHV3 in mouse peritoneal macrophages and have observed virus replication both in macrophages of resistant and susceptible mouse strains. The reasons for the discrepancy between the data of Bang and Warwick and those of the other laboratories are not understood.

The PEC population is a mixture of lymphocytes, macrophages and small numbers of additional cells [5] . Since the lymphocytes by a variety of mechanisms, e.g. by interferon production, may massively interfere with virus replication, we feel that PEC are inferior for certain studies, and that for studies of virus macrophage interactions in the MHV3 system a culture of pure target ceils is preferable. BM macrophages prepared as described in this study are pure macrophage cultures. Our data have shown that in A/J BM macrophage no CPE is formed at virus doses that readily cause a massive CPE in cultures of C57BL/6 mice.

Thus our data using BM macrophages have reiterated the results of Virelizier and Allison [3] that there are genetic differences in the in vitro replication of MHV3 in macrophages and that these differences reflect the in vivo situation, However, the block of replication in macrophages of A/J mice is not absolute in that a CPE is observed when infecting with very high doses of MHV3.

Furthermore, our data have documented the usefulness of the BM macrophage system of C57BL/6 mice for titrations of MHV3. Although we have not explicitly compared our system to other titration systems [12] [13] [14] it appears obvious that the BM system represents a "physiological" system for studies of the interactions between MHV3 and macrophages. It is particularly useful since it consists of 100% macrophages and thus negative interference with other cell types (such as interferon production by lymphocytes) can be excluded.

Microbiology and Immunology

We are most grateful to Ms. M. Kasamasch for excellent editorial assistance.