J Neurol (1992) 239 : 387-388
Journal of
Neurology © Springer-Verlag1992
Cerebrospinal fluid cytokines in AIDS dementia complex Oreste Perrella 1, Pietro B. Carrieri 2, Domenico Guarnaccia 1, and Mario Soscia 1 1D. Cotugno Hospital for Infectious Diseases, Naples, Italy 2Institute of Neurology, 2nd Medical School, University of Naples, Naples, Italy Received August 19, 1991 / Accepted December 23, 1991
Summary. We evaluated cerebrospinal fluid (CSF) and serum concentrations of interleukin-l-alpha (IL-1alpha), interleukin-6 (IL-6) and tumour necrosis factoralpha (TNF-alpha) in 30 patients with AIDS dementia complex (ADC), and in 20 HIV-seronegative subjects with other neurological diseases (OND). CSF TNFalpha, IL-l-alpha and IL-6 were more frequently detectable in A D C patients than in O N D subjects. These cytokines were also detectable in CSF of A D C patients with minimal symptoms. In contrast, the majority of both A D C and O N D patients did not contain detectable serum levels of cytokines. Our data support the notion of intrathecal synthesis of cytokines in A D C patients and raise the possibility that activated macrophages may play a significant role in the pathogenesis of ADC. Key words: Cerebrospinal fluid - Cytokines - AIDS dementia complex
Introduction Patients with human immunodeficiency virus-1 (HIV-1) frequently develop neurological disorders. Some of these complications are due to opportunistic infections, but others, such as AIDS dementia complex (ADC), occur apparently in the absence of direct infection of neurons by HIV-1 [14]. The A D C is the most frequent neurological complication in AIDS patients and some symptoms of A D C may appear before any systemic abnormalities [8]. Several studies have reported a possible role of macrophage-derived cytokines in the pathogenesis of cerebral involvement in HIV-1 infection [3, 6, 10, 13], but the mechanism is unclear. Both astrocytes and microglial cells have been found to produce a tumour necrosis factor-alpha (TNF-alpha)-like factor that has an immunomodulatory role within the cytokines network and may cause neurological damage [4]. Recently, Giulian et al. [5] reported that macrophages and microglia infected Correspondence to: O.Perrella, Via E.A.Mario, 35, 1-80131
Naples, Italy
with HIV-1 release a neurotoxic factor that kills rodent spinal neurons and chick ciliary neurons in culture. The present study was designed to demonstrate whether the cerebrospinal fluid (CSF) detection of cytokines in A D C patients in various stages of disease may predict the cerebral involvement before systemic abnormalities. Patients and methods We studied 30 patients (10 female, 20 male; mean age 30 years; range 19-41 years), affected by HIV-1 infection and classified according to the criteria of Centers for Diseases Control [2]. All patients had cerebral involvement and were diagnosed as ADC by clinical and neuroimaging data when other causes of dementia had been excluded [9]. The ADC patients were divided, according to Price and Brew [11], as follows: 15 with equivocal or subclinical symptoms (stage 0.5) and 15 in stage 2 or 3. Twenty subjects (12 male, 8 female; mean age 33 years; range 20-46 years), HIV-1 seronegative, served as controls and had one of the following other neurological diseases (OND): tension headache, meningioma, hereditary degenerative ataxia, metabolic polyneuropathy or degenerative disk problems. Paired CSF and serum were obtained simultaneously from all patients. CSF samples were evaluated for glucose, protein and cell counts. Bacteriological and fungal cultures were also performed. Serum and CSF TNF-alpha, interleukin-l-alpha (IL-l-alpha) and interleukin-6 (IL-6) were simultaneously detected by three different enzyme-linked immunosorbent assay (ELISA) kits from Genzyme (Boston, Mass., USA), according to the manufacturer's instructions. Both serum and CSF were diluted 1 : 1. The levels in serum and CSF were measured in duplicate. The sensitivity of each ELISA is as follows: TNF-alpha 7.5 pg/m.; IL-l-alpha 1.5 pg/ml; IL-6 20 pg/ml. Statistical analysis were done using the Fisher exact test and Pearson correlations. Results The results are summarized in Table 1. TNF-alpha was detected in the CSF of 14 A D C patients in stage 2 or 3, in 8 A D C patients in stage 0.5, and in none of the OND patients. The numbers of patients with detectable serum levels of TNF-alpha were: A D C 9 of 30, OND 6 of 20. CSF IL-l-alpha levels were detected in 11 of 15 A D C patients in stage 2 or 3, in 7 of 15 A D C patients in
