The growth of human HIV-1 infected U937 cells in immune-deprived mice

Biomed

Original

(1995) 49, 358-363 0 Elsevier, Paris

article

The growth

of human HIV-l in immune-deprived

IV Chernukhin’, ‘Siberian

& Phurmacother

Branch

of the Russian Academy ‘Institute of Molecular (Received

infected mice

AA Chepurnov*,

KV Gaidu12

of Medical Sciences, Timakov street, Biology 633159, Koltsovo, Novosibirsk

3 November

1994;

accepted

U937 cells

16 December

r 152, 630117 Novosibirsk, Region, Russia

Russia:

1994)

Summary - We report in vivo growth of human promonocytic cells infected with HIV-l presented in new mouse model. Cloned U937 cells chronically infected with HIV-l were grafted in (CBA*C57B1/6)FI mice deprived of immunity by thymectomia and total body irradiation with subsequent marrow reconstitution. Nine weeks after cell inoculation, HIV-l-positive cells were found only in mice that received an additional single dose of cyclophosphamide (100 mg/kg bw) prior to transplantation, whereas, in mice without further immune deprivation, the complete elimination of cells bearing viral antigen occured already on the seventh day after transplantation. The approach described may be suitable for in vivo development of antiviral drugs against latent infection in macrophage-like cells which represents a serious problem in therapy of AIDS in humans. animal

model

I U937

cells / chronically

infected

cells

immune

INTRODUCTION HIV infection in monocyte/macrophages in humans is currently regarded as a crucial factor in pathogenesis of AIDS. In particular, this type of cell is suggested to be a reservoir of latentlydeveloping viral infection [5, 6, 8, 91 resulting however in HIV dissemination to helper T cells and then AIDS after induction of the virus replication under cell-activating stimuli [15]. Besides low rates of virus replication in these cells, monocyte/macrophages appear to be relatively more resistant to the cytopathic effects of HIV infection than T cells and, therefore, are suggested to cause HIV persistence in vivo [5, 71. At the same time, monocytes infected with HIV become functionally impaired [ 181 which may account in part for the immunopathogenesis of AIDS. Thus, HIV-infected monocyte/macrophage-like cells are actually important to be targeting for an appropriate therapeutic strategy against AIDS infection in this type of cell. Human promonocytic U937 cells [ 193 appear to allow establishment of chronic HIV infection with

deprivation

a protential for virus persistence in vitro. Derived from this line, chronically virus-infected sublines are commonly used as an in vitro model to observe the interaction between HIV and monocytes and also applied in research for anti-HIV therapeutic agents in vitro [3, 4, 11, 12, 16, 171. In this study, we have established in vivo chronic HIV-l infection in U937 cells engrafted in immune-deprived mice. A clone of U937 cells was tolerated to mouse immunity by cloning in vitro and adapted therefore to grow in the mice. Under an in vitro observation, besides marked changes in morphologic and functional characters of the cloned cells, they demonstrated a greater susceptibility to HIV-l infection than the original cells, presenting the chronic virus infection. This event allowed us to perform the heterotransplantation of the human promonocytic cells infected with HIV in the immune-deprived mice which further demonstrated virus infection over several months. In this communication, we examine the character of in vivo HIV-l infection in U937 cells and the possibility for use of this mouse model for anti AIDS-therapeutic research.

Growth

MATERIALS

of human

HIV-I

AND METHODS

Cells U937 cells, a human promonocytic cell line [19], were used in the study. To obtain future reproducible adaptivity for growth in mice, the cells were cloned by limited dilution method with 50 fold-excess of murine lymphocytes immune-activated in vivo by U937 cell immunization. Identified by cytogenetic and immunomorphologic analysis, proliferating U937 clones were taken for further confluent growth in RPM1 1640 medium supplemented with 10% FCS, 2mM glutamine and 50 p/ml gentamycine.

infected U937 cells mice

359

HIV antigen determination Production of HIV-1 specific determinants on cells was monitored during a period of cell incubation and in the mouse peritoneal cell specimens by indirect immunofluorescence as well. A human polyclonal serum with high-titer reactivity with gag, pol and env determinants (Institute of Molecular Biology, Koltsovo, Russia) and antihuman IG goat antibodies conjugated to fluoresceine isothiocyanate (FITC) were used as primary and secondary antibodies, respectively. An enzyme-linked immunosorption assay (ELISA) was used to detect HIV1 p24 in culture fluids and in the mouse sera using anti-p24 monoclonal antibodies (Institute of Molecular Biology, Koltsovo, Russia).

