Journal of Neuroimmunology, 28 (1990) 97-109 Elsevier
97
YNI 00944
Soluble interleukin-2 receptor and soluble CD8 in serum and cerebrospinal flUid during human immunodeficiency virus-associated neurologic disease Diane E. Griffin, Justin C. M c A r t h u r and David R. Cornblath Departments of Neurology and Medicine, The Johns Hopkins University School of Medicine, Baltimore, MD 21205, U.S.A. (Received 31 October 1989) (Revised, received 7 December 1989) (Accepted 7 December 1989)
Key words: Human immunodeficiency virus; Interleukin-2 receptor; CD8-positive T lymphocyte; Cerebrospinal fluid
Summary We have measured levels of soluble interleukin-2 receptor (slL-2R) and soluble CD8 (sCD8) in serum and cerebrospinal fluid (CSF) of 127 human immunodeficiency virus (HIV)-seropositive and 51 HIVseronegative individuals. Serum levels of slL-2R and sCD8 were higher in HIV ÷ than in HIVindividuals. HIV ÷ individuals were grouped by neurological status: asymptomatic, abnormal on neuropsychological screening, HIV-related meningitis, inflammatory demyelinating polyneuropathy, opportunistic central nervous system (CNS) infections and HIV-related dementia, myelopathy or sensory neuropathy. Serum levels of slL-2R and sCD8 were higher in all HIV ÷ categories compared to HIV- individuals. Patients with HIV-related meningitis had higher levels of slL-2R and sCD8 than asymptomatic HIV ÷ individuals, and inflammatory polyneuropathy patients had higher levels of sCD8. CSF levels of sCD8 were higher in all categories of HIV + than in HIV- individuals. Patients with HIV-related meningitis, inflammatory neuropathy and opportunistic infections had higher levels than asymptomatic individuals. Examination of the time course showed that serum and CSF levels of slL-2R and sCD8 increased to very high levels during acute HIV infections. Serum levels then declined over several months to relatively stable elevated levels. By 1-2 years after HIV infection slL-2R was relatively low in CSF, while sCD8 remained elevated with a gradual decrease over the subsequent years of follow-up.
Introduction A variety of neurologic diseases have been recognized as a consequence of infection with the Address for correspondence: Diane E. Griffin, M.D., Ph.D., Department of Neurology, Meyer 6-181, The Johns Hopkins Hospital, Baltimore, MD 21205, U.S.A.
human immunodeficiency virus (HIV). Neurologic diseases occur at all stages of HIV infection and involve both the peripheral and central nervous systems (McArthur, 1987). Certain diseases (e.g. the inflammatory demyelinating polyneuropathies and transient meningoencephalitis) are most common early in infection when T cell function is relatively preserved and immunosuppression is not
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98 prominent (Cornblath et al., 1987; Hollander and Stringari, 1987; Parry, 1988). Other neurologic diseases (e.g. dementia, myelopathy, sensory neuropathies, and opportunistic central nervous system (CNS) infections) tend to occur later in infection with other systemic manifestations of the acquired immunodeficiency syndrome (AIDS) are likely to be present (Snider et al., 1983; Levy et al., 1985; Petito et al., 1985; Cornblath and McArthur, 1988; Price et al., 1988). HIV has been isolated from the cerebrospinal fluid (CSF) at all stages of disease (Ho et al., 1985; Hollander and Levy, 1987; Sonnerborg et al., 1988) but has been convincingly identified in brain only in scattered cells, most of which are macrophages (Gabuzda et al., 1986; Koenig et al., 1986; Wiley et al., 1986; Pumarola-Sune et al., 1987; Vazeux et al., 1987). Therefore, the mechanisms by which neurologic impairment is produced during HIV infection have not yet been clearly identified (Johnson et al., 1988). The immune system is likely to play an important role in HIV-associated neurologic disease. Virus is probably transported across the b l o o d brain barrier into the CNS inside inflammatory cells. Within the nervous system infected or reactive immune cells may produce products which are toxic to neural cells or myelin. Abnormalities of CSF are common in infected individuals both with and without neurologic disease (Anderson et al., 1988; Appleman et al., 1988; Hollander, 1988; McArthur et al., 1988). Previous characterization of the inflammatory cells in the CSF of HIVseropositive individuals has shown that the mononuclear cells are primarily T lymphocytes and that CD8-positive cells are more predominant in the CSF than in the peripheral blood (McArthur et al., 1989b). One approach to assessing the participation of the immune system in CNS disease associated with HIV infection is the measurement of soluble products of immune cells released as a consequence of immune reactions. Previous studies have shown that acute systemic viral infections (e.g. measles and infectious mononucleosis) are associated with immune activation and the appearance of increased amounts of soluble interleukin-2 receptor (sIL-2R) and soluble CD8 (sCD8) in serum (Griffin et al., 1989; Tomkinson et al., 1989).
During direct infection of the CNS slL-2R and sCD8 are increased in CSF while in immunemediated CNS disease, induced by prior virus infection, sIL-2R is not increased but sCD8 is (Boutin et al., 1987; Griffin et al., 1989). To evaluate the level of activation of T cells both in the peripheral blood and within the CNS during HIV infection we have measured the levels of sCD8 and sIL-2R in the serum and CSF of HIVseropositive individuals with various neuropsychological abnormalities. They were compared to neurologically normal HIV-seropositive individuals and to HIV-seronegative subjects with neurological diseases.
