- Email: [email protected]
Changes in lymphocyte and macrophage subsets due to morphine and ethanol treatment during a retrovirus infection causing murine aids
Life Sciences, Vol. 43, pp.v-xi Printed in the U.S.A.
Peragmon Press
CHANGES IN LYMPHOCYTEAND MACROPHAGE SUBSETS DUE TO MORPHINE AWD ETHANOL TREATMENTDURING.A RETROVIRUS INFECTION CAUSING MURINE AIDS Ronald Ross Watsonl, Rao Ii.Prabhalal,Hamid R. Darbanl, M. Danial Yahyal, and Thomas L. Smith2s3 Department of lPamily and Community Medicine, and 2Pharmacology, University of Arizona and 3VeteransAdministrationMedical Center, Tucson, A% 85724.
Infection by LP-BMS murine leukemia virus (MuLV) suppressed significantlythe percentage of peripheral blood cells showing surface markers for macrophages,lymphocytesand activated lymphoid cells. Chronic administrationof a 7% (36% calories) ethanol diet or Injection of 1.9 mg/mouse/dayof morphine for a 7 day period were followed by 3 week periods of abstinenceand then 1 week periods of consumptionof 5% ethanol diets or morphine injection to female C57BL/6 mice resulted in changes in the numbers of macrophagesand lymphocytesubsets. The number of lymphocytesof various subsets were not significantlychanged by the ethanol exposure except those showing activationmarkers which were reduced. The percentage of peripheral blood cells showing markers for macrophage functions and their activationwere significantlyreduced after "binge" use of ethanol. Ethanol retarded suppressionof cells by retroviral infection. However by 25 weeks of infection there was a 8.6% survival in the ethanol fed mice infected with retrovlruswhich was much less than virally infected controls (45.0%). Morphine treatment also increased the percentageof cells with markers for macrophages and activated macrophagesin virally infected mice, while suppressing them in uninfectedmice. The second and third morphine injection series suppressedlymphocyteT-helper and T-suppressorcells, but not total T cells. However, suppressionby morphine was significantly less during retroviraldisease than suppressioncaused by the virus only. At 25 weeks of infection 44.8% of morphine treated, infected mice survived. Morphine treatment also caused deaths such that the survival in morphine treated, retrovirallyinfectedwas higher than would have been expected if the death rate in virally infected, and morphine injected animals were combined during combined treatment. Thus these drugs of abuse can modulate peripheralblood lymphoid subsets, suppressioncaused by retroviralinfection,and survival. Cofactors may be involved in progressionto AIDS (AcquiredImmune Deficiency Syndrome) after HIV (Ruman ImmunodeficiencyVirus) infection (1) via immunomodulation(2). Possible cofactorswhich are immunosuppressive include alcohol (3-6) and morphine (7). Ethanol use has major effects on disease resistancedepending upon the amount and the duration consumed. Alcohol-inducedcirrhosis suppressescellular immune functions as well as resistanceto viral pathogens (3). There is evidence that ethanol acts as an immunosuppressiveagent In vitro and in long-term exposure without liver 0024-3205188$3.00 + .OO Copyright (c) 1988 Pergamon Press plc
iri
Drugs, Immunomodulation and Murine AIDS
Vol. 43, No. 6, 1988
disease (4). Morphine (7) also routinely suppresses cellular immune functions and reduces resistance to various pathogens. Retroviral infection in mice is an exciting system to study the effects of cofactors on immune functions. It has many characteristics of AIDS such that it has been suggested as an excellent murine model for infection with HIV in man (8,9). Therefore the present investigation examines the effects of ethanol and morphine treatments on changes in numbers of regulatory lymphocytes and monocytes/macrophages during murine retroviral infection. Materials and Methods Mice and Treatments. Three to four-week-old C57BL/6 female mice, weighing approximately 15 g, were obtained from Simonsen Laboratories, Inc., Gilroy,-CA:- They were routinely provided with Purina laboratory chow and water ad libitum. Thirty mice were injected with morphine sulfate intraperitoneally every day for 7 days during the same weeks other groups of animals described above were exposed to ethanol. The morphine was given at a daily dose of 75 mg/Kg body weight by intraperitoneal injection. For the initial ethanol exposure 30 mice were subjected to a Carnation Slender liquid diet containing 7% (v/v) ethanol (36% of calories) for the initial 7 days of the study as described previously (10). Mice fed ethanol were labeled "E", while morphine injected were designated "M". Retrovirally infected mice were abbreviated as "I," while mice not exposed to drugs were controls, "C". Thus virally infected controls (not exposed to drugs of abuse) were "C-I". Then E mice were again provided the chow diet for 3 weeks before being fed a 5% ethanol diet for 1 week which was 3 weeks post viral infection. Some mice were sacrificed and the remainder received the chow diet for 3 additional weeks followed by the 5% ethanol diet for 1 week when more were sacrificed at week 8 post infection. Uninfected mice in groups of 20 animals were treated identically with each drug of abuse. C57BL/6 mice chosen because it had previously been observed to consume sufficient amounts of ethanol in a liquid diet to develop functional tolerance and physical dependence (10). Physical dependence to ethanol was achieved as evidenced by the high incidence of convulsions and tremors 4 to 8 hr after the removal of dietary ethanol. Mice used developed signs of ataxia several days before sacrifice. In other experiments similarly treated mice exhibited signs of withdrawal upon cessation of the ethanol diet (unpublished data--T. Smith). Mice were sacrificed by ether anesthesia, and spleen and heparinixed blood were collected immediately. LP-BM5 Murine Leukemia (MuLV) Infection. The retrovirus mixture, LP-BM5, was originally isolated by Latariet and Duulan (11) and nroduces non-neoplastic iymphoproliferative disease in adult mice (12). The ecotropic component of the LP-BM5 MuLV mixture seems to function to promote replication of the poorly replicating, pathogenic Mink Cell Focus-forming component. MuLV LP-BM5 was obtained from a non-thymic lymphoma which had been induced by irradiation of mice. It is a cluster of viruses which include a B-tropic ecotropic virus and a B-tropic mink cell focus-inducing virus (12,131. The latter virus alone does not cause immunological disease, but mice infected with a non-pathogenic ecotropic virus and then infected with the cell focus-inducing virus do. Thus, this virus or one that passages with it is the etiologic agent in the disease with the ecotropic virus functioning to spread it in mouse tissues. After the first week of exposure to drugs of abuse, the animals (30 mice/group) were injected intraperitoneally with 0.1 ml of LP-BM5 murine leukemia inocula which was a kind gift of Dr. R.A. Yetter (Microbiology Research, Veterans Administration Hospital, Baltimore, MD). Twenty mice not exposed to drugs of abuse were infected as controls. Mice were sacrificed at 4 and 8 weeks post injection with LP-BM5 MuLV.