Virus infection A clone of HIV-l (EVK/IRA-3) obtained from Ivanovsky’s Institute of Virology (Moscow. Russia) was used in the study. It was adapted by passages in short-term culture of MT-4 cell line. 2* IO7 U937 cells were treated with DEAE dextran (25 Kg/ml for 30 min at 37°C) were collected by centrifuging, and resuspended in virus-containing medium at 2*106/ml density followed by incubation for 18 hours at 37°C. After washing, the cells were chased from initial 0.7*10h/ml concentration at 37°C in 5% CO> air for 10 days before use. To determine the reproducibility of virus-infecting U937 cells, de norm reinfection of MT4 cells was performed by cocultivation at 1OO:l ratio.

Mice Female (CBA*C57B1/6)Fl 4-6 wk mice were maintained on a convential diet in infectious vivarium. The immunosuppression was developed by thymectomia, 850r total body irradiation and reconstitution with 5* 1O6 syngenic bone marrow cells 6 hours after irradiation (thymectomia, ThEc; total body irradiation, Ir; bone marrow reconstitution, BMR). Additionally, a single cyclophosphamide (Cy) injection was performed at the dose of 100 mg/kg of body weight 25 hours prior to cell inoculation. 20*106 cloned U947 HIV-l-infected cells were collected and suspended in 0.5 ml of RPMI1640 medium and were injected into the peritoneal cavity 14 days after irradiation. At one week-intervals, the mice were sacrificed and the specimens of peritoneal cells and sera were collected. The human origin of cell patterns was confirmed by cytogenetic analysis with standard techniques including the staining of metaphase chromosomes with wright stain.

RESULTS To gain a reproducible heterotransplantation of human macrophage-like U937 tumor cells in mice, we carried out: immune deprivation of mice; development of further transplanted cells with low sensitivity to host immunity. For this purpose, we carried out in vitro cloning of U937 cells in the presence of mouse immunereactive lymphocytes obtained from future recipient-line mice immunized with the same cells. This procedure allowed us to obtain a clone of U937 cells resistant to mouse immunity and growing in vivo, designated here as U7Fl cells. Further, U7Fl cell xenograft gave rise to 55% reproducible peritoneal tumor growth during 8 to 10 weeks in Fl mice deprived of immunity by ThEc, Ir, BMR, whereas the cells of the original line were grafted only in 1 to 2% of the same mice. An increase in the number of tumor-bearing mice to 85 to 95% was registered after an additional peritoneal Cy injection 24 hours prior to cell inoculation. Similar recults were shown in human CEM cells grafted in mice [ 101. When tested, U7Fl cells appeared to demonstrate a greater susceptibility to HIV-l infection in contrast to the parent cells in vitro. A large relative increase in p24 protein level yielded by U7Fl cells in one week old medium culture supernatant as well as a higher percentage of viruspositive cells in the same cultures in comparison to the original line were registered (fig 1). These data are also correlated well with reverse transcriptase activity. Furthermore, U7Fl cells presented a chronic HIV-l infection.

360

IV Chernukhin

0

3

5 DAYS

7

10

et al

0

-~ 1

2

3

4

5 6 WEEKS

7

6

9

10

Fig 1. ~24 Ag percent change, as calculated from data measured in culture medium supernatant fluids by ELISA. and a percentage of FITC-stained cells bearing viral determinants, estimated at different times post viral exposure. HIV-l infected cells were plated at a density of 0.7*106/ml. Samples were removed immediately (day 0) and on day 3, 5, 7, 10 and were assayed using anti-p24 monoclonal antibodies, or human polyclonal serum reacting with gag, pol and env determinants, respectively. Solid lines show the percent changes in p24 level evaluated from the data obtained at a time point and day. 0, and the broken lines represent the percentage of HIV-positive cells in the same cultures. Open symbols depict the data for U937 cells, and solid symbols represent U7Fl cell cultures. The data presented were obtained from a single representative experiment.

4

One and a half to two weeks after irradiation, the growing 20*10h UF7Fl HIV-infected cells were peritoneally injected into immune-deprived (CBA*C57B1/6)Fl mice. The mice were then maintained within the confines of infectious vivarium at p-4 condition on a convential mouse diet. Mortality associated with immune deprivation was lower than 3% for each treatment mode. The mice were killed at one-week intervals, serum and peritoneal cell specimens were obtained. The cells were determined as human cells by cytogenetic analysis and tested for HIV-positive cells by immunofluorescent staining (fig 2). The results of the experiments are given in figure 3. From 22 to 28% of U7Fl cells obtained from the mice expressed viral determinants within two weeks. In lower amounts, these cells can be observed during the next seven weeks. Such cells occurred only in the mice exposed to Cy, whereas mice treated with ThEc, Ir, BMR alone did not show viral antigen-bearing cells. To confirm reproducibility of viral infection of cells obtained from mice, we applied MT-4 cell