Materials and methods Patients
HIV-seropositive individuals came from two sources: inpatient and outpatient services of the Johns Hopkins Hospital and the Johns Hopkins University neuropsychological component of the Multicenter AIDS Cohort Study (MACS). The MACS neuropsychological study includes longitudinal neurological and neuropsychological testing in combination with directed physical examination, questionnaires, CD4 cell counts, CSF analysis and, in some subjects, magnetic resonance imaging. It is described in detail elsewhere (McArthur et al., 1989a). All patients were evaluated and samples collected between January, 1986 and February, 1989 except for two patients with additional samples from 1985. Informed consent was obtained from all individuals and the studies were approved by the Joint Council for Clinical Investigation of the Johns Hopkins Medical Institutions. Samples were frozen at - 2 0 ° C until analysis. A total of 177 individuals were studied. 167 paired serum and CSF samples were obtained from 123 HIV-seropositive individuals and 44 paired serum and CSF samples were obtained from HIV-seronegative individuals. Twelve unpaired serum and 11 CSF samples were obtained from these same individuals plus an additional four seropositive and seven seronegative persons. Of the HIV-seropositive individuals, 27 had no systemic symptoms (i.e. CDC groups I I / I I I ) (Centers for Disease Control, 1987) and were neu-
99
rologically normal (asymptomatic), 33 were systemically asymptomatic but had abnormal values on at least one component of a battery of neurologic and neuropsychological screening tests used in the MACS (NP positive), five had inflammatory demyelinating neuropathies (Cornblath et al., 1987), five had acute or chronic meningitis presumed due to HIV, 21 had HIV-related dementia (Navia et al., 1986), five had HIV-related myelopathy or vacuolar myelopathy (Pet±to et al., 1985), four had sensory neuropathy, 18 had presumed or proven CNS infections (11 cryptococcal meningitis, three toxoplasmosis, three syphilis, and one zoster meningitis) and 12 had various other neurologic problems including headache, metabolic encephalopathy, drug-induced psychosis and facial paralysis. Three patients moved from one clinical category to another during the course of follow-up. Presence or absence of a CSF pleocytosis (> 5 cells) did not alter the classification. Blood-brain barrier function was assessed using the formula of Tibbling et al. (1977) based on serum and CSF albumin. Values greater than 6.0 were considered abnormal. The 44 HIV-seronegative individuals had a variety of neurologic problems including 20 with amyotrophic lateral sclerosis, nine with acute or chronic inflammatory demyelinating neuropathies, three with dementia of the Alzheimer's type, and 12 with various other non-inflammatory neurological diseases.
Assays Soluble CD8 and slL-2R were assayed by enzyme immunoassay (T Cell Sciences, Cambridge, MA, U.S.A.). Assays were performed according to the manufacturer's instructions. Data are expressed as units/ml using standards supplied by the manufacturer. Statistics Analysis was performed using Student's t-test or Spearman's rank correlation (CD4 counts; blood-brain barrier function), employing Statview 512 software (Brainpower, Calabasas, CA, U.S.A.). Our primary analysis determined whether HIVseropositive subjects differed from HIV-seronegative subjects. In a secondary analysis HIVseropositive subjects were classified by neurologic
disease (117 individuals) or by time after seroconversion (33 individuals) and compared.
Results
The levels of slL-2R and sCD8 in serum and CSF were measured to assess the evidence for systemic immune activation and activation of T cells within the CNS during HIV infection (Table 1). Levels of both slL-2R and sCD8 were elevated in the serum of HIV-seropositive patients compared to the HIV-seronegative controls. In the HIV-seropositive group CSF levels of sCD8 were higher and levels of slL-2R were marginally lower than in the HIV-seronegative group. To determine whether HIV-seropositive individuals with varying neurological disorders had different levels of slL-2R or sCD8 in serum or CSF, individuals were grouped according to neurologic status into six categories: (1) systemically asymptomatic, neurologically normal (N = 27), (2) one or more abnormalities on neurological and neuropsychological screening tests ( N = 33), (3) HIV-related meningitis (N = 5), (4) inflammatory demyelinating polyneuropathy (N = 5), (5) HIVrelated neurological diseases occurring late in the course of infection including dementia, myelopathy and sensory neuropathy (N = 30), (6) opportunistic CNS infections (N = 18). Categories were chosen to represent the spectrum of neurologic diseases associated with early and late stages of HIV infection (Johnson et al., 1988). Each HIVTABLE 1 S O L U B L E IL-2 R E C E P T O R A N D S O L U B L E CD8 IN T H E SERUM AND CSF OF HIV-SEROPOSITIVE AND SERONEGATIVE INDIVIDUALS EVALUATED FOR N E U R O L O G I C DISEASE Patient groups HIV- (N) slL-2 receptor Serum 4 3 1 ± 3 2 a (41) CSF 7 8 ± 17 (41) sCD8 Serum 5 0 8 ± 8 9 (41) CSF 5 1 ± 1 0 (43) a Standard error of the mean.
