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Circulating gp120 alters the blood–brain barrier permeability in HIV-1 gp120 transgenic mice
Neuroscience Letters 330 (2002) 299–301 www.elsevier.com/locate/neulet
Circulating gp120 alters the blood–brain barrier permeability in HIV-1 gp120 transgenic mice Chiara Cioni, Pasquale Annunziata* Istituto di Scienze Neurologiche, Universita` di Siena, Viale Bracci, 2, 53100 Siena, Italy Received 30 May 2002; received in revised form 2 July 2002; accepted 19 July 2002
Abstract The mechanism underlying invasion of the central nervous system by HIV-1 is unclear. We recently demonstrated blood–brain barrier changes in a model of HIV-1 gp120 transgenic mice. To test whether this alteration was intrinsic to the brain endothelium of transgenic mice or depended on circulating gp120, we used brain endothelial cultures from gp120 transgenic and non-transgenic mice and exposed them to serum from gp120 transgenic or non-transgenic mice. We measured permeability to albumin as a marker of functional endothelial integrity. A significant increase in permeability (up to 47%) was observed in transgenic and non-transgenic cultures exposed to serum samples from transgenic but not to those from non-transgenic mice. This permeability was neutralized after immunoabsorption of sera with antigp120 monoclonal antibody. These findings demonstrate that the blood–brain barrier alteration in HIV-1 gp120 transgenic mice is due to circulating gp120. q 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Blood–brain barrier; Endothelium; HIV-1; Envelope proteins; AIDS; Transgenic models
Alteration of the blood–brain barrier (BBB) function is a fundamental early step in invasion of the central nervous system by HIV-1. The mechanism underlying this invasion is unclear and subject to investigation. We recently demonstrated that the BBB of HIV-1 gp120 transgenic mice that secrete circulating HIV-1 envelope protein gp120 levels similar to those found in AIDS patients [4] is altered and that the endothelium of cerebral microvessels expresses adhesion molecules as a sign of activation [10]. It is not yet known whether this alteration is intrinsic to brain vessel endothelium of transgenic mice or depends on circulating gp120. To answer this question, we cultured brain endothelial cells from gp120 transgenic and non-transgenic mice and exposed them to serum of gp120 transgenic and nontransgenic mice. We measured permeability to albumin as a marker of the functional integrity of the endothelium. Brain endothelial cultures were prepared from microvessels isolated from brains [6] of 3-month-old transgenic (C57BL/6 £ CB6F1) and non-transgenic (C57BL/6) mice as previously described [1]. Circulating gp120 protein was measured in serum samples obtained from two transgenic mice (numbers 749 and 751) by capture ELISA [3]. * Corresponding author. Tel.: 139-577-233482; fax: 139-57740327. E-mail address: [email protected] (P. Annunziata).
Endothelial cells were trypsinized (0.1% trypsin in Hank’s solution) for 15 min at 37 8C and seeded at a density of 7.5 £ 10 5/cm 2 in growth medium (Medium 199, aminoacid solution containing 30% foetal bovine serum) on TranswellCOL membrane inserts (0.4 mm pore size, collagen treated, transparent membranes; Costar, Cambridge, MA) precoated with bovine fibronectin (25 mg/ml) and then placed in double-chamber microplates. Cultures at 37 8C were exposed for 24 h to 15% non-transgenic and transgenic mice sera previously heated to 56 8C for 30 min to inactivate complement. In some experiments, transgenic mice serum samples (containing 25 and 33 pM gp120, respectively) were immunoabsorbed with a higher molar concentration (660 and 1320 pM, respectively) of anti-gp120 monoclonal antibody (Intracel, Cambridge, MA) and of an isotypematched