J Neural Transm DOI 10.1007/s00702-014-1359-z
NEUROLOGY AND PRECLINICAL NEUROLOGICAL STUDIES - SHORT COMMUNICATION
Receptor-mediated transfer of IgG and albumin at cerebrospinal fluid interfaces Sepp Seyfert • Felicitas Ehlen • Fabian Klostermann
Received: 24 July 2014 / Accepted: 22 December 2014 Ó Springer-Verlag Wien 2015
Abstract Receptor-mediated transfer of IgG and albumin has been suggested to occur also at cerebrospinal fluid interfaces. We point out findings of statistically unchanging IgG/albumin ratios along the lumbar CSF column which propose that such transfer should be absent or very small at cerebrospinal fluid interfaces. Keywords Receptor-mediated transcytotic transfer IgG Albumin Cerebrospinal fluid interfaces
Background Receptor-mediated transcytotic transfer of IgG and albumin at interfaces between blood and brain has been repeatedly reported (Strazielle and Ghersi-Egea 2013; Deane et al. 2005; Zhang and Pardridge 2001). Detection of specific receptors in mammalian choroid plexus epithelium and cerebral small vessel endothelium suggested such transfer also at cerebrospinal fluid (CSF) interfaces (Schlachetzki et al.2002; Kristoffersen 1997). The suggestion seems, however, not to tie in easily with the results of large volume CSF analyses in patients, as may be briefly presented. The correlation between CSF/serum concentration quotients of proteins with their hydrodynamic diameter is distinctive for protein influx by diffusion and efflux by bulk flow (Felgenhauer 1974). It defines both mechanisms as dominant passages of IgG and albumin into and out of the
S. Seyfert (&) F. Ehlen F. Klostermann Department of Neurology, Campus Benjamin Franklin, Charite´, Universita¨tsmedizin Berlin, Hindenburgdamm 30, 12203 Berlin, Germany e-mail: [email protected]
CSF. An additional passage by receptor-mediated transfer should, to comply with the correlation, imitate the dominant passage effects or should be small. An imitation appears unlikely because of differences of the moleculespecific passage paths and driving forces, diffusion occurring through intercellular junction pores and following blood-CSF protein concentration gradients while transcytotic transfer follows receptor affinity (Chaudhury et al. 2006; Hochwald et al. 1969), which probably also entails different kinetics. However, a small receptor-mediated transfer could have been missed by the correlation, especially if it was confined to the brain as the reports seem to suggest. In this scenario, receptor-mediated transfer would unfold local effects on the CSF IgG and albumin composition which may be intraindividually tested by comparing sequential aliquots representing different portions of the CSF column. In contrast to the assumption of brain-located receptor-mediated transfer, unchanging CSF IgG/albumin ratios have been casuistically described (Blennow et al.1993; Wurster 1988; Wikkelso et al.1983; Rice et al. 1982). This has prompted us to systematically analyze a larger dataset and to provide statistically valid information on this issue.
Patients and methods We analyzed CSF samples of 23 consecutive patients with normal pressure hydrocephalus (11 male, 12 female patients, 52–87 years old). Per subject, clinically indicated large volume CSF was collected in 10 sequential aliquots during a single lumbar puncture in a relaxed sitting position. Patients had given written informed consent to the study protocol before inclusion into the study approved by the local ethics committee.
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The aliquots were evaluated within 30 min with cell count, quantification of IgG and albumin in CSF and a concurrent serum sample with a Behring laser nephelometer analyser and isoelectric focussing on MacroPAG with silver stain. Interlaboratory assessments showed mean albumin and IgG measurement imprecision below 5 % and reliable isoelectric focussing. IgG/albumin ratios were assessed for each aliquot. To test whether the IgG/albumin ratios varied over the 10 aliquots per subject, an ANOVA for repeated measures was performed with the within-subject factor ‘aliquot sequence’ (10 levels: aliquot 1–10). To test whether protein CSF concentrations mattered, the ANOVA additionally included the between-subject factor ‘group’ (2 levels: patients with normal (IgG\56 mg/L, albumin\400 mg/L) versus pathologic values). We considered the null hypothesis ‘‘aliquot sequence is no factor’’ as likely and expected a negative ANOVA result (indicating unchanged IgG/albumin ratios in subsequent aliquots). The power of the analysis with a common p value of 0.05 for the Type I error would have been insufficient to accept the correctness of the null hypothesis, in case of negative test results. However, by raising the p value to unusual 0.5 the likelihood of falsely identifying ‘aliquot sequence’ as factor was increased, biasing the statistical procedure against the null hypothesis. If even under this condition, the ANOVA failed to identify ‘aliquot sequence’ as a factor, the correctness of the null hypothesis could be accepted on the basis of commonly demanded probabilities, since thus the statistical power was at 0.861, yielding a Type II error probability of 0.139.