p HIV + ( N ) 965 ± 52 (174) 4 8 ± 6 (169)
< 0.0001 0.049
1 7 7 8 ± 2 3 0 (177) 2 1 0 ± 28 (174)
0.0088 0.0053
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HIV-seropositive Fig. 1. Soluble interleukin-2 receptor in the serum of HIV-seropositiveindividuals with different categories of neurologic disease and HIV-seronegativeindividuals with other neurologic disease (OND). Levels in all groups of HIV-seropositiveindividuals were higher than in OND controls: Asx, asymptomatic, p = 0.004; NP +, abnormal neuropsychological testing, p = 0.0001; Men, meningitis, p < 0.0001; IDP, inflammatory demyelinating polyneuropathy, p = 0.024; DMN, dementia (O), myelopathy (zx), sensory neuropathy (o), p 48
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Time after admission Fig. 5. Serum and CSF levels of soluble IL-2 receptor and CD8 associated with acute meningoencephalitis in a single patient during HIV seroconversion. Hatched area indicates levels found in normal individuals.
tendency toward greater variability and higher values in those with abnormal barrier function (Table 2). CD4 T cell counts were available at one or more sampling times for 73 individuals representing all categories of HIV seropositive patients. There were no significant correlations between sIL-2R levels in serum or CSF or sCD8 levels in serum, but sCD8 levels in CSF were negatively
105 correlated with CD4 counts in blood (rs = -0.215, p < 0.05).
Discussion
We have studied slL-2R and sCD8, two different parameters of immune activation, in serum and CSF during chronic infection with HIV. Both T cell products were found in increased amounts in the serum of essentially all individuals infected with HIV. The highest levels were present during the initial immune response associated with seroconversion and a mononucleosis-like syndrome accompanied by acute self-limited neurologic disease. The levels of these T cell products have been related to the wide spectrum of CNS disease found in association with HIV infection. CSF levels of slL-2R were similar or lower than levels found in the CSF of individuals with non-HIV-associated neurological diseases. In contrast, sCD8 was often elevated in the CSF during HIV infection suggesting local T cell effector activity. Levels were even higher during HIV-associated meningitis and inflammatory demyelinating polyneuropathy. Activation of the immune system is common in viral infection and undoubtedly reflects the normal host response to a new antigenic challenge. Multiple components of the immune system are mobilized for proliferation of virus-specific lymphocytes leading to generation of effector cells and cellular products (lymphokines, antibody) designed to eliminate virus from tissue, plasma and interstitial fluid (Doherty and Allan, 1986). In acute viral infections, such as measles and infectious mononucleosis, this period of immunologic activation is transient and the immune system returns to a baseline activity within weeks after clearance of active infection (Griffin et al., 1989; Tomkinson et al., 1989). However, coincident with this period of immune activation multiple in vivo and in vitro abnormalities of immune function, such as suppressed skin test responses and mitogen-induced lymphoproliferative responses, are evident (Mange et al., 1974; Hirsch et al., 1985; Junker et al., 1986; Tamashiro et al., 1987). In HIV infection virus is not eliminated by the immune system and these studies suggest that this persistence of infection leads to chronic immuno-
logic activation as evidenced by continually elevated serum levels of immunoglobulin (Edelman and Zolla-Pazner, 1989), slL-2R and sCD8. This prolonged period of immune activation may be related to the immunologic abnormalities which are often present during HIV infection well before CD4-1ymphocytes have been depleted (Ascher and Sheppard, 1988; Edelman and Zolla-Pazner, 1989). Soluble IL-2R is produced as a result of binding of IL-2 to the high affinity (aft) receptor for IL-2 which may be present on both B cells and T cells. Binding of IL-2 simultaneously induces cell proliferation and release of a 45 kDa form of the a-chain of the IL-2 receptor (Rubin et al., 1985). Normal serum contains low levels of slL-2R (Greene et al., 1986) and levels have been reported by others to be elevated during HIV infection (Price et al., 1984; Sathi and Naher, 1986; Lang et al., 1988). In acute measles virus infection slL-2R becomes elevated before the appearance of virusspecific antibody and gradually declines over weeks after recovery (Griffin et al., 1989). In HIV infection slL-2R also becomes elevated at the time of the immune response to infection (seroconversion), declines somewhat after the most acute phase but then remains chronically elevated. The function of the soluble form of the IL-2 receptor is not known, but has been postulated to provide negative feedback for lymphocyte proliferation by binding to free IL-2 (Rubin et al., 1985) or by removing receptor from the cell surface (Diamantstein et al., 1986). Chronic elevation could contribute to depressed lymphoproliferative responses. Plasma levels of slL-2R are also elevated during transplant rejection (Colvin et al., 1987), IL-2 administration (Lotze et al., 1987), presumed autoimmune diseases such as multiple sclerosis (Greenberg et al., 1988) and rheumatoid arthritis (Keystone et al., 1988), and B and T cell malignancies (Pizzolo et al., 1987). CSF levels of slL-2R are elevated compared to asymptomatic patients only during the earliest acute stages of infection associated with meningitis and then again in association with inflammatory neuropathy. Elevated CSF levels have also been reported during herpes simplex virus encephalitis (Boutin et al., 1987) and a variety of other CNS infections (Griffin et al., 1989). These data suggest that after the earliest phases of dis-