388 Table 1. Number of positive CSF and serum samples (range in pg/ml) among the total number of CSF and serum samples tested for tumour necrosis factor-alpha (TNF-alpha), interleukin-l-alpha (IL-l-alpha) and interleukin-6 (IL-6) in patients with AIDS dementia complex (ADC) and with other neurological diseases (OND). * P < 0.01, with respect to OND patients
TNF-alpha IL-l-alpha IL-6
ADC (stages 2-3)
ADC (stage 0.5)
OND
CSF
Serum
CSF
Serum
CSF
Serum
14 / 15" (15-190) 11 / 15" (10-85) 13 / 15" (20-320)
6 / 15 (7.5-20) 3 / 15 (1.5-7) 0 / 15
8 / 15" (7.5-40) 7 / 15" (7-70) 12 / 15" (20-140)
3 / 15 (7.5-15) 2 ! 15 (1.5-4) 0/15
0 / 20
6 / 20 (7.5-15) 3 / 20 (1.5-5) 0 / 20
stage 0.5, and in 1 of 20 O N D patients. T h e n u m b e r s of patients with I L - l - a l p h a detectable levels in serum were: A D C 5 of 30, O N D 3 of 20. CSF IL-6 was detectable in 13 of 15 A D C patients in stage 2 or 3, in 12 of 15 A D C patients in the earlier stage, and in 0 of 20 O N D patients. T h e n u m b e r s of patients with detectable s e r u m levels of IL-6 were: A D C 0 of 30, O N D 0 of 20. N o correlation was f o u n d b e t w e e n C S F cell c o u n t or protein c o n c e n t r a t i o n and CSF cytokine levels.
Discussion Mildly elevated C S F cell c o u n t or protein c o n c e n t r a t i o n , impaired b r a i n - b l o o d barrier, identification of oligoclonal bands, and intrathecal p r o d u c t i o n of beta-2-microglobulin, n e o p t e r i n and a n t i b o d y to H I V - 1 have b e e n described in neurologically a s y m p t o m a t i c subjects, in the early stages of chronic H I V - 1 infection. T h e present study shows that detectable levels of cytokines m a y occur intrathecally in A D C patients with subclinical symptoms. T h e s e cytokines, released f r o m infected cells, m a y have a d a m a g i n g effect on the n e r v o u s system [1], and H I V gene p r o d u c t s would have h a r m f u l effects on uninfected tissue. T h e chronically infected m a c r o p h a g e s are an i m p o r t a n t source for H I V - 1 diffusion in the central n e r v o u s system and can p r o d u c e elevated concentrations of cytokines and o t h e r soluble factors [12]. M o r e over the cytokines m a y play a role in the conversion of a latent or chronic infection to a productive one, with a transactivating m e c h a n i s m w h e r e b y a D N A - b i n d i n g protein binds to the N F k B site of the H I V - L T R [7]. M a n y conditions characterized by i n f l a m m a t i o n of brain tissue are associated with m a c r o p h a g e activation and with proliferation of astrocytes and microglial cells: these cells m a y secrete cytokines. In our data, the lack of correlation b e t w e e n C S F pleocytosis and cytokines suggests that CSF cytokine levels m a y be derived f r o m m a c r o p h a g e s or astrocytes of the central nervous system, but n o t f r o m cells in the CSF. In conclusion, we think that CSF cytokine accumulation m a y be a useful m a r k e r for diagnostic evaluation o f cerebral i n v o l v e m e n t in H I V - 1 infection, b e f o r e any systemic abnormalities arise. F u r t h e r studies will n e e d to
1 / 20 (7.5) 0 / 20
clarify the relationship b e t w e e n CSF cytokine accumulations and the clinical course of A D C .
Acknowledgement. Our research program in AIDS is supported by the Istituto Superiore di Sanitfi (Italy), research no. 6283-004.