Results The CSF protein levels were normal in 14 and pathologic in 9 patients. CSF cell counts were B5/lL. CSF with oligoclonal bands or [500 erythrocytes/lL were not encountered. Over the series of 10 aliquots, the CSF IgG/ albumin ratios remained unchanged, as shown in Table 1 Table 1 CSF values of 23 patients with normal pressure hydrocephalus
The intraindividual IgG/ albumin ratios remained statistically unchanged between aliquots 1 and 10
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and Fig. 1. The practical absence of change over aliquots 1–10 was also confirmed by the statistical results: Neither ‘aliquot sequence’ (F9,13 = 0.884; p = 0.549) nor ‘group’ (F1,21 = 0.353; p = 0.559) turned out to influence individual IgG/albumin ratios, and there was no interaction between the test factors.
Discussion If diffusion and receptor-mediated transfer with their different anatomic paths, driving forces, and probably kinetics coexisted at CSF interfaces, two results would be expected in the current CSF aliquot analysis along the lumbar CSF column: Firstly, an uneven mixing pattern of the probably different IgG/albumin ratios of both protein passage paths, and secondly, a caudal dilution of the IgG/albumin ratio from a cranial receptor-mediated transfer as described of brain-derived proteins (Reiber 2001). The slowed lumbosacral CSF turnover should in addition diminish the influence of a cranial receptor-mediated transfer in the caudal CSF. An example of such an effect is the caudally decreasing IgG Index in patients with apparent supralumbar intrathecal IgG synthesis (Wurster 1988). The IgG/albumin ratios of CSF aliquots in our patients remained statistically unchanged throughout the intraindividual collection sequence. The findings of unchanged IgG/ albumin ratios conform to and extend the cited observations. Taken together, the findings should represent a consistent sample which should argue against a significant receptor-mediated transfer of IgG and albumin at CSF interfaces. The findings being alike in healthy subjects (Blennow et al. 1993) and patients with normal or pathologic CSF may indicate them as physiologic. We are aware that the presented data are suggestive only and do not exclude a minute supralumbar contribution of a receptor-mediated protein transfer at CSF interfaces. Definite clarity could be achieved by comparing IgG/albumin ratios in intraindividual concurrent ventricular and lumbar CSF aliquots. Reliable reports of such data were not found.
Number of patients
23
CSF values
Median
Range
CSF aliquot volume (all aliquots) mL
3.3
2.3–5
Individual total CSF volume mL CSF albumin concentration (first aliquot) mg/L
33.1 252
26.7–40 138–811
CSF albumin concentration (tenth aliquot) mg/L
222
93–507
CSF IgG concentration (first aliquot) mg/L
40
15–73
CSF IgG concentration (tenth aliquot) mg/L
29
10–50
CSF IgG/albumin ratios (first aliquot)
0.108
0.081–0.186
CSF IgG/albumin ratios (tenth aliquot)
0.109
0.084–0.182
Receptor-mediated transfer of IgG and albumin
References
Fig. 1 IgG/albumin ratios in CSF aliquots, sequentially sampled per subject in one LP. The dots show the mean values of 23 patients, the bars show the standard error of the means. Note that the ratios remained practically unchanged over the 10 CSF aliquots representing sequential levels along the lumbar CSF column
Testing of ventricular CSF was not done in our patients because shunt operations were not accepted, or took place later and included preoperative hydration, risking an altered ventricular CSF protein steady state. Conflict of interest of interest.
The authors declare that they have no conflict
Ethical standard The study has been approved by the local ethics committee and has been performed in accordance with the ethical standards laid down in the 1964 Declaration of Helsinki and its later amendments.