106
ease IL-2-driven cell proliferation is not occurring to a significant extent within the CNS. Soluble CD8 is produced during the interaction of CD8-positive T cells with HLA class I-bearing target cells, such as virus-infected ceils and correlates well with the presence in the periphery of activated CD8-1ymphocytes (Tomkinson et al., 1989). CD8 is present as an associative recognition molecule in the CD3-T cell receptor complex on the cytotoxic/suppressor subpopulation of T cells (Emmrich et al., 1987; Takada and Engleman, 1987; Blue et al., 1988). Target cell interaction results in activation of the CD8-positive effector cell, phosphorylation of CD8 and release of a 27 kDa soluble form of CD8 from the surface of the cell (Fujimoto et al., 1983, 1984; Acres et al., 1987). In acute viral infection serum sCD8 is not increased until antiviral antibody appears and returns to normal shortly after virus clearance is completed (Griffin et al., 1989). Prolonged elevation of sCD8 in HIV infection is consistent with lack of virus clearance, and suggests ongoing cytotoxic T lymphocyte activity presumably directed against HIV-infected CD4-positive T cells and macrophages. The numbers of CD8-positive cells increase early in infection (Nicholson et al., 1984) and T cells cytotoxic for HIV-infected cells are present in the blood and CSF of infected individuals (Plata et al., 1987; Walker et al., 1987, 1988; Sethi et al., 1988). Elevated serum sCD8 suggests that effector-target cell interactions continue to occur throughout infection, including late stages of disease. However, increased suppressor cell activity has also been documented in HIV infection (Nicholson et al., 1984; Ascher and Sheppard, 1988) and could also account for increased circulating sCD8 (Pui et al., 1988). Either is consistent with documented abnormalities of CD8positive T cells in HIV infection which suggest chronic activation and/or immaturity (Nicholson et al., 1984; Salazar-Gonzalez et al., 1985). Markedly elevated levels during inflammatory demyelinating polyneuropathy are consistent with observed infiltration of nerve with CD8-positive T cells (Cornblath et al., 1990) and may reflect cytotoxic T cell attack either on normal ceils (autoimmune disease) or on HIV-infected cells within the nerve. CSF levels of sCD8 are elevated in most phases
of the infection, but are most elevated relatively early in infection during meningitis and inflammatory neuropathy, and then again when opportunistic infections occur. An asymptomatic pleocytosis is common during early HIV infection (Andersson et al., 1988; Hollander, 1988; McArthur et al., 1988) with a relatively increased proportion of CD8-positive cells in CSF compared to blood (McArthur et al., 1989b). These findings may reflect increased trafficking of activated CD8-positive T cells into the CNS or a specific, targeted response to HIV infection within the CNS. If the latter is the case the target cell for the CD8-positive cell has not yet been definitively identified. The low levels of sCD8 in CSF of patients with dementia, myelopathy or sensory neuropathy suggest that the poorly understood late neurologic complications of HIV infection are not associated with increased cytotoxic T cell activity in the CNS.
Acknowledgements Supported in part by grants NS26643, AI72634, NS23639, and RR00722 from the National Institutes of Health. The authors thank Ola Seines for his evaluation of patients in the neuropsychological portion of the study, B. Frank Polk and Alfred J. Saah for overall coordination of the Multicenter AIDS Cohort Study; Steve Holland, The Amyotropic Lateral Sclerosis Study Group and multiple members of the Johns Hopkins Housestaff for assistance in collecting samples of blood and CSF; Linda Kelly for preparation of the manuscript; and John Humpal and Will Howard for technical assistance.
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Price, H.E., Kermani-Arab, V. and Fahey, J.L. (1984) Depressed interleukin-2 receptor expression in acquired immune deficiency and lymphadenopathy syndromes. J. Immunol. 133, 1313-1317. Price, R.W., Brew, B., Sidtis, J., Rosenblum, M., Scheck, A.C. and Cleary, P. (1988) The brain in AIDS: central nervous system HIV-1 infection and AIDS dementia complex. Science 239, 586-592. Pui, C.H., Ip, S.H., Dodge, R.K., Carrabis, S., Brown, M., Crist, W.M., Berard, C.W., Kung, P., Dahl, G.V. and Murphy, S.B. (1988) Serum levels of CD8 antigen in childhood lymphoid malignancies: a possible indicator of increased suppressor cell activity in poor-risk patients. Blood 72, 1015-1021. Pumarola-Sune, T., Navia, B.A., Cordon-Cardo, C., Cho, E.S. and Price, R.W. (1987) HIV antigen in the brains of patients with the AIDS dementia complex. Ann. Neurol. 