References 1. Brosnan CF, Selmaj K, Raine CS (1988) Hypothesis: a role for tumor necrosis factor in immune mediated demyelination and its relevance to multiple sclerosis. J Neuroimmunol 18 : 87-94 2. Centers for Disease Control (1985) Revision of the case definition of acquired immunodeficiency syndrome for national reporting - United States. MMWR 34 : 373-374 3. Gallo P, De Rossi A, Amadori A, Tavolato B, Chieco-Bianchi L (1988) Central nervous system involvement in HIV-infection. AIDS Res Hum Retrovirus 4:211-221 4. Gallo P, Piccinno MG, Krzalic L, Tavolato B (1989) Tumor necrosis factor-alpha and neurological diseases. J Neuroimmunol 23 : 41-44 5. Giulian D, Vaca K, Noonan CA (1990) Secretion of neurotoxin by mononuclear phagocytes infected with HIV-1. Science 250 : 1593-1596 6. Koyanagy Y, O'Brien WA, Zhao JQ, Golde DW, Gasson JC, Chen CS (1988) Cytokines alter production of HIV-1 from primary mononuclear phagocytes. Science 241 : 1678 7. Levy RM, Bredsen DE (1988) Central nervous system dysfunction in AIDS. J AIDS 1 : 41-64 8. Navia BA, Price RW (1987) The acquired immunodeficiency syndrome dementia complex as the presenting or sole manifestation of human immunodeficiency virus infection. Arch Neurol 44: 65-69 9. Navia BA, Jordan BD, Price RW (1986) The AIDS dementia complex. I. Clinical features. Ann Neurol 19: 517-524 10. Osborn L, Kunkel S, Nabel GS (1989) Tumor necrosis factoralpha and activation of the nuclear factor KB. Proc Natl Acad Sci USA 86 : 2336-2340 11. Price RW, Brew BJ (1988) The AIDS dementia complex. J Infect Dis 158 : 1079-1083 12. Pulliam L, Herudler BG, Tang NM, Grath MS (1991) Human immunodeficiency virus-infected macrophages produce soluble factors that cause histological and neurochemical alteration in cultured human brains. J Clin Invest 87 : 503-512 13. Rosenberg ZF, Fauci AS (1989) Immunology of AIDS: approaches to understanding the immunopathogenesis of HIV infection. Res Clin Lab 19 : 189-209 14. Snider WD, Simpson DM, Nielsen S, Gold JWM, Metroka CE, Posner JB (1983) Neurological complications of acquired immune deficiency syndrome: analysis of 50 patients. Ann Neurol 14 : 403-418
Journal of
Neurology © Springer-Verlag1992
Cerebrospinal fluid cytokines in AIDS dementia complex Oreste Perrella 1, Pietro B. Carrieri 2, Domenico Guarnaccia 1, and Mario Soscia 1 1D. Cotugno Hospital for Infectious Diseases, Naples, Italy 2Institute of Neurology, 2nd Medical School, University of Naples, Naples, Italy Received August 19, 1991 / Accepted December 23, 1991
Summary. We evaluated cerebrospinal fluid (CSF) and serum concentrations of interleukin-l-alpha (IL-1alpha), interleukin-6 (IL-6) and tumour necrosis factoralpha (TNF-alpha) in 30 patients with AIDS dementia complex (ADC), and in 20 HIV-seronegative subjects with other neurological diseases (OND). CSF TNFalpha, IL-l-alpha and IL-6 were more frequently detectable in A D C patients than in O N D subjects. These cytokines were also detectable in CSF of A D C patients with minimal symptoms. In contrast, the majority of both A D C and O N D patients did not contain detectable serum levels of cytokines. Our data support the notion of intrathecal synthesis of cytokines in A D C patients and raise the possibility that activated macrophages may play a significant role in the pathogenesis of ADC. Key words: Cerebrospinal fluid - Cytokines - AIDS dementia complex
Introduction Patients with human immunodeficiency virus-1 (HIV-1) frequently develop neurological disorders. Some of these complications are due to opportunistic infections, but others, such as AIDS dementia complex (ADC), occur apparently in the absence of direct infection of neurons by HIV-1 [14]. The A D C is the most frequent neurological complication in AIDS patients and some symptoms of A D C may appear before any systemic abnormalities [8]. Several studies have reported a possible role of macrophage-derived cytokines in the pathogenesis of cerebral involvement in HIV-1 infection [3, 6, 10, 13], but the mechanism is unclear. Both astrocytes and microglial cells have been found to produce a tumour necrosis factor-alpha (TNF-alpha)-like factor that has an immunomodulatory role within the cytokines network and may cause neurological damage [4]. Recently, Giulian et al. [5] reported that macrophages and microglia infected Correspondence to: O.Perrella, Via E.A.Mario, 35, 1-80131