mouse monoclonal antibody (Ancell, Bayport, MN) for 2 h at room temperature, diluted to 19 ng/ml and incubated with separate cultures at a 15% final concentration. Permeability to biotin-labelled albumin was measured and calculated by a previously described method [2]. Briefly, after washing, the brain endothelial cultures were incubated with biotin-labelled albumin (4 mg/ml) for 1.5 h at 37 8C with shaking, then, after several washings with PBS–0.05% Tween-20, peroxidase-conjugated streptavidin (1:10,000 in PBS) was added for 45 min at room temperature and furthermore, after washing, 0.1% o-phenylendia-
0304-3940/02/$ - see front matter q 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S03 04 - 394 0( 0 2) 00 81 4- 5
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C. Cioni, P. Annunziata / Neuroscience Letters 330 (2002) 299–301
mine in pH 4.5 citrate buffer–0.015% H2O2 was added for 10 min at room temperature. After blocking with H2SO4, the colour that developed was analyzed by spectrophotometry at 492 nm. Permeability to albumin was calculated as a percentage passage according to the following formula: Absorbance of the samples in lower chamber 2 background values £ 100 Absorbance of biotin 2 labelled albumin in upper chamber 2 background values
Background values were obtained from absorbance of medium alone. In transgenic mouse brain endothelial cultures exposed to normal mouse serum, no increase in permeability to albumin was found; by contrast, a significant increase in permeability (up to 47%) was observed on exposure to serum samples from transgenic mice. This elevated permeability was neutralized after immunoabsorption of sera with antigp120 monoclonal antibody but not with isotype control (Fig. 1). In brain endothelial cultures from non-transgenic mice, transgenic but not non-transgenic mouse serum caused increased permeability to albumin, which was completely blocked by anti-gp120 monoclonal antibody but not by isotype-matched control antibody (Fig. 2). In transgenic or non-transgenic cultures not exposed to mouse sera, albumin passage was less than 1%, indicating similar functional integrity of the endothelium. Our findings demonstrate that changes in brain endothelium permeability found in vivo in HIV-1 gp120 transgenic mice are exclusively due to the action of circulating gp120 protein, confirming our previous in vitro data obtained with recombinant gp120 protein in rat brain endothelium cultures [2]. Circulating gp120 levels have been detected in the sera of AIDS patients [7] and may be due to protein shedding from the envelope of mature HIV-1 [5]. Impairment of the BBB has been found in autopsy brain tissues from AIDS patients with [9] and without dementia [8]. Gp120 acts as a proin-
Fig. 1. Albumin permeability of HIV-1 gp120 transgenic mouse brain endothelial cultures exposed for 24 h to non-transgenic mouse serum (NTS) and to gp120 transgenic mouse serum (TS) in the presence or absence of anti-gp120 monoclonal antibody at doses of 1 mg/ml (Mab 1) and 2 mg/ml (Mab 2) or isotype control antibody (2 mg/ml) (Iso). UNS, unstimulated cultures. Values are expressed as means ^ SE of two independent experiments (n ¼ 4). In each experiment, each point was analyzed in duplicate except for Mab 1, Mab 2 and Iso (n ¼ 2).
Fig. 2. Albumin permeability of non-transgenic mouse brain endothelial cultures exposed for 24 h to non-transgenic mouse serum (NTS) and to gp120 transgenic mouse serum (TS) in the presence or absence of anti-gp120 monoclonal antibody at doses of 1 mg/ml (Mab 1) and 2 mg/ml (Mab 2) or isotype control antibody (2 mg/ml) (Iso). UNS, unstimulated cultures. Values are expressed as means ^ SE of two independent experiments (n ¼ 4). In each experiment, each point was analyzed in duplicate except for Mab 1, Mab 2 and Iso (n ¼ 2).