Vol. 43, No. 6, 1988
Drugs, Immunomodulationand Murine AIDS
Vii
Lymphoid Cell Identification. Identificationof T cells isolated from the peripheral blood was carried out as described elsewhere (14). Briefly, heparinixedblood was removed from the brachial artery. Isolated lymphocytesat 5 x lo6 cells/ml in 0.2 ml RPM1 1640 containing 2% Neonatal Calf Serum (NCS) were stained with 5 ul of the appropriateprimary monoclonal antibody (MoAb) in 6 x 50 mm test tubes. These include anti-mouseL3T4 for T-helper cells, anti-Lyt-23as a measure of T-suppressor cells, and anti-IL2-receptor(interleukin-2receptor)as a measure of activation of lymphocytes,anti-Thy-las a measure of total T-cells, anti-MAC-las a marker for total macrophages,anti-MAC-2indicating activatedmacrophages,and B cells measured with anti-IgM (anti-mu chain specific). A conjugate control contained 5 ul of the antibody suspension buffer. The tubes were agitated, sealed with paraffin and incubated on ice for 30 min. The cell-MoAb suspensionwas carefully layered over 0.5 ml of NCS in 6 x 50 mm test tubes and centrifugedat 200 x g for 5 min at 4oC. The supernatantwas decanted and 0.2 ml of diluted fluoresceinconjugated goat anti-rat immunoglobulinwas added. This solutionwas agitated and incubated on ice for 30 min. After incubation,the cell-antibodysolution was again overlaid on to 0.2 ml of NCS and centrifugedas before. The fluoresceinlabeled conjugatewas carefully removed with a Pasteur pipette and any remaining conjugate decanted. Routinely 5000 cells were counted on the FACS flow cytometer. Data are expressed as mean 2 S.D. of the number of observationsindicated. Student T test was used to determine significance. Results Spleen. Ethanol use lowered body weight at 4 and 8 weeks post-infection,while morphine use and retrovirallyinfection did not. At 4 weeks the spleen/bodyweight ratio for C, controls, (0.0035 + 0.0031, CI, infected controls (0.053 + 0.0031, E, ethanol fed (0.0014T 0.00044), EI, ethanol-fed,infected (8.0034 + 0.0005), M, morphine injected-(0.0024+ 0.00101, and MI, morphine injected,-virallyinfected (0.0082 + O.OOll)-micewere similar to values obtained at 8 weeks, except that MI mice were lower than C. T-Cell Subsets. As shown in Table I LP-BM5 MuLV infection suppressed (p< 0.01) the percentageof peripheralblood cells with markers for T-helper, T-suppressor,B cells and activated cells showing IL-2R marker, but not total T-cells. Ethanol increased the percentageof T-helper cells in virally infected mice compared to CI (Table I). However, ethanol consumptiondid not affect the percentageof any lymphocytesubsets except those with the activationmarkers, IL-ZR, in uninfectedmice, while morphine suppressedthem. Similar effects were seen with the T-suppressorcells with both compounds. Neither drug of abuse significantlyaffected total T cells in uninfectedmice, while both tended to suppress total T cells in infected mice. Both morphine and ethanol suppressedthe percentageof activated lymphocytesidentifiedby IL-2R on their surfaces in uninfectedmice (p< 0.05). Morphine had a tendency to increase IL-2R bearing cells during infection compared to CI (Table I>. Essentially identical results were observed at 8 weeks post infection (data not shown). Then the T-helper/T-suppressor(H/S) ratio was 1.6 for C and 2.1 for I mice. The H/S ratio for E and the EI mice were 1.85 and 2.15, respectively. However the H/S ratio for M and MI mice were 2.75 and 2.4 respectively. Macrophage Subsets. As shown in Table II, LP-BM5 MuLV infection reduced (p< 0.01) the percentage of peripheralblood cells with markers for
Vol. 43, No. 6, 1988
Drugs, Immunomodulationand Murine AIDS
Viii
TABLE I Effects of Morphine or Dietary Ethanol on Frequency of Lymphocytes Identified by Surface Markers 4 Weeks Post LP-BMS Murine Leukemia Virus Infection1
Treatment
Control
virus Infection
t
T-helper
36.Ot4.0 23.el.9" -
Ethanol
Morphine t
39.e2.0#
40.0&2.8*# 26.0+_2.6*18.1+2.3*
T-suppressor/ 21.0+0.8 ll.Otl.l* 20.5+1.5# 17.8+2.3*# cytotoxic Total T cell
68.e2.9
B-cell (Mu positive)
20.5t7.3
6.6+2.5X 20.3+1.3# -
3.3+2.4#
IL2 receptor
26.5+2.1 -
6.3+_2.0* 9.3+1.5*
6.0+2.6*
71.e4.6
67.e2.0
t
61.Ot7.2# -
9.3+1.0*
73.0+3.0
8.6+0.8*
62.3t4.91
10.8+1.6# 12.8+1.1# 6.6+_2.6* lO.Otl.O*
lMean t S.D. (NM41 *SigniTicantlydifferent from control - virus (p< 0.05) #Significantlydifferent from control + virus (p< 0.05)