Blennow K, Fredman P, Wallin A, Gottfries CG, Langstrom G, Svennerholm L (1993) Protein analysis in cerebrospinal fluid. I. influence of concentration gradients for proteins on cerebrospinal fluid/serum albumin ratio. Eur Neurol 33:126–128 Chaudhury C, Brooks CL, Carter DC, Robinson JM, Anderson CL (2006) Albumin binding to FcRn: distinct from the FcRn–IgG interaction. Biochemistry 45:4983–4990 Deane R, Sagare A, Hamm K et al (2005) IgG-assisted age-dependent clearance of Alzheimer’s amyloid beta peptide by the blood– brain barrier neonatal Fc receptor. J Neurosci 25:11495–11503 Felgenhauer K (1974) Protein size and cerebrospinal fluid composition. Klin Wschr 52:1158–1164 Hochwald GM, Wallenstein MC, Mathews ES (1969) Exchange of proteins between blood and spinal subarachnoid fluid. Am J Physiol 217:348–353 Kristoffersen EK (1997) FcRn and IgG in human choroid plexus. Scand J Immunol 45:447 Reiber H (2001) Dynamics of brain-derived proteins in cerebrospinal fluid. Clin Chim Acta 310:173–186 Rice GPA, Armstrong H, Ebers GC (1982) Variation in immunoglobulin G and albumin concentrations during lumbar CSF removal: a reappraisal. Neurology 32:893–894 Schlachetzki F, Zhu C, Pardridge WM (2002) Expression of the neonatal Fc receptor (FcRn) at the blood–brain barrier. J Neurochem 81:203–206 Strazielle N, Ghersi-Egea JF (2013) Physiology of blood-brain interfaces in relation to brain disposition of small compounds and macromolecules. Mol Pharm 10:1473–1491 Wikkelso C, Andersson M, von Essen C, Ro¨nnba¨ck L, Blomstrand C (1983) Separation of cerebrospinal fluid-enriched proteins. J Neurol Sci 60:419–429 Wurster U (1988) Protein gradients in the cerebrospinal fluid and the calculation of intracerebral IgG synthesis. J Neuroimmunol 20:233–235 Zhang Y, Pardridge WM (2001) Mediated efflux of IgG molecules from brain to blood across the blood–brain barrier. J Neuroimmunol 114:168–172
123
NEUROLOGY AND PRECLINICAL NEUROLOGICAL STUDIES - SHORT COMMUNICATION
Receptor-mediated transfer of IgG and albumin at cerebrospinal fluid interfaces Sepp Seyfert • Felicitas Ehlen • Fabian Klostermann
Received: 24 July 2014 / Accepted: 22 December 2014 Ó Springer-Verlag Wien 2015
Abstract Receptor-mediated transfer of IgG and albumin has been suggested to occur also at cerebrospinal fluid interfaces. We point out findings of statistically unchanging IgG/albumin ratios along the lumbar CSF column which propose that such transfer should be absent or very small at cerebrospinal fluid interfaces. Keywords Receptor-mediated transcytotic transfer IgG Albumin Cerebrospinal fluid interfaces
Background Receptor-mediated transcytotic transfer of IgG and albumin at interfaces between blood and brain has been repeatedly reported (Strazielle and Ghersi-Egea 2013; Deane et al. 2005; Zhang and Pardridge 2001). Detection of specific receptors in mammalian choroid plexus epithelium and cerebral small vessel endothelium suggested such transfer also at cerebrospinal fluid (CSF) interfaces (Schlachetzki et al.2002; Kristoffersen 1997). The suggestion seems, however, not to tie in easily with the results of large volume CSF analyses in patients, as may be briefly presented. The correlation between CSF/serum concentration quotients of proteins with their hydrodynamic diameter is distinctive for protein influx by diffusion and efflux by bulk flow (Felgenhauer 1974). It defines both mechanisms as dominant passages of IgG and albumin into and out of the
S. Seyfert (&) F. Ehlen F. Klostermann Department of Neurology, Campus Benjamin Franklin, Charite´, Universita¨tsmedizin Berlin, Hindenburgdamm 30, 12203 Berlin, Germany e-mail: [email protected]
CSF. An additional passage by receptor-mediated transfer should, to comply with the correlation, imitate the dominant passage effects or should be small. An imitation appears unlikely because of differences of the moleculespecific passage paths and driving forces, diffusion occurring through intercellular junction pores and following blood-CSF protein concentration gradients while transcytotic transfer follows receptor affinity (Chaudhury et al. 2006; Hochwald et al. 1969), which probably also entails different kinetics. However, a small receptor-mediated transfer could have been missed by the correlation, especially if it was confined to the brain as the reports seem to suggest. In this scenario, receptor-mediated transfer would unfold local effects on the CSF IgG and albumin composition which may be intraindividually tested by comparing sequential aliquots representing different portions of the CSF column. In contrast to the assumption of brain-located receptor-mediated transfer, unchanging CSF IgG/albumin ratios have been casuistically described (Blennow et al.1993; Wurster 1988; Wikkelso et al.1983; Rice et al. 1982). This has prompted us to systematically analyze a larger dataset and to provide statistically valid information on this issue.
