- Email: [email protected]
II status in anti-HIV-1 -negative intravenous drug users receiving methadone
Immune Function and Anti-HTLV-I/II Status in Anti-HIV-l-Negative Intravenous Drug Users Receiving Methadone NANCY G. KLIMAS, M.D., NANCY T. BLANEY, Ph.D., ROBERT 0. MORGAN, Ph.D., DALE CHITWOOD, Ph.D., KAREN MILLES, Ph.D., Miami, Florida, HELEN LEE, Ph.D., North Chicago, Illinois, MARY ANN FLETCHER, Ph.D., Miami, Florida
PURPOSE: The study objective was to evaluate the effects of long-term methadone use and human T-cell leukemia virus (HTLV) types I and H seropositivity on the distribution of lymphocyte subsets and on lymphocyte function as measured in vitro in intravenous drug users seronegative for human immunodeficiency virus type 1 (HLV-1). PATIENTS AND MEX!RODs: Anti-~-l-negative
intravenous drug users receiving methadone maintenance therapy (n = 24) were studied in a Veterans Administration drug abuse treatment center. These subjects were compared to 38 ageand sex-matched control subjects who did not abuse drugs. HIV-l and HTLV serostatus was determined by repetitive enzyme-linked immuuosorbent assay and confirmed by immunoblot. Lymphocyte subsets were determined by twocolor flow cytometry. Lymphocyte function was measured by proliferative response to plant mitogens and by natural killer (NH) cell-mediated cytotoxicity to a tumor cell target. RESULTS: &$ificant
differences
were
seen in
lymphocyte phenotype in the methadone-treated group, with elevations in the T-cell helper subset CD4+CD29+; in CD8 and CD8+I2+ cells, suppressor/cytotoxic T lymphocytes, and activated suppressor/cytotoxic T cells; and in CD2+CD26+ cells and activated total T lymphocytes. Lymphocyte function was suppressed in the methadone group, with poor responses to pokeweed mitogen and phytohemagglutiuin in culture. Moreover, NH-cell cytotoxicity was significantly reduced in the methadone group. From the Center for the Biopsychosocial Study of AIDS (NGK. NTB. ROM, DC, KM, MAF). and Miami Veterans Administration Medical Center (NGK, MAF), University of Miami School of Medicine, Miami, Florida, and 1 Abbott Laboratories (HL), North Chicago, Illinois. This work was supported in part by Grant P50-MH/DA42455 from the National Institute of Mental Health. Requests for reprints should be addressed to Nancy G. Klimas. M.D.. Department of Medicine-R42, University of Miami School of Medicine, P.O. Box 016960, Miami, Florida 33101. Manuscript submitted March 2. 1990, and accepted in revised form August 14, 1990.
None of these immunologic differences were attributable to HTLV serostatus. CONCLUSION: The immune abnormalities seen suggest that a clinically significant degree of immune impairment exists in methadone-treated intravenous drug users. However, these abnormalities could not be explained by the presence of other retroviruses in this HIV-l-negative study group, as there was no significant difference in immune function when HTLV-seropositive patients were compared to HTLV-seronegative subjects treated with methadone.
ntravenous drug users are at high risk for human immunodeficiency virus type 1 (HIV-l) infection due to injection-associated behaviors [1,2]. Moreover, heroin and other street drugs may modulate immunocompetence [3-51, possibly contributing to the high incidence of bacterial and viral infection and cancers in opiate addicts [4,6,7]. Such opiateinduced immunomodulation would bode poorly for HIV-l infection in intravenous drug users [5]. Finally, intravenous drug users pose a risk for heterosexual transmission of HIV-l infection [2], thus promoting the spread of HIV-l infection to those who are not intravenous drug users. Given these imperatives for reducing HIV-l infection risk among intravenous drug users, it is not surprising that methadone treatment has emerged as a potentially important prevention modality [8,9], with more funding planned for methadone programs. Hence, it is of considerable importance to ascertain that methadone treatment is not in itself immunosuppressive lest it paradoxically elevate the risk of HIV-l infection. Studies have been reported suggesting that methadone can be immunosuppressive [lo-121; that it does not affect immune function [13]; or that it can reverse some of the immunologic effects of heroin [4]. Results from our laboratory showed that intravenous drug users in methadone treatment evidenced B-cell dysfunction and polyclonal B-cell activation, despite their anti-HIV-1-seronegative
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status [14]. Consequently, because of the ambiguity of the literature, and because this opiate is being used with increasing frequency in a population at high risk for exposure to HIV-l, the first aim of the present study was to clarify the immunologic impact of methadone treatment, using a more comprehensive panel of immune markers than typifies previous research. However, an understanding of the immunologic impact of methadone is complicated by the confounding role of human T-cell leukemia virus (HTLV), a family of pathogenic retroviruses [15]. Two subtypes are recognized. HTLV-I is associated with serious illnesses in a small minority of infected individuals, including hematologic malignancy and neurologic disease [ 161. HTLV-II has been isolated rarely in hairy cell leukemia, but is, perhaps, more properly categorized as an orphan virus [17]. These viruses are evidently passed via shared needles, although there is considerable geographic variation, as indicated by the rather high prevalence of HTLV, more frequently HTLV-II than HTLV-I, infection among intravenous drug users in certain urban areas of the United States [18,19], while low or absent in much of Europe [19-211. The impact of co-infection with HTLV in HIV-l-infected intravenous drug users is apparently serious. A threefold increase in mortality has been reported [22]. Although data on the immunologic impact of asymptomatic HTLV infection are limited [23,24], recent research with methadone users [25] has associated HTLV seropositivity with abnormalities in CD4 lymphocyte subpopulations. This suggests that some of the immunomodulation observed with methadone may actually be attributable to HTLV infection. Given the relevance of this finding for both HIV-l infection and public policy concerning methadone treatment, replication of these findings and further investigation of HTLV and immune function during methadone treatment are clearly warranted, especially because of the high prevalence of HTLV infection among intravenous drug users in the U.S. The second aim of the present study, then, included assessment of whether HTLV abnormalities are attributable to, or independent of, HTLV seropositivity.