macrophagee,activated macrophages and Ia bearing cells. Ethanol in the diet and morphine injection both reduced the percentage of these cells (p< 0.05). EI mice had a trend toward a lower percentage of each of these cell types compared to CI, or E (Table II). However morphine injection during infection increased the percentagecompared to morphine treated or virally infected controls (Table II). Extremely similar results were seen at 8 weeks post infection with both compounds (data not shown). The only significantdifferencewas that the total T cells of MI mice were lower than c (p< 0.05). Then the activated macrophage/macrophageratios were very similar to the 8 weeks values. The ratio after 8 weeks of infection was 1.05 for C, and 0.7 for CI. The ratios were different (p< 0.05) for E (1.6) and EI mice (1.11, and for M (1.4) and MI mice (0.65). These treatments also significantlydecreased the number of Iat cells. The greatest degree of decrease was observed in mice infected with retrovirusalone. Morphine alone or in combinationwith retroviralinfection produced a lesser, yet significant,decrease (Table II). Mouse Mortality. After 25 weeks of infection there had been mortality in the CI mice with 45.0% surviving. Among the controls, not treated with a drug of abuse or infected, none of the mice died by 25 weeks. However, in the EI mice there was greater mortality with 8.6% surviving. There were deaths among E mice with 41.7% surviving. In these mice deaths occurred at first two ethanol exposures when mice were small, while in the EI mice deaths continued throughout the experimentparticularlyfrom 20-25 weeks after infection. The difference in survival was less for EI mice (p< 0.05). Among the MI animals at 25 weeks post infection 44.8% survived.
Vol. 43, No. 6, 1988
Drugs, Immunomodulationand Murine AIDS
iX
There were deaths after morphine expoeure and 38.4% survived of M mice at 25 weeks post-infectionwhich was not significantlydifferent.
TABLE II Effects of Morphine and Dietary Ethanol on Frequency of Macrophages Identifiedby Surface Markers 4 Weeks Post LP-BM5 Murine Leukemia Virus Infection1
Treatment
Control
Ethanol
Virus Infection
+
+
Macro hage $ (MACl)
25.5f9.8
+
7.321.7" 11.0+_1.2*4.C~2.0*# 5.1+1.9* 18.5+2.0# -
Activated Macro hages 28.5+12.6 5.3+1.8* 17.6+2.0# 4.5+1.7* (MAC2P 1 Ia+
Morphine
7.%1.6* 12.5+2.5x -
19.2+6.58 5.5+0.5* 9.8+1.9*# 3.5+1.9*# 14.5+2.2 12.0+2.0# -
lMean + S.D. (N=4) *Signi?icantlydifferent from control - virus (p ~0.05) #Significantlydifferert from control + virus (p ~0.05)
Discussion Survival was less in the virus-infected,ethanol-fedmice than was expected if deaths due to exposure to ethanol, and virus, were combined without interaction(18%). On the other hand, survival in mice injected with morphine and retrovirus-infected was higher than would have been expected from the mortality estimated to be due to combinationof survival rates which was caused by morphine injection,and by virus infection alone (17.3%). The altered survivalmay be in response to several immunological changes. Even though the LP-BM5 MuLV infected mice are more susceptibleto infectiousdiseases (8,9,12,13),the mice in clean animal facilitiesas in our study are presumed to be relativelyunexposed to pathogens compared to humans infected with HIV (2) which would affect the evaluationof disease resistance. In addition, virus-stimulatedlymphoid cells yield increased lymphomas and expanded lymphoid tissues, bringing premature death (8,9,12,13). Thus suppressedor enhanced stimulationof lymphoid cells, particularlyB and T cells, would be expected to change survival (8,9). Finally, recent in vitro work shows that immunologicalstimulationof cells increased the likelihoodthat the retroviruswill be transmittedto other cells (15). Lymphoid cell activationmeasured by the percentagewith activationmarkers were changed by our treatments. Thus any of the above mechanisms could have been significantlyinvolved in death rate changes. Others have shown that T-cells are critical to developmentof the murine disease with death, even in the presence of continued viral infection (13). Nude mice without mature T-cells did not die as early a8 their immunologicallycompetentmice did (12). Thus, ethanol which increased
X
Drugs, Immunomodulation and Murine AIDS
Vol. 43, No. 6, 1988
T-helper and T-suppressor cells, and reduced total T-cells in virally infected mice compared to CI may exacerbate immunological changes caused by retroviral infection. Morphine may not have these effects as it tended to reduce total T-cells, while relatively increasing macrophages in infected mice. The increase in activated macrophages due to morphine occurred concomitantly with an increase in macrophages so that the ratio was similar to infected mice and lower than controls, and particularly ethanol mice. The effect of morphine to increase the percentage of activated lymphocytes and macrophages could increase the cells which can be infected by retrovirus. LP-BM5 MuLV infects T-cells and such changes suggest that drugs of abuse modulate aspects of the cellular immune systems, particularly in retrovirally infected mice. They could be cofactors in modifying the progression to AIDS by retroviruses. We found that macrophage subsets in the peripheral blood were significantly affected by repeated, short-term consumption of ethanol, confirming our work with spleen cells in rats chronically fed ethanol over months without