21, 490-496. Rubin, L.A., Kurman, C.C., Fritz, M.E., Biddison, W.E., Boutin, B., Yarchoan, R. and Nelson, D.L. (1985) Soluble interleukin 2 receptors are released from activated human lymphoid cells in vitro. J. Immunol. 135, 3172-3177. Salazar-Gonzalez, J.F., Moody, D.J., Giorgi, J.V., MartinezMaza, O.., Mitsuyasu, R.T. and Fahey, J.L. (1985) Reduced ecto-5'-nucleosidase activity and enhanced OKT10 and HLA-DR expression on CD8 (T suppressor/cytotoxic) lymphocytes in the acquired immune deficiency syndrome: evidence of CD8 cell immaturity. J. Immunol. 135, 17781785. Selnes, O.A., Miller, E., McArthur, J., Gordon, B., Munoz, A. and Becker, J.T. (1990) HIV-1 infection: no evidence of cognitive decline during the asymptomatic stages. Neurology (in press). Sethi, K.K. and Naher, H. (1986) Elevated titers of cell-free interleukin-2 receptor in serum and cerebrospinal fluid specimens of patients with acquired immunodeficiency syndrome. Immunol. Lett. 13, 179-184. Sethi, K.K., Naher, H. and Stroehmann, I. (1988) Phenotypic heterogeneity of cerebrospinal fluid-derived HIV-specific and HLA-restricted cytotoxic T-cell clones. Nature 335, 178-181. Sonnerborg, A.B., Ehrnst, A.C., Bergdahl, S.K.M., Pehrson, P.O., Skoldenberg, B.R. and Strannegard, O.O. (1988) HIV isolation from cerebrospinal fluid in relation to immunological deficiency and neurological symptoms. AIDS 2, 89-93. Snider, W.D., Simpson, D.M., Nielsen, S., Gold, J.W.M., Metroka, C.E. and Posner, J.B. (1983) Neurological complications of acquired immune deficiency syndrome: analysis of 50 patients. Ann. Neurol. 14, 403-418. Takada, S. and Engleman, E.G. (1987) Evidence for an association between CD8 molecules and the T cell receptor complex on cytotoxic T cells. J. Immunol. 139, 3231-3225. Tamashiro, V.G, Perez, H.H. and Griffin, D.E. (1987) Prospective study of the magnitude and duration of changes in tuberculin reactivity during complicated and uncomplicated measles. Pediatr. Infect. Dis. J. 6, 451-454. Tibbling, G., Link, H. and Ohman, S. (1977) Principles of
109 albumin and IgG analyses in neurological disorders. I. Establishment of reference values. Scand. J. Clin. Invest. 37, 385-390. Tomkinson, B.E., Brown, M.C., Ip, S.H., Carrabis, S. and Sullivan, J.L. (1989) Soluble CD8 during T cell activation. J. Immunol. 142, 2230-2236. Vazeux, R., Brousse, N., Jarry, A., Henin, D., Marche, C., Vedrenne, C., Mikol, J., Wolff, M., Michon, C., Rozenbaum, W., Bureau, J.-F., Montagnier, L. and Brahic, M. (1988) AIDS subacute encephalitis: identification of HIVinfected cells. Am. J. Pathol. 126, 403-410. Walker, B.D., Chakrabarti, S., Moss, B., Paradis, T.J., Flynn, T., Durno, A.G., Blumberg, R.S., Kaplan, J.C., Hirsch,
M.S. and Schooley, R.T. (1987) HIV-specific cytotoxic T lymphocytes in seropositive individuals. Nature 328, 345348. Walker, B.D,. Flexner, C., Paradis, T.J., Fuller, T.C., Hirsch, M.S., Schooley, R.T. and Moss, B. (1988) HIV-1 reverse transcriptase is a target for cytotoxic T lymphocytes in infected individuals. Science 240, 64-66. Wiley, C.A., Schrier, R.D., Nelson, J.A., Lampert, P.W. and Oldstone, M.B.A. (1986) Cellular localization of human immunodeficiency virus infection within the brains of acquired immune deficiency syndrome patients. Proc. Natl. Acad. Sci. U.S.A. 83, 7089-7093.
97
YNI 00944
Soluble interleukin-2 receptor and soluble CD8 in serum and cerebrospinal flUid during human immunodeficiency virus-associated neurologic disease Diane E. Griffin, Justin C. M c A r t h u r and David R. Cornblath Departments of Neurology and Medicine, The Johns Hopkins University School of Medicine, Baltimore, MD 21205, U.S.A. (Received 31 October 1989) (Revised, received 7 December 1989) (Accepted 7 December 1989)
Key words: Human immunodeficiency virus; Interleukin-2 receptor; CD8-positive T lymphocyte; Cerebrospinal fluid
Summary We have measured levels of soluble interleukin-2 receptor (slL-2R) and soluble CD8 (sCD8) in serum and cerebrospinal fluid (CSF) of 127 human immunodeficiency virus (HIV)-seropositive and 51 HIVseronegative individuals. Serum levels of slL-2R and sCD8 were higher in HIV ÷ than in HIVindividuals. HIV ÷ individuals were grouped by neurological status: asymptomatic, abnormal on neuropsychological screening, HIV-related meningitis, inflammatory demyelinating polyneuropathy, opportunistic central nervous system (CNS) infections and HIV-related dementia, myelopathy or sensory neuropathy. Serum levels of slL-2R and sCD8 were higher in all HIV ÷ categories compared to HIV- individuals. Patients with HIV-related meningitis had higher levels of slL-2R and sCD8 than asymptomatic HIV ÷ individuals, and inflammatory polyneuropathy patients had higher levels of sCD8. CSF levels of sCD8 were higher in all categories of HIV + than in HIV- individuals. Patients with HIV-related meningitis, inflammatory neuropathy and opportunistic infections had higher levels than asymptomatic individuals. Examination of the time course showed that serum and CSF levels of slL-2R and sCD8 increased to very high levels during acute HIV infections. Serum levels then declined over several months to relatively stable elevated levels. By 1-2 years after HIV infection slL-2R was relatively low in CSF, while sCD8 remained elevated with a gradual decrease over the subsequent years of follow-up.