PATIENTS AND METHODS Design
Comprehensive measures of immune function of anti-HIV-l-negative intravenous drug users in methadone treatment were compared to those of anti-HIV-l-negative control subjects. HTLV-I/II serostatus and drug-using histories were also determined. Anti-HIV-l-negative subjects were selected because, in order to understand the immunologic 164
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effects of HIV-l in intravenous drug users, it is first necessary to evaluate the immunologic status of intravenous drug users prior to infection with this lymphotropic virus. Patients
Anti-HIV-l-negative intravenous drug users undergoing methadone maintenance treatment (n = 24), all males, were recruited from the Miami Veterans Administration outpatient Drug and Rehabilitation Unit. Subjects were white (n = 14), black (n = 7), or Hispanic (n = 3), with a mean age of 42, moderately well educated (some high school: n = 3; high school or some college: n = 21), but with limited employment (full time: n = 9; part time: n = 3; unemployed: n = 12) and income (below $10,000: n = 15; $10,000 and above: n = 9). A physician performed a complete physical examination and obtained a medical history in all subjects. An extensive substance-abuse history was obtained by a structured interview devised and extensively field-tested in Miami [1,23,26,27]. Toxicology screens and blood chemistries (SMAC 23) were done. Subjects were excluded if they had acquired immunodeficiency syndrome (AIDS) or AIDS-related complex as defined by Centers for Disease Control class IV criteria, if there was evidence of acute infectious illness, immunosuppressive illness, or positive toxicology screen, or if they were taking immunosuppressive non-recreational drugs. Anti-HIV-l-negative, non-substance-abusing men (n = 38), matched by age, served as the comparison group. Blood Samples
To control for diurnal variations, all blood samples were obtained from fasting subjects between 7:30 and lo:30 AM. Peripheral venous blood samples were collected into appropriate tubes: heparin tubes for in uitro functional assays and flow cytometry (Vacutainefl-sodium heparin, Becton-Dickinson, Rutherford, New Jersey); EDTA for white blood cell and differential counts (CBC). Whole blood samples were held no longer than 24 hours at room temperature before preparation and fixation for flow cytometry assay or more than 4 hours for CBC or for functional assays. For serum samples, peripheral venous blood was collected and then allowed to clot at 23OC for 30 minutes, at which time the serum was separated from the clot and stored at 20°C. Flow Cytometry
A single-laser flow cytometer (EPICS C, Coulter Instruments Laboratories, Hialeah, Florida) was used with a whole-blood, two-color analysis proce90
dure as previously described to determine the distribution of lymphocyte phenotypes [28]. The following pairs of fluorescein isothiocyanate (FITC)or phycoerythrin (PE)-conjugated monoclonal antibodies (Coulter Immunology, Hialeah, Florida) were selected: Tll-FITC for CD2 or sheep erythrocyte receptor-bearing cells [29] and Tal-PE to measure a surface marker associated with T memory cell activation (CD26) [30]; T4-FITC for CD4 or helper/inducer cells [31] combined either with 4B4PE (CD29) to measure the subset of CD4 that helps B cells to respond to antigen stimulus with immunoglobulin synthesis (321, or with 2H4-PE (CD45RA), which activates CD8 cells to act as either suppressor cells or cytotoxic cells [33]; T8FITC (CD8) or suppressor/cytotoxic T cells [34] plus IZ-PE to measure HLA-DR activation antigen [35] expression on CD8; Bl-FITC (CD20) for determining B cells [36]; Mo2-FITC for CD14 or monocytes [37] combined with IQ-PE to determine the degree of expression of the HLA-DR class II activation antigen on monocytes; NKH.l-PE (CD56), which defines the entire pool of mononuclear cells with natural killer (NK) activity [38]. Isotypic controls were mouse IgGl, IgG2, or IgM (Coulter Immunology). Peripheral lymphocyte counts were calculated by multiplying the total white blood cell count and percentage of lymphocytes as determined from a Cell Dyne 1500@ (Sequoia-Turner, Mountain View, California). Estimates of absolute numbers of the lymphocytes or mononuclear cell populations positive for the respective surface markers were determined by multiplying peripheral lymphocyte or mononuclear cell counts by percentage positive cells for each surface marker [39]. Lymphocyte Proliferation Assay Lymphocyte proliferation to mitogen stimulation was measured using a whole-blood procedure [39]. Briefly, 100 PL of diluted heparinized blood (1:5) was dispensed in triplicate to the wells of a U-bottom microtiter plate (Costar, Cambridge, Massachusetts). Mitogens were tested at the following levels: phytohemagglutinin (PHA; Wellcome Diagnostics, Dartford, England) 10 Fg/mL and pokeweed mitogen (PWM; Gibco, Chagrin Falls, Ohio) diluted 1:40. Plates were incubated for 72 hours. During the last 6 hours of the appropriate incubation time, cultures were pulsed with 25 PL tritiated thymidine (New England Nuclear, Boston, Massachusetts)-1 PCi per well. Cultures were harvested onto glass filter paper discs (Titertek@ Cell Harvester, Flow Laboratories, McLean, Virginia), immersed in scintillation fluid, and counted on a beta-scintillation counter (LKB Instruments,
Rockville, Maryland). Results were expressed as mean net counts per minute (cpm) incorporated per 100,000 lymphocytes. NK-Cell Cytotoxicity NK-cell function was evaluated by determining cytotoxicity using the whole-blood chromium release assay as outlined in detail by Baron et al [40] and Fletcher et al [39]. The target cell line utilized was the NK-sensitive erythroleukemic K562 cell line. The assay was done in triplicate, at four targetto-effector cell ratios with a 4-hour incubation. The percent cytotoxicity at the four target-to-effector ratios and the number of CD56+ cells per unit of blood were used to express the results as percent cytotoxicity at a target-to-effector cell (CD56+) ratio of 1:l. Calculations were performed using an AT&T computer program written in-house. Kinetic lytic units per NK cell (KLU/NK cell) were calculated as previously indicated [40] in order to estimate the maximum number of targets lysed by each NK cell (CD56+) during the 4-hour assay. Viral Serology Antibodies to HIV-l were measured using the enzyme-linked immunosorbent assay (ELISA) and Abbott (North Chicago, Illinois) reagents. Reactive samples were repeated and doubly positive samples were confirmed by protein immunoblot. The presence of HIV-l viremia (~24) was determined using an ELISA method and Abbott reagents. Antibodies to HTLV were measured by ELISA using Abbott reagents. Repeatedly positive samples were confirmed by protein immunoblot and radioimmunoprecipitation assay (RIPA) using the HaT102B2 cell line as source of antigen and criteria as previously described [19]. Due to the high level of cross reactivity, the serologic methods of identification used do not distinguish between the subtypes I and II. Data Analysis Non-parametric rank order analyses were used to evaluate between-group differences. Given the large number of potential statistical tests, a combined multivariate and univariate strategy was used to help control the experiment-wide type I error rate. The immunologic parameters were clustered into two subgroups: (1) T-cell subsets expressed as percentages of the overall numbers of lymphocytes; and (2) measures of NK-cell function. A non-parametric multivariate analysis was performed on the T-cell subsets to determine whether this cluster evidenced statistically significant between-group differences. The non-parametric multivariate procedure used is an extension of the
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TABLE I Anti-HIV-l-Negative, Anti-HTLV-I/II-Negative and -Positive Methadone Clinic Subjects: Selected immunologic Markers* Anti-HTLV-Negative (n = 16)
Marker CD$+/mms CD4+% CD4+CD29+/mm3 CD4+CD29+% CD4+CD45RA+/mmJ CD4+CD45RA+% ;;zT(,mm3
1.041.0(849.1/1,295.1)~ 0.40 (0.39/0X) 715.1(475.4/1.105.0) 0.29(0.25/0.36) 212.8(134.9/530.1) 0.10 (0.07/0.18) 907.4(595.2/1.021.4) 0.33(0.27/0.44) 175.6(116.8/240.0) 0.08(0.04/0.11) 549.8(418.2/1.358.4) 0.28 (0.16/0.49) 333.4(280.5/409.9) 0.15(0.12/0.17) 25.9(14.8/30.4) 0.35(0.21/0.60)
CD8+P+/mmJ CD8+12+% CD2+CD26+/mm3 CD2+CD26+% CD56+/mmJ CD56+% NK % cytotoxicity KLU/NK All between-group comparisons t Midian (25%/75%).
l
were statistically
non-significant
using Kruskal-Wallis
analyses of variance
Kruskal-Wallis one-way analysis based on ranks [41,42]. Chi-square values for performing hypothesis tests are easily obtainable using the statistical analysis system (SAS) [43] and following procedures outlined by Zwick [44]. Univariate KruskalWallis analyses were performed to help interpret the multivariate result. In addition, NKH.l cell counts, NKH.l percentages, and the measures of T-, B-, and NK-cell function were collected on a subset of the overall laboratory control group. Only univariate analyses were performed on these measures. Since these subsets were only partially overlapping, univariate analyses were done on these measures, An experiment-wide type I error rate (ew) of 0.05 was used to identify statistically reliable results. An ew = 0.10 was used to identify marginally reliable results. Since multiple analyses were performed, Sidak’s [45] a-adjustment was used to calculate a-levels for the individual analyses, yielding individual analysis a-values of 0.009 and 0.017 corresponding to the ew values of 0.05 and 0.10, respectively. Other probability is represented where meaningful. RESULTS Serology
The intravenous drug users receiving methadone treatment were both anti-HIV-l-negative and negative for p24 antigen. However, nine (31%) of the 29 intravenous drug users were positive for antibodies to HTLV-I/II (repeatedly ELISA-positive and confirmed by protein immunoblot and RIPA). Neither the anti-HTLV-I/II-negative nor the anti-HTLV-I/II-positive methadone clinic subjects in this study differed from the laboratory controls in their total lymphocyte counts (data not shown). In addition, the anti-HTLV-I/II-negative and -pas166
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1.146.1(902.5/1,699.9) 0.50(0.42/0.53) 731.3 (501.6/975.7) 0.30(0.22/0.34) 172.7 (121.7/599.8) 0.09(0.06/0.24) 818.9 (624.5/1,038.7) 0.34(0.31/0.40) 125.1 (87.1/209.8) 0.07(0.04/0.09) 4?4.7(364.3/884.1) 0.24(0.15/0.36) 250.2(164.0/334.5) 0.11 (0.07/0.14) 12.0(11.1/21.6) 0.37(0.20/0.68) on ranks. Results for T- and B-cell function are presented
in Table IV.
