Vol. 21, No.3
INFECTION AND IMMUNITY, Sept. 1978, p. 852-861
0019-9567/78/0021-0852$02.00/0 Copyright © 1978 American Society for Microbiology
Printed in U.S.A.
Cerebrospinal Fluid and Serum Immunoglobulins and Antibody Titers in Mumps Meningitis and Aseptic Meningitis of Other Etiology A. FRYDgN,'* H. LINK,2 AND E. NORRBY3 Diseases' and Neurology,2 University Hospital, Linkoping, and Department of Departments of Infectious Virology,3 Karolinska Institutet, Stockholm, Sweden
Received for publication 13 June 1978
Cerebrospinal fluid (CSF) and serum from 19 patients with mumps meningitis and 19 patients with meningitis of other etiology were investigated on two or more occasions for at least 1 month after onset. Intrathecal synthesis of immunoglobulin (Ig) G was found in 55%, of IgA in 26%, and of IgM in 24% of the patients. Oligoclonal Ig was demonstrable by agarose gel electrophoresis in 37% of the patients, mostly already during the first week after onset, and could persist for years. Mumps virus antibody synthesis within the central nervous system occurred in 37% of the mumps meningitis patients. The inflammatory reaction within the central nervous system as reflected by mononuclear pleocytosis, Ig synthesis, and oligoclonal Ig was not correlated to the clinical course. The bloodbrain barrier was evaluated by determination ofthe CSF total protein, CSF/serum albumin ratio, and CSF/serum a2-macroglobulin ratio. A significant correlation was found among these three parameters. Persistence of the elevated CSF/serum albumin ratio seems to influence prognosis, and this parameter is recommended for evaluation of the blood-brain barrier function. Evidence has accumulated indicating that the central nervous system (CNS), from an immunological point' of view, must be considered partly as a separate entity. Thus, in the case of mumps meningitis, increased T and decreased B lymphocyte populations have been found in cerebrospinal fluid (CSF) as compared to blood (3). Decreased responses to mitogens (4, 10), but increased responses to mumps virus on stimulation of CSF, compared to blood lymphocytes, have also been observed in mumps meningitis patients (5). These different immunological reactions of CSF in comparison with blood lymphocytes occur although the CSF lymphocytes have been shown to derive from blood (25). Elevated immunoglobulin (Ig) levels in CSF, partly due to synthesis within the CNS, and intrathecally synthesized oligoclonal Ig have been demonstrated in aseptic meningitis (14). The importance of the cell-mediated immune response in viral CNS infection is experimentally documented, whereas the significance of the humoral immune response is not yet clarified (6, 7). No investigation has hitherto been published in which the Ig in CSF and serum have been analyzed in consecutive samples during the course of aseptic meningitis. Thus, the frequency of Ig abnormalities and occurrence of oligoclonal 852
CSF Ig in aseptic meningitis is not known, nor is the time of appearance and the persistence of these abnormalities. The aim of the present paper is to investigate the occurrence of Ig abnormalities in the form of elevated Ig levels and of locally synthesized polyclonal or oligoclonal Ig in CNS during the acute phase and convalescence of aseptic meningitis caused by mumps virus and, for comparison, by other infectious agents. In the patients with mumps meningitis, antibody titers against mumps virus antigen were determined in parallel, and variations in CSF and serum titers were correlated to Ig alterations. The blood-brain barrier function was investigated by determination of the CSF total protein and of the CSF/serum ratios of albumin and of a2-macroglobulin. These laboratory parameters were correlated to severity of the disease. MATERIALS AND METHODS Thirty-eight patients with acute aseptic meningitis treated at the Department of Infectious Diseases were studied. Most patients were treated during the hospital stay and then followed up by one of us (A.F.). The diagnosis was based on clinical findings, leukocytes in CSF exceeding 5 x 106/liter in 37 of the 38 patients, and the course of the disease with recovery without antibiotic treatment. The patients were divided into two groups. Group