liver damage (4). The number of macrophages including activated ones decreased (Table II> as well as B cells after dietary ethanol exposure. In a separate study, ethanol-fed mice had reduced Kuppfer cell functions in vivo including reduced uptake of latex particles. The retrovirus infection after 1 month also suppressed phagocytosis, as did dietary ethanol (McCusky, Darban, and Watson, unpublished data). In distinction to young mice exposed to 7% ethanol (121, 5% dietary ethanol did aot suppress lymphocyte subsets, except those with activation markers (IL-2R). This may reflect the increased maturity of the latter mice, and the lower level of ethanol in the diet at the time subsets were measured. It suggests that a short-term exposure needs to be done early in life or at a high level to change T-lymphocyte development reflected in alterations in the percentage of regulatory cells. Changes have been reported in the functioning as well as in numbers of lymphoid cells and their different parameters of immunity by long-term ethanol use with its frequent organ damage (3,5,6). The chronic, occasional exposure to dietary ethanol during retroviral infection significantly affected the numbers of various T-subsets in the peripheral blood, while not affecting uninfected mice. This may be the first demonstration of a dietary component acting to uniquely alter T-cells during a retroviral infection. Thus ethanol immuaomodulation could be a cofactor in the progression of the infection if these changes alter infectious disease resistance which infection with LP-BM5 MuLV already suppresses (13) in addition to changing lymphoid cell division and associated pathogenesis. Changes in numbers aad activation of T cells and macrophages are indicative of potentially decreased host defenses. The fact that ethanol exposure, similar to long binges in man, caused significant functional changes during retroviral infection needs more detailed studies. The levels of ethanol in the blood was similar to levels attainable in man and are associated with withdrawal signs in the mice. The loss of T-lymphocytes observed with high, short-term alcohol consumption is supported by the finding of a significantly low weight of thymus and a significantly low cell count in the spleens aad thymus glands of ethanol consuming rats (4). There was also a significant increase of the T-helper/T-suppressor ratio. Therefore the existing evidence indicates that high levels of ethanol and/or its' metabolites has the potential including during retrovirus infection, to alter the numbers of cells which could participate in cellular immune responses.
Vol. 43, No. 6, 1988
Drugs, Immunomodulationand Murine AIDS
Xi
LP-BM5 MuLV infection resulted in suppressionin the numbers of T-helper cells and macrophagesconfirmingprevious results (8,9,12-14). We found in addition that activated macrophageswere reduced by the infection. The loss of both macrophages,as well as activated macrophages (MAC-21 and lymphocytes(IL-2R positive cells), may be critical in the eventual death and altered immune functions associatedwith the virus. It can explain changes in immunoregulationand reduction in disease resistanceto pathogens caused by this retrovirus(8,9). Acknowledgments Supported by grant from Phi Beta Psi Sorority and Wallace Genetics, Inc. to R.R.W. and a Veterans Administrationresearch grant to T.L.S. Reading of T subsets on EPICS flow cytometerwas carried out as a courtesy by M.J. Hicks, M.D. Appreciationis expressed to Dr. T. Burks and Dr. F. Porreca in providing advice and the morphine used. We appreciateDr. E. Hersh suggestingthe LP-BM5 model, and Dr. R.A. Yetter for supplying the virus inocula. References L. SIEGEL. J. Subst. Abuse Treat. 3 271-274 (1986). A.S. FAUCI. Science. 239 617-622 (i988). R.R. MACGREGOR. J. AmerMed. Assn. 256 1474-1479 (1986). S.I. MUFTI, R. PRABHALA, S. MORIGUCHI7.G. SIPES, and R.R. WATSON. Immunopharmacology. 15:85-94 (1988). 5. R.R. WATSON. In: NuGition Disease Resistance,and Immune Function, R.R. Watson (Ed.) M. DelckerInc., pp. 313-329 (1984). 6. R.R. WATSON, J.C. JACKSON, R. HARTMANN,D. SAMPLINER,D. MOBLEY, and C. ESRELSON. Alcohol. Clin. Exper. Res. 9 248-254 (1985). 7. M.D. YAHYA, and R.R. WATSON. Life sci. 4i 2503-2510 (1987). 8. D.E. MOSIW, R.A. YETTER, and H.C. MORSETIII. J. Exp. Med. 161 766-784 (1985). 9. R.R. WATSON. Life Sci. 42 (1988). J. Pharmacol.Exp. Ther. -199 158-170 10. R.F. RITZMANN, and B. TmOFF. (1976). 11. R. LATARJET and J.F. DUPLAN. Int. J. Radiat. Biol. 5 339-344 (1962). 12. D.E. MOSIER, R.A. YETTER, and H.C. MORSE, III. J. Egp. Med. -165 1737-1782 (1987). 13. R.M.L. BULLER, R.A. YETTER, T.N. FREDRICKSON,and H.C. MORSE, III. J. Virology. 61 383-387 (1987). 14. R.R. WATSON7H.L. GENSLER, G.T. BOWDBN, and S. MORIGUCHI. Cancer Lett. 36 181-187 (1987). 15. EL. DOROTHY, B. YOFFE, C.G. BOSWORTH, F.B. HOLLJNGFR,and R.R. RICH. FASEB J. 2 251-255 (1988). 1. 2. 3. 4.