Introduction A variety of neurologic diseases have been recognized as a consequence of infection with the Address for correspondence: Diane E. Griffin, M.D., Ph.D., Department of Neurology, Meyer 6-181, The Johns Hopkins Hospital, Baltimore, MD 21205, U.S.A.
human immunodeficiency virus (HIV). Neurologic diseases occur at all stages of HIV infection and involve both the peripheral and central nervous systems (McArthur, 1987). Certain diseases (e.g. the inflammatory demyelinating polyneuropathies and transient meningoencephalitis) are most common early in infection when T cell function is relatively preserved and immunosuppression is not
0165-5728/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
98 prominent (Cornblath et al., 1987; Hollander and Stringari, 1987; Parry, 1988). Other neurologic diseases (e.g. dementia, myelopathy, sensory neuropathies, and opportunistic central nervous system (CNS) infections) tend to occur later in infection with other systemic manifestations of the acquired immunodeficiency syndrome (AIDS) are likely to be present (Snider et al., 1983; Levy et al., 1985; Petito et al., 1985; Cornblath and McArthur, 1988; Price et al., 1988). HIV has been isolated from the cerebrospinal fluid (CSF) at all stages of disease (Ho et al., 1985; Hollander and Levy, 1987; Sonnerborg et al., 1988) but has been convincingly identified in brain only in scattered cells, most of which are macrophages (Gabuzda et al., 1986; Koenig et al., 1986; Wiley et al., 1986; Pumarola-Sune et al., 1987; Vazeux et al., 1987). Therefore, the mechanisms by which neurologic impairment is produced during HIV infection have not yet been clearly identified (Johnson et al., 1988). The immune system is likely to play an important role in HIV-associated neurologic disease. Virus is probably transported across the b l o o d brain barrier into the CNS inside inflammatory cells. Within the nervous system infected or reactive immune cells may produce products which are toxic to neural cells or myelin. Abnormalities of CSF are common in infected individuals both with and without neurologic disease (Anderson et al., 1988; Appleman et al., 1988; Hollander, 1988; McArthur et al., 1988). Previous characterization of the inflammatory cells in the CSF of HIVseropositive individuals has shown that the mononuclear cells are primarily T lymphocytes and that CD8-positive cells are more predominant in the CSF than in the peripheral blood (McArthur et al., 1989b). One approach to assessing the participation of the immune system in CNS disease associated with HIV infection is the measurement of soluble products of immune cells released as a consequence of immune reactions. Previous studies have shown that acute systemic viral infections (e.g. measles and infectious mononucleosis) are associated with immune activation and the appearance of increased amounts of soluble interleukin-2 receptor (sIL-2R) and soluble CD8 (sCD8) in serum (Griffin et al., 1989; Tomkinson et al., 1989).
During direct infection of the CNS slL-2R and sCD8 are increased in CSF while in immunemediated CNS disease, induced by prior virus infection, sIL-2R is not increased but sCD8 is (Boutin et al., 1987; Griffin et al., 1989). To evaluate the level of activation of T cells both in the peripheral blood and within the CNS during HIV infection we have measured the levels of sCD8 and sIL-2R in the serum and CSF of HIVseropositive individuals with various neuropsychological abnormalities. They were compared to neurologically normal HIV-seropositive individuals and to HIV-seronegative subjects with neurological diseases.
Materials and methods Patients
HIV-seropositive individuals came from two sources: inpatient and outpatient services of the Johns Hopkins Hospital and the Johns Hopkins University neuropsychological component of the Multicenter AIDS Cohort Study (MACS). The MACS neuropsychological study includes longitudinal neurological and neuropsychological testing in combination with directed physical examination, questionnaires, CD4 cell counts, CSF analysis and, in some subjects, magnetic resonance imaging. It is described in detail elsewhere (McArthur et al., 1989a). All patients were evaluated and samples collected between January, 1986 and February, 1989 except for two patients with additional samples from 1985. Informed consent was obtained from all individuals and the studies were approved by the Joint Council for Clinical Investigation of the Johns Hopkins Medical Institutions. Samples were frozen at - 2 0 ° C until analysis. A total of 177 individuals were studied. 167 paired serum and CSF samples were obtained from 123 HIV-seropositive individuals and 44 paired serum and CSF samples were obtained from HIV-seronegative individuals. Twelve unpaired serum and 11 CSF samples were obtained from these same individuals plus an additional four seropositive and seven seronegative persons. Of the HIV-seropositive individuals, 27 had no systemic symptoms (i.e. CDC groups I I / I I I ) (Centers for Disease Control, 1987) and were neu-
99
rologically normal (asymptomatic), 33 were systemically asymptomatic but had abnormal values on at least one component of a battery of neurologic and neuropsychological screening tests used in the MACS (NP positive), five had inflammatory demyelinating neuropathies (Cornblath et al., 1987), five had acute or chronic meningitis presumed due to HIV, 21 had HIV-related dementia (Navia et al., 1986), five had HIV-related myelopathy or vacuolar myelopathy (Pet±to et al., 1985), four had sensory neuropathy, 18 had presumed or proven CNS infections (11 cryptococcal meningitis, three toxoplasmosis, three syphilis, and one zoster meningitis) and 12 had various other neurologic problems including headache, metabolic encephalopathy, drug-induced psychosis and facial paralysis. Three patients moved from one clinical category to another during the course of follow-up. Presence or absence of a CSF pleocytosis (> 5 cells) did not alter the classification. Blood-brain barrier function was assessed using the formula of Tibbling et al. (1977) based on serum and CSF albumin. Values greater than 6.0 were considered abnormal. The 44 HIV-seronegative individuals had a variety of neurologic problems including 20 with amyotrophic lateral sclerosis, nine with acute or chronic inflammatory demyelinating neuropathies, three with dementia of the Alzheimer's type, and 12 with various other non-inflammatory neurological diseases.
Assays Soluble CD8 and slL-2R were assayed by enzyme immunoassay (T Cell Sciences, Cambridge, MA, U.S.A.). Assays were performed according to the manufacturer's instructions. Data are expressed as units/ml using standards supplied by the manufacturer. Statistics Analysis was performed using Student's t-test or Spearman's rank correlation (CD4 counts; blood-brain barrier function), employing Statview 512 software (Brainpower, Calabasas, CA, U.S.A.). Our primary analysis determined whether HIVseropositive subjects differed from HIV-seronegative subjects. In a secondary analysis HIVseropositive subjects were classified by neurologic
disease (117 individuals) or by time after seroconversion (33 individuals) and compared.