Patients and methods We analyzed CSF samples of 23 consecutive patients with normal pressure hydrocephalus (11 male, 12 female patients, 52–87 years old). Per subject, clinically indicated large volume CSF was collected in 10 sequential aliquots during a single lumbar puncture in a relaxed sitting position. Patients had given written informed consent to the study protocol before inclusion into the study approved by the local ethics committee.
123
S. Seyfert et al.
The aliquots were evaluated within 30 min with cell count, quantification of IgG and albumin in CSF and a concurrent serum sample with a Behring laser nephelometer analyser and isoelectric focussing on MacroPAG with silver stain. Interlaboratory assessments showed mean albumin and IgG measurement imprecision below 5 % and reliable isoelectric focussing. IgG/albumin ratios were assessed for each aliquot. To test whether the IgG/albumin ratios varied over the 10 aliquots per subject, an ANOVA for repeated measures was performed with the within-subject factor ‘aliquot sequence’ (10 levels: aliquot 1–10). To test whether protein CSF concentrations mattered, the ANOVA additionally included the between-subject factor ‘group’ (2 levels: patients with normal (IgG\56 mg/L, albumin\400 mg/L) versus pathologic values). We considered the null hypothesis ‘‘aliquot sequence is no factor’’ as likely and expected a negative ANOVA result (indicating unchanged IgG/albumin ratios in subsequent aliquots). The power of the analysis with a common p value of 0.05 for the Type I error would have been insufficient to accept the correctness of the null hypothesis, in case of negative test results. However, by raising the p value to unusual 0.5 the likelihood of falsely identifying ‘aliquot sequence’ as factor was increased, biasing the statistical procedure against the null hypothesis. If even under this condition, the ANOVA failed to identify ‘aliquot sequence’ as a factor, the correctness of the null hypothesis could be accepted on the basis of commonly demanded probabilities, since thus the statistical power was at 0.861, yielding a Type II error probability of 0.139.
Results The CSF protein levels were normal in 14 and pathologic in 9 patients. CSF cell counts were B5/lL. CSF with oligoclonal bands or [500 erythrocytes/lL were not encountered. Over the series of 10 aliquots, the CSF IgG/ albumin ratios remained unchanged, as shown in Table 1 Table 1 CSF values of 23 patients with normal pressure hydrocephalus
The intraindividual IgG/ albumin ratios remained statistically unchanged between aliquots 1 and 10
123
and Fig. 1. The practical absence of change over aliquots 1–10 was also confirmed by the statistical results: Neither ‘aliquot sequence’ (F9,13 = 0.884; p = 0.549) nor ‘group’ (F1,21 = 0.353; p = 0.559) turned out to influence individual IgG/albumin ratios, and there was no interaction between the test factors.
Discussion If diffusion and receptor-mediated transfer with their different anatomic paths, driving forces, and probably kinetics coexisted at CSF interfaces, two results would be expected in the current CSF aliquot analysis along the lumbar CSF column: Firstly, an uneven mixing pattern of the probably different IgG/albumin ratios of both protein passage paths, and secondly, a caudal dilution of the IgG/albumin ratio from a cranial receptor-mediated transfer as described of brain-derived proteins (Reiber 2001). The slowed lumbosacral CSF turnover should in addition diminish the influence of a cranial receptor-mediated transfer in the caudal CSF. An example of such an effect is the caudally decreasing IgG Index in patients with apparent supralumbar intrathecal IgG synthesis (Wurster 1988). The IgG/albumin ratios of CSF aliquots in our patients remained statistically unchanged throughout the intraindividual collection sequence. The findings of unchanged IgG/ albumin ratios conform to and extend the cited observations. Taken together, the findings should represent a consistent sample which should argue against a significant receptor-mediated transfer of IgG and albumin at CSF interfaces. The findings being alike in healthy subjects (Blennow et al. 1993) and patients with normal or pathologic CSF may indicate them as physiologic. We are aware that the presented data are suggestive only and do not exclude a minute supralumbar contribution of a receptor-mediated protein transfer at CSF interfaces. Definite clarity could be achieved by comparing IgG/albumin ratios in intraindividual concurrent ventricular and lumbar CSF aliquots. Reliable reports of such data were not found.