flammatory cytokine inducing the production of substance P by rat brain endothelium [2] and causing significant changes in permeability. The demonstration that circulating gp120 levels increase in vitro brain endothelium permeability in a transgenic mouse model for HIV-1 gp120 is further evidence that a similar mechanism may occur in HIV-positive human subjects and contributes to the pathogenesis of HIV-related neurological findings. We thank Dr O. Finco (Chiron S.p.A., Siena, Italy) for providing us with gp120 transgenic mice. [1] Annunziata, P., Cioni, C., Moschini, F., Riccucci, A. and Guazzi, G.C., Serum anti-brain endothelium antibodies and cognitive assessment in patients with Binswanger’s encephalopathy, J. Neurol. Sci., 128 (1995) 96–102. [2] Annunziata, P., Cioni, C., Toneatto, S. and Paccagnini, E., HIV-1 gp120 increases the permeability of rat brain endothelium cultures by a mechanism involving substance P, AIDS, 12 (1998) 2377–2385. [3] Coligan, J.E., Kruisbeek, A.M., Margulies, D.H., Sherac, E.M. and Strober, W., Current Protocols in Immunology, National Institute of Health, Bethesda, MD, 1995, pp. 2.1.9–2.1.11. [4] Finco, O., Nuti, S., De Magistris, M.T., Mangiavacchi, L., Aiuti, A., Forte, P., Fantoni, A., van der Putten, H. and Abrignani, S., Induction of CD4 1 T cell depletion in mice doubly transgenic for HIV gp120 and human CD4, Eur. J. Immunol., 27 (1997) 1319–1324. [5] Gelderblom, H.R., Reupke, H. and Pauli, G., Loss of envelope antigens of HTLV-III/LAV, a factor in AIDS pathogenesis? Lancet, 2 (1985) 1016–1017. [6] Mrsulja, B.B., Mrsulja, B.J., Fujimoto, T., Klatzo, I. and Spatz, M., Isolation of brain capillaries: a simplified technique, Brain Res., 110 (1976) 361–365. [7] Oh, S.-K., Cruikshank, W.W., Raina, J., Blanchard, G.C., Adler, W.H., Walker, J. and Kornfeld, H., Identification of HIV-1 envelope glycoprotein in the serum of AIDS and ARC patients, J. Acquir. Immune Defic. Syndr., 5 (1992) 251–256.
C. Cioni, P. Annunziata / Neuroscience Letters 330 (2002) 299–301 [8] Petito, C.K. and Cash, K.S., Blood–brain barrier abnormalities in the acquired immunodeficiency syndrome: immunohistochemical localization of serum proteins in postmortem brain, Ann. Neurol., 32 (1992) 658–666. [9] Power, C., Kong, P.-A., Crawford, T.O., Wesselingh, S., Glass, J.D., McArthur, J.C. and Trapp, B.D., Cerebral white matter changes in acquired immunodeficiency syndrome
301
dementia: alterations of the blood–brain barrier, Ann. Neurol., 34 (1993) 339–350. [10] Toneatto, S., Finco, O., van der Putten, H., Abrignani, S. and Annunziata, P., Evidence of blood–brain barrier alteration and activation in HIV-1 gp120 transgenic mice, AIDS, 13 (1999) 2343–2348.
Circulating gp120 alters the blood–brain barrier permeability in HIV-1 gp120 transgenic mice Chiara Cioni, Pasquale Annunziata* Istituto di Scienze Neurologiche, Universita` di Siena, Viale Bracci, 2, 53100 Siena, Italy Received 30 May 2002; received in revised form 2 July 2002; accepted 19 July 2002
Abstract The mechanism underlying invasion of the central nervous system by HIV-1 is unclear. We recently demonstrated blood–brain barrier changes in a model of HIV-1 gp120 transgenic mice. To test whether this alteration was intrinsic to the brain endothelium of transgenic mice or depended on circulating gp120, we used brain endothelial cultures from gp120 transgenic and non-transgenic mice and exposed them to serum from gp120 transgenic or non-transgenic mice. We measured permeability to albumin as a marker of functional endothelial integrity. A significant increase in permeability (up to 47%) was observed in transgenic and non-transgenic cultures exposed to serum samples from transgenic but not to those from non-transgenic mice. This permeability was neutralized after immunoabsorption of sera with antigp120 monoclonal antibody. These findings demonstrate that the blood–brain barrier alteration in HIV-1 gp120 transgenic mice is due to circulating gp120. q 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Blood–brain barrier; Endothelium; HIV-1; Envelope proteins; AIDS; Transgenic models
Alteration of the blood–brain barrier (BBB) function is a fundamental early step in invasion of the central nervous system by HIV-1. The mechanism underlying this invasion is unclear and subject to investigation. We recently demonstrated that the BBB of HIV-1 gp120 transgenic mice that secrete circulating HIV-1 envelope protein gp120 levels similar to those found in AIDS patients [4] is altered and that the endothelium of cerebral microvessels expresses adhesion molecules as a sign of activation [10]. It is not yet known whether this alteration is intrinsic to brain vessel endothelium of transgenic mice or depends on circulating gp120. To answer this question, we cultured brain endothelial cells from gp120 transgenic and non-transgenic mice and exposed them to serum of gp120 transgenic and nontransgenic mice. We measured permeability to albumin as a marker of the functional integrity of the endothelium. Brain endothelial cultures were prepared from microvessels isolated from brains [6] of 3-month-old transgenic (C57BL/6 £ CB6F1) and non-transgenic (C57BL/6) mice as previously described [1]. Circulating gp120 protein was measured in serum samples obtained from two transgenic mice (numbers 749 and 751) by capture ELISA [3]. * Corresponding author. Tel.: 139-577-233482; fax: 139-57740327. E-mail address: [email protected] (P. Annunziata).