Results
The levels of slL-2R and sCD8 in serum and CSF were measured to assess the evidence for systemic immune activation and activation of T cells within the CNS during HIV infection (Table 1). Levels of both slL-2R and sCD8 were elevated in the serum of HIV-seropositive patients compared to the HIV-seronegative controls. In the HIV-seropositive group CSF levels of sCD8 were higher and levels of slL-2R were marginally lower than in the HIV-seronegative group. To determine whether HIV-seropositive individuals with varying neurological disorders had different levels of slL-2R or sCD8 in serum or CSF, individuals were grouped according to neurologic status into six categories: (1) systemically asymptomatic, neurologically normal (N = 27), (2) one or more abnormalities on neurological and neuropsychological screening tests ( N = 33), (3) HIV-related meningitis (N = 5), (4) inflammatory demyelinating polyneuropathy (N = 5), (5) HIVrelated neurological diseases occurring late in the course of infection including dementia, myelopathy and sensory neuropathy (N = 30), (6) opportunistic CNS infections (N = 18). Categories were chosen to represent the spectrum of neurologic diseases associated with early and late stages of HIV infection (Johnson et al., 1988). Each HIVTABLE 1 S O L U B L E IL-2 R E C E P T O R A N D S O L U B L E CD8 IN T H E SERUM AND CSF OF HIV-SEROPOSITIVE AND SERONEGATIVE INDIVIDUALS EVALUATED FOR N E U R O L O G I C DISEASE Patient groups HIV- (N) slL-2 receptor Serum 4 3 1 ± 3 2 a (41) CSF 7 8 ± 17 (41) sCD8 Serum 5 0 8 ± 8 9 (41) CSF 5 1 ± 1 0 (43) a Standard error of the mean.
p HIV + ( N ) 965 ± 52 (174) 4 8 ± 6 (169)
< 0.0001 0.049
1 7 7 8 ± 2 3 0 (177) 2 1 0 ± 28 (174)
0.0088 0.0053
100 60
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HIV-seropositive Fig. 1. Soluble interleukin-2 receptor in the serum of HIV-seropositiveindividuals with different categories of neurologic disease and HIV-seronegativeindividuals with other neurologic disease (OND). Levels in all groups of HIV-seropositiveindividuals were higher than in OND controls: Asx, asymptomatic, p = 0.004; NP +, abnormal neuropsychological testing, p = 0.0001; Men, meningitis, p < 0.0001; IDP, inflammatory demyelinating polyneuropathy, p = 0.024; DMN, dementia (O), myelopathy (zx), sensory neuropathy (o), p 48
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Time after admission Fig. 5. Serum and CSF levels of soluble IL-2 receptor and CD8 associated with acute meningoencephalitis in a single patient during HIV seroconversion. Hatched area indicates levels found in normal individuals.
tendency toward greater variability and higher values in those with abnormal barrier function (Table 2). CD4 T cell counts were available at one or more sampling times for 73 individuals representing all categories of HIV seropositive patients. There were no significant correlations between sIL-2R levels in serum or CSF or sCD8 levels in serum, but sCD8 levels in CSF were negatively
105 correlated with CD4 counts in blood (rs = -0.215, p < 0.05).
Discussion
We have studied slL-2R and sCD8, two different parameters of immune activation, in serum and CSF during chronic infection with HIV. Both T cell products were found in increased amounts in the serum of essentially all individuals infected with HIV. The highest levels were present during the initial immune response associated with seroconversion and a mononucleosis-like syndrome accompanied by acute self-limited neurologic disease. The levels of these T cell products have been related to the wide spectrum of CNS disease found in association with HIV infection. CSF levels of slL-2R were similar or lower than levels found in the CSF of individuals with non-HIV-associated neurological diseases. In contrast, sCD8 was often elevated in the CSF during HIV infection suggesting local T cell effector activity. Levels were even higher during HIV-associated meningitis and inflammatory demyelinating polyneuropathy. Activation of the immune system is common in viral infection and undoubtedly reflects the normal host response to a new antigenic challenge. Multiple components of the immune system are mobilized for proliferation of virus-specific lymphocytes leading to generation of effector cells and cellular products (lymphokines, antibody) designed to eliminate virus from tissue, plasma and interstitial fluid (Doherty and Allan, 1986). In acute viral infections, such as measles and infectious mononucleosis, this period of immunologic activation is transient and the immune system returns to a baseline activity within weeks after clearance of active infection (Griffin et al., 1989; Tomkinson et al., 1989). However, coincident with this period of immune activation multiple in vivo and in vitro abnormalities of immune function, such as suppressed skin test responses and mitogen-induced lymphoproliferative responses, are evident (Mange et al., 1974; Hirsch et al., 1985; Junker et al., 1986; Tamashiro et al., 1987). In HIV infection virus is not eliminated by the immune system and these studies suggest that this persistence of infection leads to chronic immuno-