itive methadone clinic subjects did not differ in terms of their percentages of various lymphocyte subsets, or in their measures of NK-cell function (Table I). Consequently, for the lymphocyte enumeration and NK-cell function comparisons presented in the next two paragraphs (Tables II and III), the anti-HTLV-I/II-negative and -positive methadone subjects are combined into a single methadone group. For the T- and B-cell function analyses, the anti-HTLV distinction is retained. Lymphocyte Enumeration
As indicated in Table II, the methadone group (anti-HTLV-I/II-negative and -positive subjects combined) had higher percentages than the laboratory controls for the following markers: CD4+CD29+; CD8; CD8+12+; and CD2 cells coexpressing CD26, a marker expressed on activated memory T cells. The lab control subjects and methadone clinic patients did not differ significantly in percentages of either CD4 cells or CD4+CD45R+ subset. In addition, there were no differences among the three groups in percentage of NKH.l+ celIs (data not shown). The absolute counts presented in Table II reflect the differences seen in the percentages. Functional Assays NK-CELL FUNCTION: As indicated in Table III, methadone subjects (regardless of anti-HTLV-I/II serostatus) had lower levels of NK percent cytotoxicity, higher levels of lytic units, and lower levels of KLU/NK cell than the laboratory controls. As indicated before, the anti-HTLV-I/II-negative and -positive methadone groups did not differ in any of these markers. Given the lack of group differences in the NKH.l enumeration measures (both
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TABLE II
T-Cell Subsets In Male Intravenous Drug Users Receiving Methadone: Subsets Expressed as Percentages and Absolute Counts of Total lymphocytes Methadone Clinic Anti-HIV-l-Negative (n = 24)
Marker
0.45 (0.39/0.53)’ 1,138(849/1,330)
CD456 AbsoluteCD4 CD4+CD29+% AbsoluteCD4+CD29+ CD4+CD45RA+% AbsoluteCD4+CD45RA+ CD8% AbsoluteCD8 CD8+12+% AbsoluteCD8+12+ CD2+CD26+% AbsoluteCD2+CD26+
I
Multivariaterank
ordertestP$=
(n=38) 0.43(0.39/0.49)
1,013(741/1,217) 0.21(0.17/0.25) 496 (326/612) 0.13 (0.09/0.22) 2841190/527) 0.25 (d.21j0.31j
0.30(0.24/0.34) 715(475/1,030)
0.10 (0.06/0.20) 197 (129/542) 0.34(0.28/0.42)
904(601/1,031)
530(447/757) 0.03(0.02/0.05) 74(44/118) 0.16(0.13/0.20) 344(257/510)
0.07(0.04/0.11)
159 (99/226) 0.26(0.16/0.40)
522(418/1.198)
35.58(df=
Comparison* NS p
’
NS p
6), p
Kruskal-Watlis analyses of variance on ranks. + Median (25%/75X). t Multivariate test performed on percentage measures only.
l
TABLE Ill NK&ll
Function In Male Intravenous Drug Users Recelvlng Methadone
Marker ;;;;;totoxicity
Methadone Clinic Anti-HIV-l-Negative (n = 24)
Lab Controls (n=38)
23.2 (11.5/29.2)+ 0.35(0.21/0.60)
47.4(25.4/52.4) 1.31(0.58/1.69)
Unlvarlate Comparison* p <0.002 p <0.008
* Kruskal-Wallis analyses of variance on ranks. t Median (25%/75%).
absolute counts and percentages), these results suggest that it is NK-cell function that is most affected by methadone. T- AND B-CELL FUNCTION: As indicated in Table IV, for T-cell function (PHA response) there was an overall group difference between the laboratory controls and the two groups of methadone-treated subjects (anti-HTLV-I/II-negative and -positive). Interestingly, no differences were observed between the laboratory control subjects and the anti-HTLVI/II-positive methadone subjects. Also, there were no differences between the anti-HTLV-I/II-positive and -negative methadone groups (p
22% of a non-clinic sample. In general, no association was found between use of a variety of drugs (e.g., heroin, cocaine, speedballing) and the immunologic markers. In addition, there were no apparent associations between anti-HTLV seropositivity and either type of drug use or frequency of injection. Alcohol use did show significant associations with the T- and B-cell functional parameters, but did not differ between the anti-HTLV-negative and -positive groups as reported elsewhere by Klimas et al [46]. However, the levels of use among the methadone-treated sample were not greater than those found in other normal populations. Alcohol use data for the laboratory controls were not available. Eighty-nine percent of the anti-HTLV-negative group reported cigarette smoking compared to 57% of the anti-HTLV-positive group. This difference was not statistically significant (p = 0.11, Fisher’s exact test [two-tailed]), possibly due to the small sample size. However, smoking was not significantly correlated (at p
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TABLEIV T-and B-CellFunction in MaleIntravenous Drug Users ReceivingMethadone I Unlvarlate Methadone
Antl-HTLVPosltlve
Anti-HTLVNegative
Marker PWM net cpm
Cllnlc
2,938.0
‘2,38;;/84$4.0’*
PHA net cpm
(29,512.b/78.136.0) * Median (25%/ 75%). t Kruskal-Wallis analyses of variance
11,252.0
(36,603.OjlliO94.0)
on ranks. Note: no differences
Antl-HTLVPositive Versus -Negative
Laboratory Controls Versus Anti-HTLVNegative
Laboratory Controls
All Groups
12,864.5
p <0.005
p
p
<0.002
p x0.02
p <0.12
p
<0.005
(6,509$~;,~51.0)
(2,80~~$‘$~7.0’
Comparlsonst
(54,947.d/bli,246.0)
were found between the HTLV-l/II-positive
methadone
treatment
subjects and the laboratory
controls.