Peragmon Press
CHANGES IN LYMPHOCYTEAND MACROPHAGE SUBSETS DUE TO MORPHINE AWD ETHANOL TREATMENTDURING.A RETROVIRUS INFECTION CAUSING MURINE AIDS Ronald Ross Watsonl, Rao Ii.Prabhalal,Hamid R. Darbanl, M. Danial Yahyal, and Thomas L. Smith2s3 Department of lPamily and Community Medicine, and 2Pharmacology, University of Arizona and 3VeteransAdministrationMedical Center, Tucson, A% 85724.
Infection by LP-BMS murine leukemia virus (MuLV) suppressed significantlythe percentage of peripheral blood cells showing surface markers for macrophages,lymphocytesand activated lymphoid cells. Chronic administrationof a 7% (36% calories) ethanol diet or Injection of 1.9 mg/mouse/dayof morphine for a 7 day period were followed by 3 week periods of abstinenceand then 1 week periods of consumptionof 5% ethanol diets or morphine injection to female C57BL/6 mice resulted in changes in the numbers of macrophagesand lymphocytesubsets. The number of lymphocytesof various subsets were not significantlychanged by the ethanol exposure except those showing activationmarkers which were reduced. The percentage of peripheral blood cells showing markers for macrophage functions and their activationwere significantlyreduced after "binge" use of ethanol. Ethanol retarded suppressionof cells by retroviral infection. However by 25 weeks of infection there was a 8.6% survival in the ethanol fed mice infected with retrovlruswhich was much less than virally infected controls (45.0%). Morphine treatment also increased the percentageof cells with markers for macrophages and activated macrophagesin virally infected mice, while suppressing them in uninfectedmice. The second and third morphine injection series suppressedlymphocyteT-helper and T-suppressorcells, but not total T cells. However, suppressionby morphine was significantly less during retroviraldisease than suppressioncaused by the virus only. At 25 weeks of infection 44.8% of morphine treated, infected mice survived. Morphine treatment also caused deaths such that the survival in morphine treated, retrovirallyinfectedwas higher than would have been expected if the death rate in virally infected, and morphine injected animals were combined during combined treatment. Thus these drugs of abuse can modulate peripheralblood lymphoid subsets, suppressioncaused by retroviralinfection,and survival. Cofactors may be involved in progressionto AIDS (AcquiredImmune Deficiency Syndrome) after HIV (Ruman ImmunodeficiencyVirus) infection (1) via immunomodulation(2). Possible cofactorswhich are immunosuppressive include alcohol (3-6) and morphine (7). Ethanol use has major effects on disease resistancedepending upon the amount and the duration consumed. Alcohol-inducedcirrhosis suppressescellular immune functions as well as resistanceto viral pathogens (3). There is evidence that ethanol acts as an immunosuppressiveagent In vitro and in long-term exposure without liver 0024-3205188$3.00 + .OO Copyright (c) 1988 Pergamon Press plc
iri
Drugs, Immunomodulation and Murine AIDS
Vol. 43, No. 6, 1988
disease (4). Morphine (7) also routinely suppresses cellular immune functions and reduces resistance to various pathogens. Retroviral infection in mice is an exciting system to study the effects of cofactors on immune functions. It has many characteristics of AIDS such that it has been suggested as an excellent murine model for infection with HIV in man (8,9). Therefore the present investigation examines the effects of ethanol and morphine treatments on changes in numbers of regulatory lymphocytes and monocytes/macrophages during murine retroviral infection. Materials and Methods Mice and Treatments. Three to four-week-old C57BL/6 female mice, weighing approximately 15 g, were obtained from Simonsen Laboratories, Inc., Gilroy,-CA:- They were routinely provided with Purina laboratory chow and water ad libitum. Thirty mice were injected with morphine sulfate intraperitoneally every day for 7 days during the same weeks other groups of animals described above were exposed to ethanol. The morphine was given at a daily dose of 75 mg/Kg body weight by intraperitoneal injection. For the initial ethanol exposure 30 mice were subjected to a Carnation Slender liquid diet containing 7% (v/v) ethanol (36% of calories) for the initial 7 days of the study as described previously (10). Mice fed ethanol were labeled "E", while morphine injected were designated "M". Retrovirally infected mice were abbreviated as "I," while mice not exposed to drugs were controls, "C". Thus virally infected controls (not exposed to drugs of abuse) were "C-I". Then E mice were again provided the chow diet for 3 weeks before being fed a 5% ethanol diet for 1 week which was 3 weeks post viral infection. Some mice were sacrificed and the remainder received the chow diet for 3 additional weeks followed by the 5% ethanol diet for 1 week when more were sacrificed at week 8 post infection. Uninfected mice in groups of 20 animals were treated identically with each drug of abuse. C57BL/6 mice chosen because it had previously been observed to consume sufficient amounts of ethanol in a liquid diet to develop functional tolerance and physical dependence (10). Physical dependence to ethanol was achieved as evidenced by the high incidence of convulsions and tremors 4 to 8 hr after the removal of dietary ethanol. Mice used developed signs of ataxia several days before sacrifice. In other experiments similarly treated mice exhibited signs of withdrawal upon cessation of the ethanol diet (unpublished data--T. Smith). Mice were sacrificed by ether anesthesia, and spleen and heparinixed blood were collected immediately. LP-BM5 Murine Leukemia (MuLV) Infection. The retrovirus mixture, LP-BM5, was originally isolated by Latariet and Duulan (11) and nroduces non-neoplastic iymphoproliferative disease in adult mice (12). The ecotropic component of the LP-BM5 MuLV mixture seems to function to promote replication of the poorly replicating, pathogenic Mink Cell Focus-forming component. MuLV LP-BM5 was obtained from a non-thymic lymphoma which had been induced by irradiation of mice. It is a cluster of viruses which include a B-tropic ecotropic virus and a B-tropic mink cell focus-inducing virus (12,131. The latter virus alone does not cause immunological disease, but mice infected with a non-pathogenic ecotropic virus and then infected with the cell focus-inducing virus do. Thus, this virus or one that passages with it is the etiologic agent in the disease with the ecotropic virus functioning to spread it in mouse tissues. After the first week of exposure to drugs of abuse, the animals (30 mice/group) were injected intraperitoneally with 0.1 ml of LP-BM5 murine leukemia inocula which was a kind gift of Dr. R.A. Yetter (Microbiology Research, Veterans Administration Hospital, Baltimore, MD). Twenty mice not exposed to drugs of abuse were infected as controls. Mice were sacrificed at 4 and 8 weeks post injection with LP-BM5 MuLV.