VOL. 21, 1978
CSF Ig VARIATIONS IN MENINGITIS
853
A included 19 patients 20 to 40 years old (mean, 33 Determinations of albumin, IgG, IgA, and kappa years) with serologically confirmed mumps meningitis. and lambda light chains were carried out on unconGroup B also included 19 patients, 16 to 60 years old centrated CSF and on serum by an automatic immu(mean, 32 years). In this group the etiology of menin- noprecipitation technique utilizing nephelometric gitis could be established with certainty in six patients analyses of antigen-antibody complexes in a continu(individual cases caused by herpes simplex type 1, ous flow system (Auto-analyzer II, Technicon Corp., varicella-zoster, ECHO 30, influenza A, Coxsackie B4, Inc., Tarrytown, N.Y.). a2-Macroglobulin, IgM, and and tick-borne encephalitis virus), while a probable the complement factors C3 and C4 were determined etiological agent was detected in four additional pa- by single radial immunodiffusion (20), modified as tients (Mycoplasma pneumoniae in 3 and enterovirus described (18). Antisera against albumin, IgG, IgA a2-macroglobuin 1). The etiology remained unknown in the remaining nine patients. All patients were above 15 years old lin, and kappa and lambda light chains were purchased and were informed of the purpose of the study. Only from Dakopatts (Copenhagen, Denmark), antisera those who gave full consent were included. against IgM and C3 was from Organon Teknika (Oss, CSF and serum were taken at the same time from Holland), and antiserum against C4 was from Beheach patient on three occasions, namely on the day of ringwerke (Marburg-Lahn, W. Germany). admission (specimen I), about 5 days after admission The reference values for the individual proteins in (specimen II), and 30 to 60 days after admission (spec- CSF and serum are given in Table 1. These values imen III). Because the interval between onset of men- refer to a normal material defined according to Tibingitis symptoms and admission was rather constant bling et al. (32), consisting of 56 individuals with (0 to 5 days; mean, 2 days), the day of admission was functional neurological disorders but normal findings accepted as the starting point for the investigation. at neurological examination and with normal serum Additional CSF and serum specimens (specimen IV) proteins. The CSF/serum albumin ratio and the CSF were obtained more than 3 months after onset from total protein increase with age, the upper normal those patients who displayed CSF abnormalities in values being 5.7 and 304 mg/liter, respectively, at 30 specimen III and/or persisting symptoms. years, and 9.7 and 658 mg/liter, respectively, at 60 Cell counts and determinations of total protein, IgG, years (32). The IgG level in CSF was presented by the CSFand albumin were carried out immediately after lumbar puncture on all CSF specimens. The erythrocyte IgG index equal to (CSF IgG/serum IgG):(CSF albucount in CSF did not exceed 1,000 x 106/liter and was min/serum albumin) (32). The CSF-IgG index takes 100 x 10" to 820 x 106/liter in 12 of the CSF specimens. into account the influence of fluctuations of serum IgG IgG and albumin were determined simultaneously in and of blood-brain barrier disturbances (15). An elethe CSF and in the serum, and the CSF-IgG index was vated CSF-IgG index value indicates the occurrence determined. The specimens were then stored at of IgG synthesis within the CNS (16). The IgA and -201C. A portion of the CSF was concentrated by IgM concentrations in CSF were similarity presented ultrafiltration in collodium bags (Sartorius Membran- by the CSF-IgA and CSF-IgM indexes, respectively. filter, Gottingen, W. Germany) at 40C to an IgG con- The upper normal values in our laboratory are 0.70 for centration of about 3 g/liter and then analyzed by the CSF-IgG index, and 0.62 for the CSF-IgA index. agarose gel electrophoresis in parallel with serum. All The lower limit of IgM detected with the method specimens from individual patients were then investi- used was 2 mg/liter, and IgM was therefore not degated simultaneously regarding IgA, IgM, kappa and monstrable in normal unconcentrated CSF. Although lambda light chains, a2-macroglobulin, and comple- a normal range for the CSF-IgM index has not been ment factors C3 46ic/fi, globulin) and C4 (,61E globu- defined as accurately as it has for the other Ig's, >0.6 lin). appears to be a raised value. We only evaluated >1.0 Immunochemistry. After cell counting was per- as an abnormal value. formed by phase-contrast microscopy, the CSF was The kappa/lambda ratio (17) was determined. The centrifuged. 