Recognition of the increased risk of AIDS among intravenous drug users has fostered an increased interest in oral methadone maintenance programs as a means of reducing the risk of HIV-l exposure via infected needles. However, the immunologic consequences of long-term methadone use are not well characterized; also, a substantial percentage of intravenous drug users are seropositive for another retrovirus, HTLV, which has been identified as predominantly subtype II when appropriately sensitive methods are used [ 191. Again, the immunologic sequelae of HTLV infection have been addressed in very few studies. Studies are limited on the in uiuo immunologic effects of HTLV infection, both subtypes I and II, in asymptomatic individuals. Yasuda and colleagues [24] reported elevated total T lymphocytes (CD2+) and elevated levels of immunoglobulins. In contrast, Tanaka et al [47] found lower percentages of CD3 and CD4 cells and unchanged percentages of CD8 lymphocytes when they compared HTLV-I carriers with sex- and age-matched controls. Fletcher and the Transfusion Safety Study Group [48] found elevated levels of CD8+, CD8+12+, CD4+CD45RA+, and CD56+ lymphocytes in HTLV antibody-positive blood donors. DeShazo et a1 [25] assessed immunophenotypes and cellular immune function in anti-HTLV-positive and -negative intravenous drug users in a New Orleans methadone treatment program. They reported elevated CD4+CD29+ lymphocytes and poor PWM responses in the anti-HTLV-positive group, but no effect of HTLV serostatus on total number of T lymphocytes (CD2), T-cell subsets (CD4, CD4+CD45R+, CD8), B cells (CD19), or NK cells (CD56), or on NK-cell cytotoxicity or lymphoproliferative response to PHA. They reported that both
HTLV-seropositive and HTLV-seronegative drug users had depressed levels of CD4tCD45RAt lymphocytes. In contrast, the present study indicates that intravenous drug users undergoing methadone treatment in South Florida had no immunologic impairments or alterations that could be attributed to HTLV serostatus. Both anti-HTLV-positive and negative methadone-treated patients had significant elevations in CD4+CD29+ lymphocytes, CD8 cells, and CD8tI2t cells, but depressed NK-cell cytotoxicity and poor lymphocyte response to mitogenie stimulation, when compared to non-drugabusing control subjects. These findings could be interpreted to demonstrate that the immunomodulatory effects of common variables in this methadone treatment population were so pronounced that no independent effects of HTLV infection could be seen. It should be noted, however, that persons recently infected with HTLV were reported to show no immunologic effects [49], and the time since infection with HTLV in our cohort was not defined. The immunologic effects observed are unlikely to be due to street drugs since prospective subjects were excluded from study if urine samples were positive for other drugs. Analysis indicates that the rather infrequent use of street drugs in this group was not related to the immunologic changes obs&ved. Although concurrent studies showed some association between psychosocial variables and immune function, there was no clear pattern of psychoimmunologic relationships that would account for the present findings [50]. Although the changes seen could result from other factors, including subclinical infections, treatment with methadone must be regarded as a likely mediator. Other types of immunomodulation by methadone have been reported. In studies of peripheral blood mononuclear cells from patients receiving methadone, there was a significant impairment of ability to generate superoxide anion. Tubaro et al [ll] re-
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subjects and the intravenous
drug
COMMENTS
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ported that morphine and, to a lesser extent, methadone reduced phagocytosis, intracellular killing, and superoxide production in polymorphonuclear leukocytes and in monocytes. In heroin addicts, both before and after methadone treatment, skin test anergy to seven recall antigens was common [4]. The present study raises concern regarding the possible consequences of methadone-mediated immunologic changes in HIV-l susceptibility. The major changes seen in T-cell subsets were proliferative and activating. The increase noted in CD4+CD29+ lymphocytes deserves further study in light of the observations of Fletcher et ~1 [51] indicating that this subset may be a preferred target of HIV-1 in early infection. The lymphocyte activation observed in men receiving methadone may also have ominous consequences. It has been repeatedly noted in the literature that HIV-l replicates in activated T cells [52]. Reduction in natural immunity as evidenced by poor in vitro cytotoxicity might lead to compromised antimicrobial defense, and perhaps to more rapid onset of secohdary infections with opportunistic pathogens, following HIV-l infection, There is evidence in the literature that intravenous drug abusers tend to have a shorter period of time from infection to onset of AIDS and to mortality [53,54]. These findings underscore the need for further studies. Currently, longitudinal studies of drug users undergoing 6 weeks of inpatient detoxification followed by methadone or nonmethadone interventions are underway.
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L, Trombini M. et a/. Immunologic status of heroin addicts: maintenance treatment. Drug Alcohol Depend 1989: 13:
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human phagocytic physiology. Int J lmmunopharmacol 1985; 7: 865-74. 12. Peterson PK. Sharp B. Geker G. Brummitt C. Kean WF. Opioid-mediated suppression of interferon gamma production by cultured peripheral blood mo= nonuclear cells. J Clin Invest 1987; 80: 824-31. 13. Shine D, Mall B, Emeson E, et al. Serological, immunological and clinical features of parenteral drug users from contrasting populations. Am J Drug Alcohol Abuse 1987; 13: 401-12. 14. Klimas NG. Baron GC, Fletcher MA. Immunology of HIV-l. In: Fields T, McCabe P, Schneiderman N, eds. Stress and coping in disease. New York: Lawrence Erlbaum. in press. 15. Broder S, Gallo RC. Human T-cell leukemia viruses (HTLV): a unique family of pathogenic retroviruses. Annu Rev lmmunol 1985; 3: 321-36. 16. Kim JH, Durack DT. Manifestations of human T-lymphotropic virus type 1 infection. Am J Med 1988; 84: 919-28. 17. Blatner WA. Human T lymphotrophic viruses and diseases of long latency. Ann Intern Med 1989; 1: 4-6. 18. RobertGuroff M, Weiss SH, Giron JA, et a/. Prevalence of antibodies to HTLV-I. -II and -III in intravenous drug abusers from an AIDS endemic region. JAMA 1986: 255: 3133-7. 19. Lee H. Swanson P, Shoarty VS. tack JA. Rosenblatt JD, Chen ISY. High rate of HTLV-II infection in seropositive drug abusers in New Orleans. Science 1989; 244: 471-5. 20. Cour MI. Palau L. Contreras EF. Perezagua C. deGuevara JL. HIV-l. HIV-2, and HTLV-I in Spanish inmates [Letter]. AIDS 1989; 3: 320-l. 21. Fuchs D. Untenveger B. Hausen A, et a/. Anti-HIV-l antibodies, anti-HTLV-I antibodies and neopterin levels in parenteral drug addicts in the Austrian Tyrol. J AIDS 1988; 1: 65-6. 22. Kramer A, Goedert JJ. Blattner WA. No evidence of HTLV-I infection in intravenous drug abusers in West Germany. J AIDS 1988; 1: 163-4. 23. Page JB, Lai S, Chitwood DD, Klimas NG, Fletcher MA. HTLV l/II seropositivity and death from AIDS among HIV-1 seropositiie intravenous drug users. Lancet 1990; 1: 1439-41. 24. Yasuda K. Sei Y. Yokoyama MM. Tanaka K. Hara A. Healthy HTLV-I carriers in Japan: the hematological and immunological characteristics. Br J Haematol
1986; 64: 195-203. 25. DeShazo RD. Chadha N. Morgan JE. et a/. Immunologic assessment of a cluster of asymptomatic HTLV-l-infected individuals in New Orleans. Am J Med
1989; 86: 65-70. 26. Page JB. Shooting scenarios and risk of HIV-l infection. Am Behav Scientist 1990; 33: 478-90. 27. Chitwood DD. Cumerford M. Drugs, sex. and AIDS risk. Am Behav Scientist 1990; 33: 465-77. 28. Fletcher MA, Azen S, Adelsberg 8. eta/. lmmunophenotyping in a multicenter study: the Transfusion Safety Study experience. Clin lmmunol Immunopathol 1989: 52: 38-47. 29. Kamoun M. Martin PJ, Hansen JA. Brown BA. Siadak AW. Nowinski RC. Identification of a human T lymphocyte surface protein associated with the Erosette receptor. J Exp Med 1981; 153: 207-12. 30. Fox DA, Husey RE, Fitzgerald KA. et a/. Tal. a novel 105 KD human T cell activation antigen defined by a monoclonal antibody. J lmmunol 1984; 133:
1250-6. 31. Reinhertz al subsets
EL, Kung PC, Goldstein G, Schlossman SF. Separation of functionof human T cells by a monoclonal antibody. Proc Natl Acad Sci USA
117-23.
1979: 76: 4061-5.
5. Donahoe RM, Falek A. Neuroimmunomodulation by opiates and other drugs of abuse: relationship to HIV infection and AIDS. In: Bridge TP. Mirsky AF. Goodwin FK. et al, eds. Psychological, neuropsychiatric and substance abuse aspects of AIDS. New York: Raven Press, 1988: 145-58. 6. Dismukes WE, Karchmer AW. Johnson RF, Doughterty WJ. Viral hepatitis associated with the illicit parenteral use of drugs. JAMA 1968; 206: 1048-52. 7. Harris PD, Garret R. Susceptibility of addicts to infection and neoplasia [Letter]. N Engi J Med 1972; 287: 310. 8. Dole VP. Implications of methadone maintenance for theories of narcotic addiction. JAMA 1988; 260: 3025-g. 9. Hubbard RL. Marsden ME, Cavanaugh E. Rachal JV. Ginzburg HM. Role of drug-abuse treatment in limiting the spread of AIDS. Rev Infect Dis 1988: IO: 377-84. 10. Singh VK, Jakubovic A, Thomas DA. Suppressive effects of methadone on human blood lymphocytes. lmmunol Lett 1980; 2: 177-80.