Endothelial cells were trypsinized (0.1% trypsin in Hank’s solution) for 15 min at 37 8C and seeded at a density of 7.5 £ 10 5/cm 2 in growth medium (Medium 199, aminoacid solution containing 30% foetal bovine serum) on TranswellCOL membrane inserts (0.4 mm pore size, collagen treated, transparent membranes; Costar, Cambridge, MA) precoated with bovine fibronectin (25 mg/ml) and then placed in double-chamber microplates. Cultures at 37 8C were exposed for 24 h to 15% non-transgenic and transgenic mice sera previously heated to 56 8C for 30 min to inactivate complement. In some experiments, transgenic mice serum samples (containing 25 and 33 pM gp120, respectively) were immunoabsorbed with a higher molar concentration (660 and 1320 pM, respectively) of anti-gp120 monoclonal antibody (Intracel, Cambridge, MA) and of an isotypematched mouse monoclonal antibody (Ancell, Bayport, MN) for 2 h at room temperature, diluted to 19 ng/ml and incubated with separate cultures at a 15% final concentration. Permeability to biotin-labelled albumin was measured and calculated by a previously described method [2]. Briefly, after washing, the brain endothelial cultures were incubated with biotin-labelled albumin (4 mg/ml) for 1.5 h at 37 8C with shaking, then, after several washings with PBS–0.05% Tween-20, peroxidase-conjugated streptavidin (1:10,000 in PBS) was added for 45 min at room temperature and furthermore, after washing, 0.1% o-phenylendia-
0304-3940/02/$ - see front matter q 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S03 04 - 394 0( 0 2) 00 81 4- 5
300
C. Cioni, P. Annunziata / Neuroscience Letters 330 (2002) 299–301
mine in pH 4.5 citrate buffer–0.015% H2O2 was added for 10 min at room temperature. After blocking with H2SO4, the colour that developed was analyzed by spectrophotometry at 492 nm. Permeability to albumin was calculated as a percentage passage according to the following formula: Absorbance of the samples in lower chamber 2 background values £ 100 Absorbance of biotin 2 labelled albumin in upper chamber 2 background values
Background values were obtained from absorbance of medium alone. In transgenic mouse brain endothelial cultures exposed to normal mouse serum, no increase in permeability to albumin was found; by contrast, a significant increase in permeability (up to 47%) was observed on exposure to serum samples from transgenic mice. This elevated permeability was neutralized after immunoabsorption of sera with antigp120 monoclonal antibody but not with isotype control (Fig. 1). In brain endothelial cultures from non-transgenic mice, transgenic but not non-transgenic mouse serum caused increased permeability to albumin, which was completely blocked by anti-gp120 monoclonal antibody but not by isotype-matched control antibody (Fig. 2). In transgenic or non-transgenic cultures not exposed to mouse sera, albumin passage was less than 1%, indicating similar functional integrity of the endothelium. Our findings demonstrate that changes in brain endothelium permeability found in vivo in HIV-1 gp120 transgenic mice are exclusively due to the action of circulating gp120 protein, confirming our previous in vitro data obtained with recombinant gp120 protein in rat brain endothelium cultures [2]. Circulating gp120 levels have been detected in the sera of AIDS patients [7] and may be due to protein shedding from the envelope of mature HIV-1 [5]. Impairment of the BBB has been found in autopsy brain tissues from AIDS patients with [9] and without dementia [8]. Gp120 acts as a proin-
Fig. 1. Albumin permeability of HIV-1 gp120 transgenic mouse brain endothelial cultures exposed for 24 h to non-transgenic mouse serum (NTS) and to gp120 transgenic mouse serum (TS) in the presence or absence of anti-gp120 monoclonal antibody at doses of 1 mg/ml (Mab 1) and 2 mg/ml (Mab 2) or isotype control antibody (2 mg/ml) (Iso). UNS, unstimulated cultures. Values are expressed as means ^ SE of two independent experiments (n ¼ 4). In each experiment, each point was analyzed in duplicate except for Mab 1, Mab 2 and Iso (n ¼ 2).