Vol. 43, No. 6, 1988
Drugs, Immunomodulationand Murine AIDS
Vii
Lymphoid Cell Identification. Identificationof T cells isolated from the peripheral blood was carried out as described elsewhere (14). Briefly, heparinixedblood was removed from the brachial artery. Isolated lymphocytesat 5 x lo6 cells/ml in 0.2 ml RPM1 1640 containing 2% Neonatal Calf Serum (NCS) were stained with 5 ul of the appropriateprimary monoclonal antibody (MoAb) in 6 x 50 mm test tubes. These include anti-mouseL3T4 for T-helper cells, anti-Lyt-23as a measure of T-suppressor cells, and anti-IL2-receptor(interleukin-2receptor)as a measure of activation of lymphocytes,anti-Thy-las a measure of total T-cells, anti-MAC-las a marker for total macrophages,anti-MAC-2indicating activatedmacrophages,and B cells measured with anti-IgM (anti-mu chain specific). A conjugate control contained 5 ul of the antibody suspension buffer. The tubes were agitated, sealed with paraffin and incubated on ice for 30 min. The cell-MoAb suspensionwas carefully layered over 0.5 ml of NCS in 6 x 50 mm test tubes and centrifugedat 200 x g for 5 min at 4oC. The supernatantwas decanted and 0.2 ml of diluted fluoresceinconjugated goat anti-rat immunoglobulinwas added. This solutionwas agitated and incubated on ice for 30 min. After incubation,the cell-antibodysolution was again overlaid on to 0.2 ml of NCS and centrifugedas before. The fluoresceinlabeled conjugatewas carefully removed with a Pasteur pipette and any remaining conjugate decanted. Routinely 5000 cells were counted on the FACS flow cytometer. Data are expressed as mean 2 S.D. of the number of observationsindicated. Student T test was used to determine significance. Results Spleen. Ethanol use lowered body weight at 4 and 8 weeks post-infection,while morphine use and retrovirallyinfection did not. At 4 weeks the spleen/bodyweight ratio for C, controls, (0.0035 + 0.0031, CI, infected controls (0.053 + 0.0031, E, ethanol fed (0.0014T 0.00044), EI, ethanol-fed,infected (8.0034 + 0.0005), M, morphine injected-(0.0024+ 0.00101, and MI, morphine injected,-virallyinfected (0.0082 + O.OOll)-micewere similar to values obtained at 8 weeks, except that MI mice were lower than C. T-Cell Subsets. As shown in Table I LP-BM5 MuLV infection suppressed (p< 0.01) the percentageof peripheralblood cells with markers for T-helper, T-suppressor,B cells and activated cells showing IL-2R marker, but not total T-cells. Ethanol increased the percentageof T-helper cells in virally infected mice compared to CI (Table I). However, ethanol consumptiondid not affect the percentageof any lymphocytesubsets except those with the activationmarkers, IL-ZR, in uninfectedmice, while morphine suppressedthem. Similar effects were seen with the T-suppressorcells with both compounds. Neither drug of abuse significantlyaffected total T cells in uninfectedmice, while both tended to suppress total T cells in infected mice. Both morphine and ethanol suppressedthe percentageof activated lymphocytesidentifiedby IL-2R on their surfaces in uninfectedmice (p< 0.05). Morphine had a tendency to increase IL-2R bearing cells during infection compared to CI (Table I>. Essentially identical results were observed at 8 weeks post infection (data not shown). Then the T-helper/T-suppressor(H/S) ratio was 1.6 for C and 2.1 for I mice. The H/S ratio for E and the EI mice were 1.85 and 2.15, respectively. However the H/S ratio for M and MI mice were 2.75 and 2.4 respectively. Macrophage Subsets. As shown in Table II, LP-BM5 MuLV infection reduced (p< 0.01) the percentage of peripheralblood cells with markers for
Vol. 43, No. 6, 1988
Drugs, Immunomodulationand Murine AIDS
Viii
TABLE I Effects of Morphine or Dietary Ethanol on Frequency of Lymphocytes Identified by Surface Markers 4 Weeks Post LP-BMS Murine Leukemia Virus Infection1
Treatment
Control
virus Infection
t
T-helper
36.Ot4.0 23.el.9" -
Ethanol
Morphine t
39.e2.0#
40.0&2.8*# 26.0+_2.6*18.1+2.3*
T-suppressor/ 21.0+0.8 ll.Otl.l* 20.5+1.5# 17.8+2.3*# cytotoxic Total T cell
68.e2.9
B-cell (Mu positive)
20.5t7.3
6.6+2.5X 20.3+1.3# -
3.3+2.4#
IL2 receptor
26.5+2.1 -
6.3+_2.0* 9.3+1.5*
6.0+2.6*
71.e4.6
67.e2.0
t
61.Ot7.2# -
9.3+1.0*
73.0+3.0
8.6+0.8*
62.3t4.91
10.8+1.6# 12.8+1.1# 6.6+_2.6* lO.Otl.O*
lMean t S.D. (NM41 *SigniTicantlydifferent from control - virus (p< 0.05) #Significantlydifferent from control + virus (p< 0.05)