32. the 33. and
Morimoto C. Letvin NL. Boyd AW, eta/. The isolation and characterization of human helper/inducer T cell subset. J lmmunol 1985; 134: 3762-9. Morimoto C. Letvin NL. Distaso JA. Aldrich W. Schlossman SF. The isolation characterization of the human suppressor inducer T cell subset. J lmmunol
1985; 134: 1508-15. 34. Reinherr EL, Kung PC, Goldstein G. Schlossman SF. A monoclonal antibody reactive with the human cytotoxic/suppressor T cell subset previously defined by a hetero antiserum TH2. J lmmunol 1980; 124: 1301-7. 35. Reinherz EL, Kung PC, Pesandro JM. Ritz J, Goldstein G, Schlossman SF. la determinants on human T cell subsets defined by monoclonal antibody: activation stimuli required for expression. J Exp Med 1979; 150: 1472-80. 38. Stashenko P, Nadler LM. Hardy R, Schlossman SF. The characterization of a human B lymphocyte specific antigen. J lmmunol 1980: 125: 1678-85. 37. Todd RF, Agthoven AV, Schlossman SF, Terhorst C. Structural analysis of diierentiation antigens MO1 and MO2 on human monocytes. Hybridoma 1982;
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ence. New York: AR Liss. in press. 47. Tanaka Y. Susumu 0, Kazuhiko N. ef al. Immunologic functions types of peripheral blood lymphocytes from human T-cell leukemia ers. J Clin lmmunol 1989; 9: 477-84.
1: 32937. 38. Hercend T. Griffin JD. Bensussan A, et al. Generation of a monoclonal antibody to a human natural killer cell clone. Characterization of two natural killer cell associated antigens, NKHla and NKH2, expressed on subsets of large granular lymphocytes. J Clin Invest 1985: 75: 932-43. 39. Fletcher MA, Baron GC. Ashman MR. Fischl MA, Klimas NG. Use of whole blood methods in assessment of immune parameters in immunodeficiency states. Diagn Clin lmmunol 1987; 5: 69-81. 40. Baron GA, Klimas NG, Fischl MA, Fletcher MA. Decreased natural cell-mediated cytotoxicity per effector cell in acquired immunodeficiency syndrome. Diagn lmmunol 1985; 3: 197-204. 41. Puri ML, Sen PK. Nonparametric methods in multivariate analysis. New York: John Wiley & Sons, 1971. 42. Siegel S. Nonparametric statistics for the behavioral sciences. New York: McGraw-Hill, 1956. 43. SAS/STAT guide for personal computers. Version 6th ed. Gary, North Carolina: SAS Institute, 1987. 44. Zwick R. Nonparametric one-way analysis of variance: a computational approach based on the Pillai-Bartlett Trace. Psycho1 Bull 1985: 97: 149-52. 45. Sidak Z. Rectangular confidence regions for the means of multivariate normal distributions. J Am Stat Assoc 1967; 62: 628-33. 46. Klimas NG, Morgan R, Blaney N. et al. Alcohol and immune function in HIV-l seropositive. HTLVI/II seronegative and positive men on methadone. In: Watson R. ed. Proceedings of the Alcohol and Immune Function in AIDS Network Confer-
48. Fletcher MA. Effects of HTLV-I/II seropositivity on lymphocyte phenotypes in blood donors. American Society of Hematology National Conference, Nov. 1989 [Abstract]. Blood 1989; 74: 246a. 49. Mosley JW. HTLV-I/II infection of blood recipients and its early effect on lymphocyte subpopulations [Abstract]. Blood 1989: 74: 178a. 50. Blaney NT, Klimas NG. Fletcher MA, Morgan RO. Mood state, social support and immune function in HIV- and HIV+ IV drug abusers, an AIDS risk group. Abstract Book, 4th International Conference on AIDS. Stockholm, Sweden, 1988; 8026: 463. 51. Fletcher MA, Klimas NG. lronson G. Laperriere A. Schneiderman N. Immunologic abnormalities in seropositive and negative gay men. Abstract Book, 5th International Conference on AIDS, Montreal, 1989: 442. 52. Nabel GJ. Activation of human immunodeficiency virus. J Lab Clin Med 1988; 111: 495-500. 53. Reua G. Lauarin A, Angarano G. et al. The natural history of HIV infection in intravenous drug users: risk of disease progression in a cohort of seroconverters. AIDS 1989; 3: 87-90. 54. Des Jarlais DC, Friedman S, Marmor M. Development of AIDS, HIV seroconversion, and potential cofactors for progression in a cohort of intravenous drug users. AIDS 1987: 1: 105-12.
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and phenovirus carri-
PURPOSE: The study objective was to evaluate the effects of long-term methadone use and human T-cell leukemia virus (HTLV) types I and H seropositivity on the distribution of lymphocyte subsets and on lymphocyte function as measured in vitro in intravenous drug users seronegative for human immunodeficiency virus type 1 (HLV-1). PATIENTS AND MEX!RODs: Anti-~-l-negative
intravenous drug users receiving methadone maintenance therapy (n = 24) were studied in a Veterans Administration drug abuse treatment center. These subjects were compared to 38 ageand sex-matched control subjects who did not abuse drugs. HIV-l and HTLV serostatus was determined by repetitive enzyme-linked immuuosorbent assay and confirmed by immunoblot. Lymphocyte subsets were determined by twocolor flow cytometry. Lymphocyte function was measured by proliferative response to plant mitogens and by natural killer (NH) cell-mediated cytotoxicity to a tumor cell target. RESULTS: &$ificant
differences
were
seen in
lymphocyte phenotype in the methadone-treated group, with elevations in the T-cell helper subset CD4+CD29+; in CD8 and CD8+I2+ cells, suppressor/cytotoxic T lymphocytes, and activated suppressor/cytotoxic T cells; and in CD2+CD26+ cells and activated total T lymphocytes. Lymphocyte function was suppressed in the methadone group, with poor responses to pokeweed mitogen and phytohemagglutiuin in culture. Moreover, NH-cell cytotoxicity was significantly reduced in the methadone group. From the Center for the Biopsychosocial Study of AIDS (NGK. NTB. ROM, DC, KM, MAF). and Miami Veterans Administration Medical Center (NGK, MAF), University of Miami School of Medicine, Miami, Florida, and 1 Abbott Laboratories (HL), North Chicago, Illinois. This work was supported in part by Grant P50-MH/DA42455 from the National Institute of Mental Health. Requests for reprints should be addressed to Nancy G. Klimas. M.D.. Department of Medicine-R42, University of Miami School of Medicine, P.O. Box 016960, Miami, Florida 33101. Manuscript submitted March 2. 1990, and accepted in revised form August 14, 1990.