I I 5 -6 WEEKS

, 7

6

9

10

Fig 3. Heterotransplantation of HIV- 1 infected human promonocytic cells in immune-deprived (CBA*C57B 1/6)Fl mice. A: Percentage of tumor-bearing mice at different times post U7Fl cell exposure. 0: data for mice without Cy treatment; 0 Cytreated mice; n percentage of Cy-treated mice in which HIVinfected cells were observed. For each 7-day interval, a group of 5 to 6 mice was assessed in four separate experiments. B: Kinetics of HIV-l spread in promonocytic U7Fl cells grafted in the same mice. Percentage of U7Fl cells reacting with antiHIV antiserum in individual mice was calculated and the averaged percent data are presented. * positive MT-4 cell reinfection test.

de NWO infection test. In each case of HIV antigen-positive U7Fl cells obtained from mice which were exposed to Cy, the reinfection of MT4 cells occurred mostly within nine weeks of the experiments. As it has been documented, the viral p24 antigenemia was observed in nude mice with a human HIV- 1-infected cell xenograft [2 11. Likewise. we attempted to provide p24-antigen

Growth

of human

HIV-l

infected

U937

cells

mice

361

Fig 2. A: Phtotomicrograph of peritoneal cells stained with Gimsa stain obtained from Fl mice transplanted with U7Fl cells (x 400), and B: Wright-stained metaphase chromosomes of the same cells confirm their human origin. C: Indirect immunofluorescence assays of cells obtained from mice. Cells were fixed on slides with methanol: acetone (l:l). Slides were exposed to anti-HIV-1 human antiserum for 30 min, and subsequently washed in phosphate buffered saline. Further, cells were stained for 30 min with antihuman IG goat antibodies conjugated to fluoresceine isothiocyanate (FITC). Cells were washed in PBS containing 1% Evans Blue diluted at 1 in 2,000. Cl: U7Fl FITC-stained cells taken from tumor-developed mice without Cy injection; C2: cells obtained from Cy-treated mice.

362

IV Chernukhin

determination in sera from Fl mice with U7Fl xenograft. Nevertheless, there were no mouse serum specimens reacting with anti-p24 monoclonal antibodies throughout the experiments. However, a higher titer reactivity of murine sera with the viral p24 peptide was determined by using ELISA in Cy-treated mice in comparison with approximate background titer, ie, that of untreated mice (data not shown). Presumably, low rates of virus replication commonly appeared in the monocytic cells [3, 41 and the host humoral immune response made the viral p24 peptide determination impossible.

DISCUSSION The data show that cloned U7Fl cells were infected with HIV-l to a greater extent than the cells of the parent line. This fact could be explained by the cell differentiation through the cloning. Of note were differences in the morphological and functional characters between the cloned and parent lines. These characters were associated mostly with mature macrophages. Most of these celles were plastic-adherent and gained capability for phagocytosis, an increase in Fc receptors and IL-l production (data not presented). Such a differentiation event occured in vitro correlated to that described for the other human tumor cells in vivo, which were grafted into nude mice or mice treated in the same manner [ 1, 10, 13, 141, and is known to result in activation of HIV replication in U937 cells with chronic infection [3, 4, 111. The event is proposed to promote engraftment of HIV-infected U7Fl cells in mice. The immunedeprived/Cy-treated mice demonstrated HIV-infection within nine weeks whereas the mice without Cy treatment did not feature any sign of infection. Although the decrease in the number of cells infected with HIV, determined by serologic criteria, was observed in more than four weeks, the reproducibility of the virus infection was, however, evident. The time-dependent decrease in HIV infection in the mice may be a consequence of the partial recovery of the host immune reaction affected by Cy-exposure. It is possible that HIV-bearing cells become the target for recovering immune cells and are subsequently eliminated, or HIV expression in the U7Fl cells could be down-regulated by a host effector mech-

er crl

anism. Such regulation might be provided by HIV-specific antibodies since the humoral immune response remained less affected by the immune suppression than cellular immunity in these mice. It is also of interest whether HIV was completely eliminated from the xenograft, or the U7Fl cells became non-replicating containing provirus. Future establishment of the mechanism responsible for the decreasing HIV expression in this xenograft system is thought to be important and may give new information on regulation of virus expression in this type of human cell. In search for successful therapy of AIDS, it may be important to establish therapeutic agents directed against virus infection in macrophagelike cells it? vivo as well as in vitro. Agents that are not active in mononuclear phagocytes are unlikely to exert significant clinical effects, no matter how promising the activity observed in vitro in T-cell-based assay systems. A development of animal models for in vivo AIDS research is carried out, in part, in mice with severe combined immunodeficiency (SCID) [20] or athymic (nude) mice [Zl] which allow the growth of human HIVinfected immune cells or fetal organ xenograft. We suggest that this model may be suitable for in vivo anti-HIV drug investigation supplementing that of the virus infection in macrophage-like cells in vitro. In this regard, it may be more useful than the existing mouse models because: i) derived from normal mice, the immune-deprived mice tolerate physical and chemical agents better than SCID or nude mice in which, besides an immunodeficiency, a low function of vital systems exists naturally; the immunedeprived mice with HIV infection may be therefore effectively exposed to a high variation of drugs and drug dose; and ii) the mice do not require sterile environment and may be housed under less stringent conditions than SCID or nude mice.