macrophagee,activated macrophages and Ia bearing cells. Ethanol in the diet and morphine injection both reduced the percentage of these cells (p< 0.05). EI mice had a trend toward a lower percentage of each of these cell types compared to CI, or E (Table II). However morphine injection during infection increased the percentagecompared to morphine treated or virally infected controls (Table II). Extremely similar results were seen at 8 weeks post infection with both compounds (data not shown). The only significantdifferencewas that the total T cells of MI mice were lower than c (p< 0.05). Then the activated macrophage/macrophageratios were very similar to the 8 weeks values. The ratio after 8 weeks of infection was 1.05 for C, and 0.7 for CI. The ratios were different (p< 0.05) for E (1.6) and EI mice (1.11, and for M (1.4) and MI mice (0.65). These treatments also significantlydecreased the number of Iat cells. The greatest degree of decrease was observed in mice infected with retrovirusalone. Morphine alone or in combinationwith retroviralinfection produced a lesser, yet significant,decrease (Table II). Mouse Mortality. After 25 weeks of infection there had been mortality in the CI mice with 45.0% surviving. Among the controls, not treated with a drug of abuse or infected, none of the mice died by 25 weeks. However, in the EI mice there was greater mortality with 8.6% surviving. There were deaths among E mice with 41.7% surviving. In these mice deaths occurred at first two ethanol exposures when mice were small, while in the EI mice deaths continued throughout the experimentparticularlyfrom 20-25 weeks after infection. The difference in survival was less for EI mice (p< 0.05). Among the MI animals at 25 weeks post infection 44.8% survived.
Vol. 43, No. 6, 1988
Drugs, Immunomodulationand Murine AIDS
iX
There were deaths after morphine expoeure and 38.4% survived of M mice at 25 weeks post-infectionwhich was not significantlydifferent.
TABLE II Effects of Morphine and Dietary Ethanol on Frequency of Macrophages Identifiedby Surface Markers 4 Weeks Post LP-BM5 Murine Leukemia Virus Infection1
Treatment
Control
Ethanol
Virus Infection
+
+
Macro hage $ (MACl)
25.5f9.8
+
7.321.7" 11.0+_1.2*4.C~2.0*# 5.1+1.9* 18.5+2.0# -
Activated Macro hages 28.5+12.6 5.3+1.8* 17.6+2.0# 4.5+1.7* (MAC2P 1 Ia+
Morphine
7.%1.6* 12.5+2.5x -
19.2+6.58 5.5+0.5* 9.8+1.9*# 3.5+1.9*# 14.5+2.2 12.0+2.0# -
lMean + S.D. (N=4) *Signi?icantlydifferent from control - virus (p ~0.05) #Significantlydifferert from control + virus (p ~0.05)
Discussion Survival was less in the virus-infected,ethanol-fedmice than was expected if deaths due to exposure to ethanol, and virus, were combined without interaction(18%). On the other hand, survival in mice injected with morphine and retrovirus-infected was higher than would have been expected from the mortality estimated to be due to combinationof survival rates which was caused by morphine injection,and by virus infection alone (17.3%). The altered survivalmay be in response to several immunological changes. Even though the LP-BM5 MuLV infected mice are more susceptibleto infectiousdiseases (8,9,12,13),the mice in clean animal facilitiesas in our study are presumed to be relativelyunexposed to pathogens compared to humans infected with HIV (2) which would affect the evaluationof disease resistance. In addition, virus-stimulatedlymphoid cells yield increased lymphomas and expanded lymphoid tissues, bringing premature death (8,9,12,13). Thus suppressedor enhanced stimulationof lymphoid cells, particularlyB and T cells, would be expected to change survival (8,9). Finally, recent in vitro work shows that immunologicalstimulationof cells increased the likelihoodthat the retroviruswill be transmittedto other cells (15). Lymphoid cell activationmeasured by the percentagewith activationmarkers were changed by our treatments. Thus any of the above mechanisms could have been significantlyinvolved in death rate changes. Others have shown that T-cells are critical to developmentof the murine disease with death, even in the presence of continued viral infection (13). Nude mice without mature T-cells did not die as early a8 their immunologicallycompetentmice did (12). Thus, ethanol which increased
X
Drugs, Immunomodulation and Murine AIDS
Vol. 43, No. 6, 1988
T-helper and T-suppressor cells, and reduced total T-cells in virally infected mice compared to CI may exacerbate immunological changes caused by retroviral infection. Morphine may not have these effects as it tended to reduce total T-cells, while relatively increasing macrophages in infected mice. The increase in activated macrophages due to morphine occurred concomitantly with an increase in macrophages so that the ratio was similar to infected mice and lower than controls, and particularly ethanol mice. The effect of morphine to increase the percentage of activated lymphocytes and macrophages could increase the cells which can be infected by retrovirus. LP-BM5 MuLV infects T-cells and such changes suggest that drugs of abuse modulate aspects of the cellular immune systems, particularly in retrovirally infected mice. They could be cofactors in modifying the progression to AIDS by retroviruses. We found that macrophage subsets in the peripheral blood were significantly affected by repeated, short-term consumption of ethanol, confirming our work with spleen cells in rats chronically fed ethanol over months without