None of these immunologic differences were attributable to HTLV serostatus. CONCLUSION: The immune abnormalities seen suggest that a clinically significant degree of immune impairment exists in methadone-treated intravenous drug users. However, these abnormalities could not be explained by the presence of other retroviruses in this HIV-l-negative study group, as there was no significant difference in immune function when HTLV-seropositive patients were compared to HTLV-seronegative subjects treated with methadone.
ntravenous drug users are at high risk for human immunodeficiency virus type 1 (HIV-l) infection due to injection-associated behaviors [1,2]. Moreover, heroin and other street drugs may modulate immunocompetence [3-51, possibly contributing to the high incidence of bacterial and viral infection and cancers in opiate addicts [4,6,7]. Such opiateinduced immunomodulation would bode poorly for HIV-l infection in intravenous drug users [5]. Finally, intravenous drug users pose a risk for heterosexual transmission of HIV-l infection [2], thus promoting the spread of HIV-l infection to those who are not intravenous drug users. Given these imperatives for reducing HIV-l infection risk among intravenous drug users, it is not surprising that methadone treatment has emerged as a potentially important prevention modality [8,9], with more funding planned for methadone programs. Hence, it is of considerable importance to ascertain that methadone treatment is not in itself immunosuppressive lest it paradoxically elevate the risk of HIV-l infection. Studies have been reported suggesting that methadone can be immunosuppressive [lo-121; that it does not affect immune function [13]; or that it can reverse some of the immunologic effects of heroin [4]. Results from our laboratory showed that intravenous drug users in methadone treatment evidenced B-cell dysfunction and polyclonal B-cell activation, despite their anti-HIV-1-seronegative
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status [14]. Consequently, because of the ambiguity of the literature, and because this opiate is being used with increasing frequency in a population at high risk for exposure to HIV-l, the first aim of the present study was to clarify the immunologic impact of methadone treatment, using a more comprehensive panel of immune markers than typifies previous research. However, an understanding of the immunologic impact of methadone is complicated by the confounding role of human T-cell leukemia virus (HTLV), a family of pathogenic retroviruses [15]. Two subtypes are recognized. HTLV-I is associated with serious illnesses in a small minority of infected individuals, including hematologic malignancy and neurologic disease [ 161. HTLV-II has been isolated rarely in hairy cell leukemia, but is, perhaps, more properly categorized as an orphan virus [17]. These viruses are evidently passed via shared needles, although there is considerable geographic variation, as indicated by the rather high prevalence of HTLV, more frequently HTLV-II than HTLV-I, infection among intravenous drug users in certain urban areas of the United States [18,19], while low or absent in much of Europe [19-211. The impact of co-infection with HTLV in HIV-l-infected intravenous drug users is apparently serious. A threefold increase in mortality has been reported [22]. Although data on the immunologic impact of asymptomatic HTLV infection are limited [23,24], recent research with methadone users [25] has associated HTLV seropositivity with abnormalities in CD4 lymphocyte subpopulations. This suggests that some of the immunomodulation observed with methadone may actually be attributable to HTLV infection. Given the relevance of this finding for both HIV-l infection and public policy concerning methadone treatment, replication of these findings and further investigation of HTLV and immune function during methadone treatment are clearly warranted, especially because of the high prevalence of HTLV infection among intravenous drug users in the U.S. The second aim of the present study, then, included assessment of whether HTLV abnormalities are attributable to, or independent of, HTLV seropositivity.
PATIENTS AND METHODS Design
Comprehensive measures of immune function of anti-HIV-l-negative intravenous drug users in methadone treatment were compared to those of anti-HIV-l-negative control subjects. HTLV-I/II serostatus and drug-using histories were also determined. Anti-HIV-l-negative subjects were selected because, in order to understand the immunologic 164
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effects of HIV-l in intravenous drug users, it is first necessary to evaluate the immunologic status of intravenous drug users prior to infection with this lymphotropic virus. Patients
Anti-HIV-l-negative intravenous drug users undergoing methadone maintenance treatment (n = 24), all males, were recruited from the Miami Veterans Administration outpatient Drug and Rehabilitation Unit. Subjects were white (n = 14), black (n = 7), or Hispanic (n = 3), with a mean age of 42, moderately well educated (some high school: n = 3; high school or some college: n = 21), but with limited employment (full time: n = 9; part time: n = 3; unemployed: n = 12) and income (below $10,000: n = 15; $10,000 and above: n = 9). A physician performed a complete physical examination and obtained a medical history in all subjects. An extensive substance-abuse history was obtained by a structured interview devised and extensively field-tested in Miami [1,23,26,27]. Toxicology screens and blood chemistries (SMAC 23) were done. Subjects were excluded if they had acquired immunodeficiency syndrome (AIDS) or AIDS-related complex as defined by Centers for Disease Control class IV criteria, if there was evidence of acute infectious illness, immunosuppressive illness, or positive toxicology screen, or if they were taking immunosuppressive non-recreational drugs. Anti-HIV-l-negative, non-substance-abusing men (n = 38), matched by age, served as the comparison group. Blood Samples
To control for diurnal variations, all blood samples were obtained from fasting subjects between 7:30 and lo:30 AM. Peripheral venous blood samples were collected into appropriate tubes: heparin tubes for in uitro functional assays and flow cytometry (Vacutainefl-sodium heparin, Becton-Dickinson, Rutherford, New Jersey); EDTA for white blood cell and differential counts (CBC). Whole blood samples were held no longer than 24 hours at room temperature before preparation and fixation for flow cytometry assay or more than 4 hours for CBC or for functional assays. For serum samples, peripheral venous blood was collected and then allowed to clot at 23OC for 30 minutes, at which time the serum was separated from the clot and stored at 20°C. Flow Cytometry
A single-laser flow cytometer (EPICS C, Coulter Instruments Laboratories, Hialeah, Florida) was used with a whole-blood, two-color analysis proce90
dure as previously described to determine the distribution of lymphocyte phenotypes [28]. The following pairs of fluorescein isothiocyanate (FITC)or phycoerythrin (PE)-conjugated monoclonal antibodies (Coulter Immunology, Hialeah, Florida) were selected: Tll-FITC for CD2 or sheep erythrocyte receptor-bearing cells [29] and Tal-PE to measure a surface marker associated with T memory cell activation (CD26) [30]; T4-FITC for CD4 or helper/inducer cells [31] combined either with 4B4PE (CD29) to measure the subset of CD4 that helps B cells to respond to antigen stimulus with immunoglobulin synthesis (321, or with 2H4-PE (CD45RA), which activates CD8 cells to act as either suppressor cells or cytotoxic cells [33]; T8FITC (CD8) or suppressor/cytotoxic T cells [34] plus IZ-PE to measure HLA-DR activation antigen [35] expression on CD8; Bl-FITC (CD20) for determining B cells [36]; Mo2-FITC for CD14 or monocytes [37] combined with IQ-PE to determine the degree of expression of the HLA-DR class II activation antigen on monocytes; NKH.l-PE (CD56), which defines the entire pool of mononuclear cells with natural killer (NK) activity [38]. Isotypic controls were mouse IgGl, IgG2, or IgM (Coulter Immunology). Peripheral lymphocyte counts were calculated by multiplying the total white blood cell count and percentage of lymphocytes as determined from a Cell Dyne 1500@ (Sequoia-Turner, Mountain View, California). Estimates of absolute numbers of the lymphocytes or mononuclear cell populations positive for the respective surface markers were determined by multiplying peripheral lymphocyte or mononuclear cell counts by percentage positive cells for each surface marker [39]. Lymphocyte Proliferation Assay Lymphocyte proliferation to mitogen stimulation was measured using a whole-blood procedure [39]. Briefly, 100 PL of diluted heparinized blood (1:5) was dispensed in triplicate to the wells of a U-bottom microtiter plate (Costar, Cambridge, Massachusetts). Mitogens were tested at the following levels: phytohemagglutinin (PHA; Wellcome Diagnostics, Dartford, England) 10 Fg/mL and pokeweed mitogen (PWM; Gibco, Chagrin Falls, Ohio) diluted 1:40. Plates were incubated for 72 hours. During the last 6 hours of the appropriate incubation time, cultures were pulsed with 25 PL tritiated thymidine (New England Nuclear, Boston, Massachusetts)-1 PCi per well. Cultures were harvested onto glass filter paper discs (Titertek@ Cell Harvester, Flow Laboratories, McLean, Virginia), immersed in scintillation fluid, and counted on a beta-scintillation counter (LKB Instruments,