Naples, Italy
with HIV-1 release a neurotoxic factor that kills rodent spinal neurons and chick ciliary neurons in culture. The present study was designed to demonstrate whether the cerebrospinal fluid (CSF) detection of cytokines in A D C patients in various stages of disease may predict the cerebral involvement before systemic abnormalities. Patients and methods We studied 30 patients (10 female, 20 male; mean age 30 years; range 19-41 years), affected by HIV-1 infection and classified according to the criteria of Centers for Diseases Control [2]. All patients had cerebral involvement and were diagnosed as ADC by clinical and neuroimaging data when other causes of dementia had been excluded [9]. The ADC patients were divided, according to Price and Brew [11], as follows: 15 with equivocal or subclinical symptoms (stage 0.5) and 15 in stage 2 or 3. Twenty subjects (12 male, 8 female; mean age 33 years; range 20-46 years), HIV-1 seronegative, served as controls and had one of the following other neurological diseases (OND): tension headache, meningioma, hereditary degenerative ataxia, metabolic polyneuropathy or degenerative disk problems. Paired CSF and serum were obtained simultaneously from all patients. CSF samples were evaluated for glucose, protein and cell counts. Bacteriological and fungal cultures were also performed. Serum and CSF TNF-alpha, interleukin-l-alpha (IL-l-alpha) and interleukin-6 (IL-6) were simultaneously detected by three different enzyme-linked immunosorbent assay (ELISA) kits from Genzyme (Boston, Mass., USA), according to the manufacturer's instructions. Both serum and CSF were diluted 1 : 1. The levels in serum and CSF were measured in duplicate. The sensitivity of each ELISA is as follows: TNF-alpha 7.5 pg/m.; IL-l-alpha 1.5 pg/ml; IL-6 20 pg/ml. Statistical analysis were done using the Fisher exact test and Pearson correlations. Results The results are summarized in Table 1. TNF-alpha was detected in the CSF of 14 A D C patients in stage 2 or 3, in 8 A D C patients in stage 0.5, and in none of the OND patients. The numbers of patients with detectable serum levels of TNF-alpha were: A D C 9 of 30, OND 6 of 20. CSF IL-l-alpha levels were detected in 11 of 15 A D C patients in stage 2 or 3, in 7 of 15 A D C patients in
388 Table 1. Number of positive CSF and serum samples (range in pg/ml) among the total number of CSF and serum samples tested for tumour necrosis factor-alpha (TNF-alpha), interleukin-l-alpha (IL-l-alpha) and interleukin-6 (IL-6) in patients with AIDS dementia complex (ADC) and with other neurological diseases (OND). * P < 0.01, with respect to OND patients
TNF-alpha IL-l-alpha IL-6
ADC (stages 2-3)
ADC (stage 0.5)
OND
CSF
Serum
CSF
Serum
CSF
Serum
14 / 15" (15-190) 11 / 15" (10-85) 13 / 15" (20-320)
6 / 15 (7.5-20) 3 / 15 (1.5-7) 0 / 15
8 / 15" (7.5-40) 7 / 15" (7-70) 12 / 15" (20-140)
3 / 15 (7.5-15) 2 ! 15 (1.5-4) 0/15
0 / 20
6 / 20 (7.5-15) 3 / 20 (1.5-5) 0 / 20
stage 0.5, and in 1 of 20 O N D patients. T h e n u m b e r s of patients with I L - l - a l p h a detectable levels in serum were: A D C 5 of 30, O N D 3 of 20. CSF IL-6 was detectable in 13 of 15 A D C patients in stage 2 or 3, in 12 of 15 A D C patients in the earlier stage, and in 0 of 20 O N D patients. T h e n u m b e r s of patients with detectable s e r u m levels of IL-6 were: A D C 0 of 30, O N D 0 of 20. N o correlation was f o u n d b e t w e e n C S F cell c o u n t or protein c o n c e n t r a t i o n and CSF cytokine levels.