liver damage (4). The number of macrophages including activated ones decreased (Table II> as well as B cells after dietary ethanol exposure. In a separate study, ethanol-fed mice had reduced Kuppfer cell functions in vivo including reduced uptake of latex particles. The retrovirus infection after 1 month also suppressed phagocytosis, as did dietary ethanol (McCusky, Darban, and Watson, unpublished data). In distinction to young mice exposed to 7% ethanol (121, 5% dietary ethanol did aot suppress lymphocyte subsets, except those with activation markers (IL-2R). This may reflect the increased maturity of the latter mice, and the lower level of ethanol in the diet at the time subsets were measured. It suggests that a short-term exposure needs to be done early in life or at a high level to change T-lymphocyte development reflected in alterations in the percentage of regulatory cells. Changes have been reported in the functioning as well as in numbers of lymphoid cells and their different parameters of immunity by long-term ethanol use with its frequent organ damage (3,5,6). The chronic, occasional exposure to dietary ethanol during retroviral infection significantly affected the numbers of various T-subsets in the peripheral blood, while not affecting uninfected mice. This may be the first demonstration of a dietary component acting to uniquely alter T-cells during a retroviral infection. Thus ethanol immuaomodulation could be a cofactor in the progression of the infection if these changes alter infectious disease resistance which infection with LP-BM5 MuLV already suppresses (13) in addition to changing lymphoid cell division and associated pathogenesis. Changes in numbers aad activation of T cells and macrophages are indicative of potentially decreased host defenses. The fact that ethanol exposure, similar to long binges in man, caused significant functional changes during retroviral infection needs more detailed studies. The levels of ethanol in the blood was similar to levels attainable in man and are associated with withdrawal signs in the mice. The loss of T-lymphocytes observed with high, short-term alcohol consumption is supported by the finding of a significantly low weight of thymus and a significantly low cell count in the spleens aad thymus glands of ethanol consuming rats (4). There was also a significant increase of the T-helper/T-suppressor ratio. Therefore the existing evidence indicates that high levels of ethanol and/or its' metabolites has the potential including during retrovirus infection, to alter the numbers of cells which could participate in cellular immune responses.
Vol. 43, No. 6, 1988
Drugs, Immunomodulationand Murine AIDS
Xi
LP-BM5 MuLV infection resulted in suppressionin the numbers of T-helper cells and macrophagesconfirmingprevious results (8,9,12-14). We found in addition that activated macrophageswere reduced by the infection. The loss of both macrophages,as well as activated macrophages (MAC-21 and lymphocytes(IL-2R positive cells), may be critical in the eventual death and altered immune functions associatedwith the virus. It can explain changes in immunoregulationand reduction in disease resistanceto pathogens caused by this retrovirus(8,9). Acknowledgments Supported by grant from Phi Beta Psi Sorority and Wallace Genetics, Inc. to R.R.W. and a Veterans Administrationresearch grant to T.L.S. Reading of T subsets on EPICS flow cytometerwas carried out as a courtesy by M.J. Hicks, M.D. Appreciationis expressed to Dr. T. Burks and Dr. F. Porreca in providing advice and the morphine used. We appreciateDr. E. Hersh suggestingthe LP-BM5 model, and Dr. R.A. Yetter for supplying the virus inocula. References L. SIEGEL. J. Subst. Abuse Treat. 3 271-274 (1986). A.S. FAUCI. Science. 239 617-622 (i988). R.R. MACGREGOR. J. AmerMed. Assn. 256 1474-1479 (1986). S.I. MUFTI, R. PRABHALA, S. MORIGUCHI7.G. SIPES, and R.R. WATSON. Immunopharmacology. 15:85-94 (1988). 5. R.R. WATSON. In: NuGition Disease Resistance,and Immune Function, R.R. Watson (Ed.) M. DelckerInc., pp. 313-329 (1984). 6. R.R. WATSON, J.C. JACKSON, R. HARTMANN,D. SAMPLINER,D. MOBLEY, and C. ESRELSON. Alcohol. Clin. Exper. Res. 9 248-254 (1985). 7. M.D. YAHYA, and R.R. WATSON. Life sci. 4i 2503-2510 (1987). 8. D.E. MOSIW, R.A. YETTER, and H.C. MORSETIII. J. Exp. Med. 161 766-784 (1985). 9. R.R. WATSON. Life Sci. 42 (1988). J. Pharmacol.Exp. Ther. -199 158-170 10. R.F. RITZMANN, and B. TmOFF. (1976). 11. R. LATARJET and J.F. DUPLAN. Int. J. Radiat. Biol. 5 339-344 (1962). 12. D.E. MOSIER, R.A. YETTER, and H.C. MORSE, III. J. Egp. Med. -165 1737-1782 (1987). 13. R.M.L. BULLER, R.A. YETTER, T.N. FREDRICKSON,and H.C. MORSE, III. J. Virology. 61 383-387 (1987). 14. R.R. WATSON7H.L. GENSLER, G.T. BOWDBN, and S. MORIGUCHI. Cancer Lett. 36 181-187 (1987). 15. EL. DOROTHY, B. YOFFE, C.G. BOSWORTH, F.B. HOLLJNGFR,and R.R. RICH. FASEB J. 2 251-255 (1988). 1. 2. 3. 4.