Rockville, Maryland). Results were expressed as mean net counts per minute (cpm) incorporated per 100,000 lymphocytes. NK-Cell Cytotoxicity NK-cell function was evaluated by determining cytotoxicity using the whole-blood chromium release assay as outlined in detail by Baron et al [40] and Fletcher et al [39]. The target cell line utilized was the NK-sensitive erythroleukemic K562 cell line. The assay was done in triplicate, at four targetto-effector cell ratios with a 4-hour incubation. The percent cytotoxicity at the four target-to-effector ratios and the number of CD56+ cells per unit of blood were used to express the results as percent cytotoxicity at a target-to-effector cell (CD56+) ratio of 1:l. Calculations were performed using an AT&T computer program written in-house. Kinetic lytic units per NK cell (KLU/NK cell) were calculated as previously indicated [40] in order to estimate the maximum number of targets lysed by each NK cell (CD56+) during the 4-hour assay. Viral Serology Antibodies to HIV-l were measured using the enzyme-linked immunosorbent assay (ELISA) and Abbott (North Chicago, Illinois) reagents. Reactive samples were repeated and doubly positive samples were confirmed by protein immunoblot. The presence of HIV-l viremia (~24) was determined using an ELISA method and Abbott reagents. Antibodies to HTLV were measured by ELISA using Abbott reagents. Repeatedly positive samples were confirmed by protein immunoblot and radioimmunoprecipitation assay (RIPA) using the HaT102B2 cell line as source of antigen and criteria as previously described [19]. Due to the high level of cross reactivity, the serologic methods of identification used do not distinguish between the subtypes I and II. Data Analysis Non-parametric rank order analyses were used to evaluate between-group differences. Given the large number of potential statistical tests, a combined multivariate and univariate strategy was used to help control the experiment-wide type I error rate. The immunologic parameters were clustered into two subgroups: (1) T-cell subsets expressed as percentages of the overall numbers of lymphocytes; and (2) measures of NK-cell function. A non-parametric multivariate analysis was performed on the T-cell subsets to determine whether this cluster evidenced statistically significant between-group differences. The non-parametric multivariate procedure used is an extension of the
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TABLE I Anti-HIV-l-Negative, Anti-HTLV-I/II-Negative and -Positive Methadone Clinic Subjects: Selected immunologic Markers* Anti-HTLV-Negative (n = 16)
Marker CD$+/mms CD4+% CD4+CD29+/mm3 CD4+CD29+% CD4+CD45RA+/mmJ CD4+CD45RA+% ;;zT(,mm3
1.041.0(849.1/1,295.1)~ 0.40 (0.39/0X) 715.1(475.4/1.105.0) 0.29(0.25/0.36) 212.8(134.9/530.1) 0.10 (0.07/0.18) 907.4(595.2/1.021.4) 0.33(0.27/0.44) 175.6(116.8/240.0) 0.08(0.04/0.11) 549.8(418.2/1.358.4) 0.28 (0.16/0.49) 333.4(280.5/409.9) 0.15(0.12/0.17) 25.9(14.8/30.4) 0.35(0.21/0.60)
CD8+P+/mmJ CD8+12+% CD2+CD26+/mm3 CD2+CD26+% CD56+/mmJ CD56+% NK % cytotoxicity KLU/NK All between-group comparisons t Midian (25%/75%).
l
were statistically
non-significant
using Kruskal-Wallis
analyses of variance
Kruskal-Wallis one-way analysis based on ranks [41,42]. Chi-square values for performing hypothesis tests are easily obtainable using the statistical analysis system (SAS) [43] and following procedures outlined by Zwick [44]. Univariate KruskalWallis analyses were performed to help interpret the multivariate result. In addition, NKH.l cell counts, NKH.l percentages, and the measures of T-, B-, and NK-cell function were collected on a subset of the overall laboratory control group. Only univariate analyses were performed on these measures. Since these subsets were only partially overlapping, univariate analyses were done on these measures, An experiment-wide type I error rate (ew) of 0.05 was used to identify statistically reliable results. An ew = 0.10 was used to identify marginally reliable results. Since multiple analyses were performed, Sidak’s [45] a-adjustment was used to calculate a-levels for the individual analyses, yielding individual analysis a-values of 0.009 and 0.017 corresponding to the ew values of 0.05 and 0.10, respectively. Other probability is represented where meaningful. RESULTS Serology
The intravenous drug users receiving methadone treatment were both anti-HIV-l-negative and negative for p24 antigen. However, nine (31%) of the 29 intravenous drug users were positive for antibodies to HTLV-I/II (repeatedly ELISA-positive and confirmed by protein immunoblot and RIPA). Neither the anti-HTLV-I/II-negative nor the anti-HTLV-I/II-positive methadone clinic subjects in this study differed from the laboratory controls in their total lymphocyte counts (data not shown). In addition, the anti-HTLV-I/II-negative and -pas166
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1.146.1(902.5/1,699.9) 0.50(0.42/0.53) 731.3 (501.6/975.7) 0.30(0.22/0.34) 172.7 (121.7/599.8) 0.09(0.06/0.24) 818.9 (624.5/1,038.7) 0.34(0.31/0.40) 125.1 (87.1/209.8) 0.07(0.04/0.09) 4?4.7(364.3/884.1) 0.24(0.15/0.36) 250.2(164.0/334.5) 0.11 (0.07/0.14) 12.0(11.1/21.6) 0.37(0.20/0.68) on ranks. Results for T- and B-cell function are presented
in Table IV.
itive methadone clinic subjects did not differ in terms of their percentages of various lymphocyte subsets, or in their measures of NK-cell function (Table I). Consequently, for the lymphocyte enumeration and NK-cell function comparisons presented in the next two paragraphs (Tables II and III), the anti-HTLV-I/II-negative and -positive methadone subjects are combined into a single methadone group. For the T- and B-cell function analyses, the anti-HTLV distinction is retained. Lymphocyte Enumeration
As indicated in Table II, the methadone group (anti-HTLV-I/II-negative and -positive subjects combined) had higher percentages than the laboratory controls for the following markers: CD4+CD29+; CD8; CD8+12+; and CD2 cells coexpressing CD26, a marker expressed on activated memory T cells. The lab control subjects and methadone clinic patients did not differ significantly in percentages of either CD4 cells or CD4+CD45R+ subset. In addition, there were no differences among the three groups in percentage of NKH.l+ celIs (data not shown). The absolute counts presented in Table II reflect the differences seen in the percentages. Functional Assays NK-CELL FUNCTION: As indicated in Table III, methadone subjects (regardless of anti-HTLV-I/II serostatus) had lower levels of NK percent cytotoxicity, higher levels of lytic units, and lower levels of KLU/NK cell than the laboratory controls. As indicated before, the anti-HTLV-I/II-negative and -positive methadone groups did not differ in any of these markers. Given the lack of group differences in the NKH.l enumeration measures (both
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TABLE II
T-Cell Subsets In Male Intravenous Drug Users Receiving Methadone: Subsets Expressed as Percentages and Absolute Counts of Total lymphocytes Methadone Clinic Anti-HIV-l-Negative (n = 24)
Marker
0.45 (0.39/0.53)’ 1,138(849/1,330)
CD456 AbsoluteCD4 CD4+CD29+% AbsoluteCD4+CD29+ CD4+CD45RA+% AbsoluteCD4+CD45RA+ CD8% AbsoluteCD8 CD8+12+% AbsoluteCD8+12+ CD2+CD26+% AbsoluteCD2+CD26+
I
Multivariaterank
ordertestP$=
(n=38) 0.43(0.39/0.49)
1,013(741/1,217) 0.21(0.17/0.25) 496 (326/612) 0.13 (0.09/0.22) 2841190/527) 0.25 (d.21j0.31j
0.30(0.24/0.34) 715(475/1,030)
0.10 (0.06/0.20) 197 (129/542) 0.34(0.28/0.42)
904(601/1,031)
530(447/757) 0.03(0.02/0.05) 74(44/118) 0.16(0.13/0.20) 344(257/510)
0.07(0.04/0.11)
159 (99/226) 0.26(0.16/0.40)
522(418/1.198)
35.58(df=
Comparison* NS p
’
NS p
6), p
Kruskal-Watlis analyses of variance on ranks. + Median (25%/75X). t Multivariate test performed on percentage measures only.