Discussion Mildly elevated C S F cell c o u n t or protein c o n c e n t r a t i o n , impaired b r a i n - b l o o d barrier, identification of oligoclonal bands, and intrathecal p r o d u c t i o n of beta-2-microglobulin, n e o p t e r i n and a n t i b o d y to H I V - 1 have b e e n described in neurologically a s y m p t o m a t i c subjects, in the early stages of chronic H I V - 1 infection. T h e present study shows that detectable levels of cytokines m a y occur intrathecally in A D C patients with subclinical symptoms. T h e s e cytokines, released f r o m infected cells, m a y have a d a m a g i n g effect on the n e r v o u s system [1], and H I V gene p r o d u c t s would have h a r m f u l effects on uninfected tissue. T h e chronically infected m a c r o p h a g e s are an i m p o r t a n t source for H I V - 1 diffusion in the central n e r v o u s system and can p r o d u c e elevated concentrations of cytokines and o t h e r soluble factors [12]. M o r e over the cytokines m a y play a role in the conversion of a latent or chronic infection to a productive one, with a transactivating m e c h a n i s m w h e r e b y a D N A - b i n d i n g protein binds to the N F k B site of the H I V - L T R [7]. M a n y conditions characterized by i n f l a m m a t i o n of brain tissue are associated with m a c r o p h a g e activation and with proliferation of astrocytes and microglial cells: these cells m a y secrete cytokines. In our data, the lack of correlation b e t w e e n C S F pleocytosis and cytokines suggests that CSF cytokine levels m a y be derived f r o m m a c r o p h a g e s or astrocytes of the central nervous system, but n o t f r o m cells in the CSF. In conclusion, we think that CSF cytokine accumulation m a y be a useful m a r k e r for diagnostic evaluation o f cerebral i n v o l v e m e n t in H I V - 1 infection, b e f o r e any systemic abnormalities arise. F u r t h e r studies will n e e d to
1 / 20 (7.5) 0 / 20
clarify the relationship b e t w e e n CSF cytokine accumulations and the clinical course of A D C .
Acknowledgement. Our research program in AIDS is supported by the Istituto Superiore di Sanitfi (Italy), research no. 6283-004.
References 1. Brosnan CF, Selmaj K, Raine CS (1988) Hypothesis: a role for tumor necrosis factor in immune mediated demyelination and its relevance to multiple sclerosis. J Neuroimmunol 18 : 87-94 2. Centers for Disease Control (1985) Revision of the case definition of acquired immunodeficiency syndrome for national reporting - United States. MMWR 34 : 373-374 3. Gallo P, De Rossi A, Amadori A, Tavolato B, Chieco-Bianchi L (1988) Central nervous system involvement in HIV-infection. AIDS Res Hum Retrovirus 4:211-221 4. Gallo P, Piccinno MG, Krzalic L, Tavolato B (1989) Tumor necrosis factor-alpha and neurological diseases. J Neuroimmunol 23 : 41-44 5. Giulian D, Vaca K, Noonan CA (1990) Secretion of neurotoxin by mononuclear phagocytes infected with HIV-1. Science 250 : 1593-1596 6. Koyanagy Y, O'Brien WA, Zhao JQ, Golde DW, Gasson JC, Chen CS (1988) Cytokines alter production of HIV-1 from primary mononuclear phagocytes. Science 241 : 1678 7. Levy RM, Bredsen DE (1988) Central nervous system dysfunction in AIDS. J AIDS 1 : 41-64 8. Navia BA, Price RW (1987) The acquired immunodeficiency syndrome dementia complex as the presenting or sole manifestation of human immunodeficiency virus infection. Arch Neurol 44: 65-69 9. Navia BA, Jordan BD, Price RW (1986) The AIDS dementia complex. I. Clinical features. Ann Neurol 19: 517-524 10. Osborn L, Kunkel S, Nabel GS (1989) Tumor necrosis factoralpha and activation of the nuclear factor KB. Proc Natl Acad Sci USA 86 : 2336-2340 11. Price RW, Brew BJ (1988) The AIDS dementia complex. J Infect Dis 158 : 1079-1083 12. Pulliam L, Herudler BG, Tang NM, Grath MS (1991) Human immunodeficiency virus-infected macrophages produce soluble factors that cause histological and neurochemical alteration in cultured human brains. J Clin Invest 87 : 503-512 13. Rosenberg ZF, Fauci AS (1989) Immunology of AIDS: approaches to understanding the immunopathogenesis of HIV infection. Res Clin Lab 19 : 189-209 14. Snider WD, Simpson DM, Nielsen S, Gold JWM, Metroka CE, Posner JB (1983) Neurological complications of acquired immune deficiency syndrome: analysis of 50 patients. Ann Neurol 14 : 403-418