logic activation as evidenced by continually elevated serum levels of immunoglobulin (Edelman and Zolla-Pazner, 1989), slL-2R and sCD8. This prolonged period of immune activation may be related to the immunologic abnormalities which are often present during HIV infection well before CD4-1ymphocytes have been depleted (Ascher and Sheppard, 1988; Edelman and Zolla-Pazner, 1989). Soluble IL-2R is produced as a result of binding of IL-2 to the high affinity (aft) receptor for IL-2 which may be present on both B cells and T cells. Binding of IL-2 simultaneously induces cell proliferation and release of a 45 kDa form of the a-chain of the IL-2 receptor (Rubin et al., 1985). Normal serum contains low levels of slL-2R (Greene et al., 1986) and levels have been reported by others to be elevated during HIV infection (Price et al., 1984; Sathi and Naher, 1986; Lang et al., 1988). In acute measles virus infection slL-2R becomes elevated before the appearance of virusspecific antibody and gradually declines over weeks after recovery (Griffin et al., 1989). In HIV infection slL-2R also becomes elevated at the time of the immune response to infection (seroconversion), declines somewhat after the most acute phase but then remains chronically elevated. The function of the soluble form of the IL-2 receptor is not known, but has been postulated to provide negative feedback for lymphocyte proliferation by binding to free IL-2 (Rubin et al., 1985) or by removing receptor from the cell surface (Diamantstein et al., 1986). Chronic elevation could contribute to depressed lymphoproliferative responses. Plasma levels of slL-2R are also elevated during transplant rejection (Colvin et al., 1987), IL-2 administration (Lotze et al., 1987), presumed autoimmune diseases such as multiple sclerosis (Greenberg et al., 1988) and rheumatoid arthritis (Keystone et al., 1988), and B and T cell malignancies (Pizzolo et al., 1987). CSF levels of slL-2R are elevated compared to asymptomatic patients only during the earliest acute stages of infection associated with meningitis and then again in association with inflammatory neuropathy. Elevated CSF levels have also been reported during herpes simplex virus encephalitis (Boutin et al., 1987) and a variety of other CNS infections (Griffin et al., 1989). These data suggest that after the earliest phases of dis-
106
ease IL-2-driven cell proliferation is not occurring to a significant extent within the CNS. Soluble CD8 is produced during the interaction of CD8-positive T cells with HLA class I-bearing target cells, such as virus-infected ceils and correlates well with the presence in the periphery of activated CD8-1ymphocytes (Tomkinson et al., 1989). CD8 is present as an associative recognition molecule in the CD3-T cell receptor complex on the cytotoxic/suppressor subpopulation of T cells (Emmrich et al., 1987; Takada and Engleman, 1987; Blue et al., 1988). Target cell interaction results in activation of the CD8-positive effector cell, phosphorylation of CD8 and release of a 27 kDa soluble form of CD8 from the surface of the cell (Fujimoto et al., 1983, 1984; Acres et al., 1987). In acute viral infection serum sCD8 is not increased until antiviral antibody appears and returns to normal shortly after virus clearance is completed (Griffin et al., 1989). Prolonged elevation of sCD8 in HIV infection is consistent with lack of virus clearance, and suggests ongoing cytotoxic T lymphocyte activity presumably directed against HIV-infected CD4-positive T cells and macrophages. The numbers of CD8-positive cells increase early in infection (Nicholson et al., 1984) and T cells cytotoxic for HIV-infected cells are present in the blood and CSF of infected individuals (Plata et al., 1987; Walker et al., 1987, 1988; Sethi et al., 1988). Elevated serum sCD8 suggests that effector-target cell interactions continue to occur throughout infection, including late stages of disease. However, increased suppressor cell activity has also been documented in HIV infection (Nicholson et al., 1984; Ascher and Sheppard, 1988) and could also account for increased circulating sCD8 (Pui et al., 1988). Either is consistent with documented abnormalities of CD8positive T cells in HIV infection which suggest chronic activation and/or immaturity (Nicholson et al., 1984; Salazar-Gonzalez et al., 1985). Markedly elevated levels during inflammatory demyelinating polyneuropathy are consistent with observed infiltration of nerve with CD8-positive T cells (Cornblath et al., 1990) and may reflect cytotoxic T cell attack either on normal ceils (autoimmune disease) or on HIV-infected cells within the nerve. CSF levels of sCD8 are elevated in most phases
of the infection, but are most elevated relatively early in infection during meningitis and inflammatory neuropathy, and then again when opportunistic infections occur. An asymptomatic pleocytosis is common during early HIV infection (Andersson et al., 1988; Hollander, 1988; McArthur et al., 1988) with a relatively increased proportion of CD8-positive cells in CSF compared to blood (McArthur et al., 1989b). These findings may reflect increased trafficking of activated CD8-positive T cells into the CNS or a specific, targeted response to HIV infection within the CNS. If the latter is the case the target cell for the CD8-positive cell has not yet been definitively identified. The low levels of sCD8 in CSF of patients with dementia, myelopathy or sensory neuropathy suggest that the poorly understood late neurologic complications of HIV infection are not associated with increased cytotoxic T cell activity in the CNS.
Acknowledgements Supported in part by grants NS26643, AI72634, NS23639, and RR00722 from the National Institutes of Health. The authors thank Ola Seines for his evaluation of patients in the neuropsychological portion of the study, B. Frank Polk and Alfred J. Saah for overall coordination of the Multicenter AIDS Cohort Study; Steve Holland, The Amyotropic Lateral Sclerosis Study Group and multiple members of the Johns Hopkins Housestaff for assistance in collecting samples of blood and CSF; Linda Kelly for preparation of the manuscript; and John Humpal and Will Howard for technical assistance.
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