REFERENCES I Clutterbuck RD, Hills CA, Hoey P, Alexander P, Powles RL, Millar JL. Studies on the development of human acute myeloid leukemia xenografts in immune-deprived mice: comparison with cells in short-term culture. Leuk Res 1985;9:1511 2 Fauci AS. The human immunodeficiency virus: infectivity and mechanisms of pathogenesis. Science 1988;238:617 3 Folks T, Justement J, Kinter A, Dinarello C. Fauci A. Cytokine-induced expression of HIV-l in a chronically infected promonocytic cell line. Science 1987;238:800

Growth

of human

HIV-I

4 Foks T, Justement J, Kinter A et al. Characterisation of a promonocyte clone chronically infected with HIV and inducible by PMA. J Immunol 1988;140:1117 5 Gartner S, Markovitz P, Markovitz DM, Kaplan MH, Gallo RC, Popovic M. The role of mononuclear phagocytes in HTLV-III/LAV infection. Science 1986;233:215 6 Gendelman HE, Narayan 0, Molineaux S. Clements JE, Gho Z. Slow, persistent replication of lentivirus: role of tissue macrophages and macrophage precursors in bone marrow. Proc Nat[ Acad Sci USA 1985;82:7086 7 Hammer S, Gillis J, Groopman J, Rose R. In vitro modification of human immunodeficiency virus infection by granulocyte-macrophage colony stimulating factor and gamma interferon. Proc Natl Acad Sci Usa 1986;83: 8734 8 Ho DD, Pomerantz RJ, Kaplan JC. Pathogenesis of infection with human immunodeficiency virus. N Engl J Med 1987;317:278 9 Ho DD, Rota TR, Hirson MS. Infection of monocytes/macrophages by human T lymphotropic virus Type III. J Clin Invest 1986;77:1712 10 Houghton PJ, Mirro J, Jr, Goorha RM, Raimondi SC, Fridland A, Houghton JA. Growth and differentiation of a human T-cell leukemia cell line, CCRF-CEM, grafted in mice. Cancer Res 1989;40:7124 11 Locardi C, Petrini C, Boccoli G et al. Increased human immunodeficiency virus (HIV) expression in chronically infected U937 cells upon in vitro differentiation by hydroxyvitamin D3: roles of interferon and tumor necrosis factor in regulation of HIV production. J Virol 1990:64: 5874

infected

U937

cells mice

363

12 Mace K., Due-Dodon M, Gazzolo L. Restriction of HIV-l replication in promonocytic cells: A role for IFN-a. Virology 1989; 168:399 13 Machado EA, Gerard DA, Lozzio CB, Lozzio BB, Mitchell JR, Golde DW. Proliferation and differentiation of human myeloid leukemia cells in immunodeficient mice: electron microscopy and cytochemistry. Blood 1984;63:1015 14 Palu G. Selbv P, Powles R, Alexander P. Spontaneous regression of human acute myeloid leukemia xenografts andphenotvnic evidence for maturation. Br J Cancer 1979:40: 731 15 PauLa CD. HIV persistence in monocytes leads to pathogenesis and AIDS. Cell Immuno1 1988;112:414 16 Poli G, Orenstein JM, Kinter A, Folks TM, Fauci AS. Interferon-a but not AZT suppresses HIV expression in chronically infected cell lines. Science 1989;224:575 17 Rooke R, Tremblay M, Wainberg MA. AZT may act postintegrationally to inhibit generation of HIV progeny virus in chronicallv infected cells. Virology 1990; 176:205 18 Roy S. Wait&erg MA. Role of the mononuclear phagocyte system in the development of acquired immunodeficiency syndrome (AIDS). J. Leucocyte Biol 1988;43:91 19 Sundsrtrdm C, Nilsson K. Establishment and characterization of a human histiocytic lymphoma cell line (U937). Int J Cancer 1976;17:565 20 Wan W. Mouse models with human immune tissue may help screen for AIDS drugs. Biotechnol News 1990;10/4 21 Wetherall NT, Eiring A. The development of HIB-1 p24 antigenemia in the athymic “nude” mouse and the implications for the assessment of antiviral therapy in viva. Int TNC Meet Animal Models AIDS, Maastricht, Abstract Amsterdam; 1989; 12