Number of patients
23
CSF values
Median
Range
CSF aliquot volume (all aliquots) mL
3.3
2.3–5
Individual total CSF volume mL CSF albumin concentration (first aliquot) mg/L
33.1 252
26.7–40 138–811
CSF albumin concentration (tenth aliquot) mg/L
222
93–507
CSF IgG concentration (first aliquot) mg/L
40
15–73
CSF IgG concentration (tenth aliquot) mg/L
29
10–50
CSF IgG/albumin ratios (first aliquot)
0.108
0.081–0.186
CSF IgG/albumin ratios (tenth aliquot)
0.109
0.084–0.182
Receptor-mediated transfer of IgG and albumin
References
Fig. 1 IgG/albumin ratios in CSF aliquots, sequentially sampled per subject in one LP. The dots show the mean values of 23 patients, the bars show the standard error of the means. Note that the ratios remained practically unchanged over the 10 CSF aliquots representing sequential levels along the lumbar CSF column
Testing of ventricular CSF was not done in our patients because shunt operations were not accepted, or took place later and included preoperative hydration, risking an altered ventricular CSF protein steady state. Conflict of interest of interest.
The authors declare that they have no conflict
Ethical standard The study has been approved by the local ethics committee and has been performed in accordance with the ethical standards laid down in the 1964 Declaration of Helsinki and its later amendments.
Blennow K, Fredman P, Wallin A, Gottfries CG, Langstrom G, Svennerholm L (1993) Protein analysis in cerebrospinal fluid. I. influence of concentration gradients for proteins on cerebrospinal fluid/serum albumin ratio. Eur Neurol 33:126–128 Chaudhury C, Brooks CL, Carter DC, Robinson JM, Anderson CL (2006) Albumin binding to FcRn: distinct from the FcRn–IgG interaction. Biochemistry 45:4983–4990 Deane R, Sagare A, Hamm K et al (2005) IgG-assisted age-dependent clearance of Alzheimer’s amyloid beta peptide by the blood– brain barrier neonatal Fc receptor. J Neurosci 25:11495–11503 Felgenhauer K (1974) Protein size and cerebrospinal fluid composition. Klin Wschr 52:1158–1164 Hochwald GM, Wallenstein MC, Mathews ES (1969) Exchange of proteins between blood and spinal subarachnoid fluid. Am J Physiol 217:348–353 Kristoffersen EK (1997) FcRn and IgG in human choroid plexus. Scand J Immunol 45:447 Reiber H (2001) Dynamics of brain-derived proteins in cerebrospinal fluid. Clin Chim Acta 310:173–186 Rice GPA, Armstrong H, Ebers GC (1982) Variation in immunoglobulin G and albumin concentrations during lumbar CSF removal: a reappraisal. Neurology 32:893–894 Schlachetzki F, Zhu C, Pardridge WM (2002) Expression of the neonatal Fc receptor (FcRn) at the blood–brain barrier. J Neurochem 81:203–206 Strazielle N, Ghersi-Egea JF (2013) Physiology of blood-brain interfaces in relation to brain disposition of small compounds and macromolecules. Mol Pharm 10:1473–1491 Wikkelso C, Andersson M, von Essen C, Ro¨nnba¨ck L, Blomstrand C (1983) Separation of cerebrospinal fluid-enriched proteins. J Neurol Sci 60:419–429 Wurster U (1988) Protein gradients in the cerebrospinal fluid and the calculation of intracerebral IgG synthesis. J Neuroimmunol 20:233–235 Zhang Y, Pardridge WM (2001) Mediated efflux of IgG molecules from brain to blood across the blood–brain barrier. J Neuroimmunol 114:168–172
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