Fig. 2. Albumin permeability of non-transgenic mouse brain endothelial cultures exposed for 24 h to non-transgenic mouse serum (NTS) and to gp120 transgenic mouse serum (TS) in the presence or absence of anti-gp120 monoclonal antibody at doses of 1 mg/ml (Mab 1) and 2 mg/ml (Mab 2) or isotype control antibody (2 mg/ml) (Iso). UNS, unstimulated cultures. Values are expressed as means ^ SE of two independent experiments (n ¼ 4). In each experiment, each point was analyzed in duplicate except for Mab 1, Mab 2 and Iso (n ¼ 2).
flammatory cytokine inducing the production of substance P by rat brain endothelium [2] and causing significant changes in permeability. The demonstration that circulating gp120 levels increase in vitro brain endothelium permeability in a transgenic mouse model for HIV-1 gp120 is further evidence that a similar mechanism may occur in HIV-positive human subjects and contributes to the pathogenesis of HIV-related neurological findings. We thank Dr O. Finco (Chiron S.p.A., Siena, Italy) for providing us with gp120 transgenic mice. [1] Annunziata, P., Cioni, C., Moschini, F., Riccucci, A. and Guazzi, G.C., Serum anti-brain endothelium antibodies and cognitive assessment in patients with Binswanger’s encephalopathy, J. Neurol. Sci., 128 (1995) 96–102. [2] Annunziata, P., Cioni, C., Toneatto, S. and Paccagnini, E., HIV-1 gp120 increases the permeability of rat brain endothelium cultures by a mechanism involving substance P, AIDS, 12 (1998) 2377–2385. [3] Coligan, J.E., Kruisbeek, A.M., Margulies, D.H., Sherac, E.M. and Strober, W., Current Protocols in Immunology, National Institute of Health, Bethesda, MD, 1995, pp. 2.1.9–2.1.11. [4] Finco, O., Nuti, S., De Magistris, M.T., Mangiavacchi, L., Aiuti, A., Forte, P., Fantoni, A., van der Putten, H. and Abrignani, S., Induction of CD4 1 T cell depletion in mice doubly transgenic for HIV gp120 and human CD4, Eur. J. Immunol., 27 (1997) 1319–1324. [5] Gelderblom, H.R., Reupke, H. and Pauli, G., Loss of envelope antigens of HTLV-III/LAV, a factor in AIDS pathogenesis? Lancet, 2 (1985) 1016–1017. [6] Mrsulja, B.B., Mrsulja, B.J., Fujimoto, T., Klatzo, I. and Spatz, M., Isolation of brain capillaries: a simplified technique, Brain Res., 110 (1976) 361–365. [7] Oh, S.-K., Cruikshank, W.W., Raina, J., Blanchard, G.C., Adler, W.H., Walker, J. and Kornfeld, H., Identification of HIV-1 envelope glycoprotein in the serum of AIDS and ARC patients, J. Acquir. Immune Defic. Syndr., 5 (1992) 251–256.
C. Cioni, P. Annunziata / Neuroscience Letters 330 (2002) 299–301 [8] Petito, C.K. and Cash, K.S., Blood–brain barrier abnormalities in the acquired immunodeficiency syndrome: immunohistochemical localization of serum proteins in postmortem brain, Ann. Neurol., 32 (1992) 658–666. [9] Power, C., Kong, P.-A., Crawford, T.O., Wesselingh, S., Glass, J.D., McArthur, J.C. and Trapp, B.D., Cerebral white matter changes in acquired immunodeficiency syndrome
301
dementia: alterations of the blood–brain barrier, Ann. Neurol., 34 (1993) 339–350. [10] Toneatto, S., Finco, O., van der Putten, H., Abrignani, S. and Annunziata, P., Evidence of blood–brain barrier alteration and activation in HIV-1 gp120 transgenic mice, AIDS, 13 (1999) 2343–2348.