l
TABLE Ill NK&ll
Function In Male Intravenous Drug Users Recelvlng Methadone
Marker ;;;;;totoxicity
Methadone Clinic Anti-HIV-l-Negative (n = 24)
Lab Controls (n=38)
23.2 (11.5/29.2)+ 0.35(0.21/0.60)
47.4(25.4/52.4) 1.31(0.58/1.69)
Unlvarlate Comparison* p <0.002 p <0.008
* Kruskal-Wallis analyses of variance on ranks. t Median (25%/75%).
absolute counts and percentages), these results suggest that it is NK-cell function that is most affected by methadone. T- AND B-CELL FUNCTION: As indicated in Table IV, for T-cell function (PHA response) there was an overall group difference between the laboratory controls and the two groups of methadone-treated subjects (anti-HTLV-I/II-negative and -positive). Interestingly, no differences were observed between the laboratory control subjects and the anti-HTLVI/II-positive methadone subjects. Also, there were no differences between the anti-HTLV-I/II-positive and -negative methadone groups (p
22% of a non-clinic sample. In general, no association was found between use of a variety of drugs (e.g., heroin, cocaine, speedballing) and the immunologic markers. In addition, there were no apparent associations between anti-HTLV seropositivity and either type of drug use or frequency of injection. Alcohol use did show significant associations with the T- and B-cell functional parameters, but did not differ between the anti-HTLV-negative and -positive groups as reported elsewhere by Klimas et al [46]. However, the levels of use among the methadone-treated sample were not greater than those found in other normal populations. Alcohol use data for the laboratory controls were not available. Eighty-nine percent of the anti-HTLV-negative group reported cigarette smoking compared to 57% of the anti-HTLV-positive group. This difference was not statistically significant (p = 0.11, Fisher’s exact test [two-tailed]), possibly due to the small sample size. However, smoking was not significantly correlated (at p
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TABLEIV T-and B-CellFunction in MaleIntravenous Drug Users ReceivingMethadone I Unlvarlate Methadone
Antl-HTLVPosltlve
Anti-HTLVNegative
Marker PWM net cpm
Cllnlc
2,938.0
‘2,38;;/84$4.0’*
PHA net cpm
(29,512.b/78.136.0) * Median (25%/ 75%). t Kruskal-Wallis analyses of variance
11,252.0
(36,603.OjlliO94.0)
on ranks. Note: no differences
Antl-HTLVPositive Versus -Negative
Laboratory Controls Versus Anti-HTLVNegative
Laboratory Controls
All Groups
12,864.5
p <0.005
p
p
<0.002
p x0.02
p <0.12
p
<0.005
(6,509$~;,~51.0)
(2,80~~$‘$~7.0’
Comparlsonst
(54,947.d/bli,246.0)
were found between the HTLV-l/II-positive
methadone
treatment
subjects and the laboratory
controls.
Recognition of the increased risk of AIDS among intravenous drug users has fostered an increased interest in oral methadone maintenance programs as a means of reducing the risk of HIV-l exposure via infected needles. However, the immunologic consequences of long-term methadone use are not well characterized; also, a substantial percentage of intravenous drug users are seropositive for another retrovirus, HTLV, which has been identified as predominantly subtype II when appropriately sensitive methods are used [ 191. Again, the immunologic sequelae of HTLV infection have been addressed in very few studies. Studies are limited on the in uiuo immunologic effects of HTLV infection, both subtypes I and II, in asymptomatic individuals. Yasuda and colleagues [24] reported elevated total T lymphocytes (CD2+) and elevated levels of immunoglobulins. In contrast, Tanaka et al [47] found lower percentages of CD3 and CD4 cells and unchanged percentages of CD8 lymphocytes when they compared HTLV-I carriers with sex- and age-matched controls. Fletcher and the Transfusion Safety Study Group [48] found elevated levels of CD8+, CD8+12+, CD4+CD45RA+, and CD56+ lymphocytes in HTLV antibody-positive blood donors. DeShazo et a1 [25] assessed immunophenotypes and cellular immune function in anti-HTLV-positive and -negative intravenous drug users in a New Orleans methadone treatment program. They reported elevated CD4+CD29+ lymphocytes and poor PWM responses in the anti-HTLV-positive group, but no effect of HTLV serostatus on total number of T lymphocytes (CD2), T-cell subsets (CD4, CD4+CD45R+, CD8), B cells (CD19), or NK cells (CD56), or on NK-cell cytotoxicity or lymphoproliferative response to PHA. They reported that both
HTLV-seropositive and HTLV-seronegative drug users had depressed levels of CD4tCD45RAt lymphocytes. In contrast, the present study indicates that intravenous drug users undergoing methadone treatment in South Florida had no immunologic impairments or alterations that could be attributed to HTLV serostatus. Both anti-HTLV-positive and negative methadone-treated patients had significant elevations in CD4+CD29+ lymphocytes, CD8 cells, and CD8tI2t cells, but depressed NK-cell cytotoxicity and poor lymphocyte response to mitogenie stimulation, when compared to non-drugabusing control subjects. These findings could be interpreted to demonstrate that the immunomodulatory effects of common variables in this methadone treatment population were so pronounced that no independent effects of HTLV infection could be seen. It should be noted, however, that persons recently infected with HTLV were reported to show no immunologic effects [49], and the time since infection with HTLV in our cohort was not defined. The immunologic effects observed are unlikely to be due to street drugs since prospective subjects were excluded from study if urine samples were positive for other drugs. Analysis indicates that the rather infrequent use of street drugs in this group was not related to the immunologic changes obs&ved. Although concurrent studies showed some association between psychosocial variables and immune function, there was no clear pattern of psychoimmunologic relationships that would account for the present findings [50]. Although the changes seen could result from other factors, including subclinical infections, treatment with methadone must be regarded as a likely mediator. Other types of immunomodulation by methadone have been reported. In studies of peripheral blood mononuclear cells from patients receiving methadone, there was a significant impairment of ability to generate superoxide anion. Tubaro et al [ll] re-
166
90
ratory control users.
subjects and the intravenous
drug
COMMENTS
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1991
The American
Journal
of Medicine
Volume
IMMUNE
ported that morphine and, to a lesser extent, methadone reduced phagocytosis, intracellular killing, and superoxide production in polymorphonuclear leukocytes and in monocytes. In heroin addicts, both before and after methadone treatment, skin test anergy to seven recall antigens was common [4]. The present study raises concern regarding the possible consequences of methadone-mediated immunologic changes in HIV-l susceptibility. The major changes seen in T-cell subsets were proliferative and activating. The increase noted in CD4+CD29+ lymphocytes deserves further study in light of the observations of Fletcher et ~1 [51] indicating that this subset may be a preferred target of HIV-1 in early infection. The lymphocyte activation observed in men receiving methadone may also have ominous consequences. It has been repeatedly noted in the literature that HIV-l replicates in activated T cells [52]. Reduction in natural immunity as evidenced by poor in vitro cytotoxicity might lead to compromised antimicrobial defense, and perhaps to more rapid onset of secohdary infections with opportunistic pathogens, following HIV-l infection, There is evidence in the literature that intravenous drug abusers tend to have a shorter period of time from infection to onset of AIDS and to mortality [53,54]. These findings underscore the need for further studies. Currently, longitudinal studies of drug users undergoing 6 weeks of inpatient detoxification followed by methadone or nonmethadone interventions are underway.
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L, Trombini M. et a/. Immunologic status of heroin addicts: maintenance treatment. Drug Alcohol Depend 1989: 13:
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and phenovirus carri-