95% confidence limits in our laboratory are 0.7 to 1.7 The total protein concentration in CSF was deter- for CSF, and 0.7 to 1.3 for serum. mined according to Lowry et al. (19). The age-dependAgarose gel (Behringwerke) electrophoresis was ent reference values calculated by Tibbling et al. (32) carried out as described previously (13). The occurwere adopted (Table 1). rence of one or more homogeneous bands in the TABLE 1. Reference values for CSF and serum proteins given as 95% confidence limits Protein
Total protein'
Albumin" IgG IgA
IgM a2-Macroglobulin
CSF (mg/liter) 304-658
Serum (g/liter)
55-400 3.8-57.8 0.15-6.7
INFECTION AND IMMUNITY, Sept. 1978, p. 852-861
0019-9567/78/0021-0852$02.00/0 Copyright © 1978 American Society for Microbiology
Printed in U.S.A.
Cerebrospinal Fluid and Serum Immunoglobulins and Antibody Titers in Mumps Meningitis and Aseptic Meningitis of Other Etiology A. FRYDgN,'* H. LINK,2 AND E. NORRBY3 Diseases' and Neurology,2 University Hospital, Linkoping, and Department of Departments of Infectious Virology,3 Karolinska Institutet, Stockholm, Sweden
Received for publication 13 June 1978
Cerebrospinal fluid (CSF) and serum from 19 patients with mumps meningitis and 19 patients with meningitis of other etiology were investigated on two or more occasions for at least 1 month after onset. Intrathecal synthesis of immunoglobulin (Ig) G was found in 55%, of IgA in 26%, and of IgM in 24% of the patients. Oligoclonal Ig was demonstrable by agarose gel electrophoresis in 37% of the patients, mostly already during the first week after onset, and could persist for years. Mumps virus antibody synthesis within the central nervous system occurred in 37% of the mumps meningitis patients. The inflammatory reaction within the central nervous system as reflected by mononuclear pleocytosis, Ig synthesis, and oligoclonal Ig was not correlated to the clinical course. The bloodbrain barrier was evaluated by determination ofthe CSF total protein, CSF/serum albumin ratio, and CSF/serum a2-macroglobulin ratio. A significant correlation was found among these three parameters. Persistence of the elevated CSF/serum albumin ratio seems to influence prognosis, and this parameter is recommended for evaluation of the blood-brain barrier function. Evidence has accumulated indicating that the central nervous system (CNS), from an immunological point' of view, must be considered partly as a separate entity. Thus, in the case of mumps meningitis, increased T and decreased B lymphocyte populations have been found in cerebrospinal fluid (CSF) as compared to blood (3). Decreased responses to mitogens (4, 10), but increased responses to mumps virus on stimulation of CSF, compared to blood lymphocytes, have also been observed in mumps meningitis patients (5). These different immunological reactions of CSF in comparison with blood lymphocytes occur although the CSF lymphocytes have been shown to derive from blood (25). Elevated immunoglobulin (Ig) levels in CSF, partly due to synthesis within the CNS, and intrathecally synthesized oligoclonal Ig have been demonstrated in aseptic meningitis (14). The importance of the cell-mediated immune response in viral CNS infection is experimentally documented, whereas the significance of the humoral immune response is not yet clarified (6, 7). No investigation has hitherto been published in which the Ig in CSF and serum have been analyzed in consecutive samples during the course of aseptic meningitis. Thus, the frequency of Ig abnormalities and occurrence of oligoclonal 852
CSF Ig in aseptic meningitis is not known, nor is the time of appearance and the persistence of these abnormalities. The aim of the present paper is to investigate the occurrence of Ig abnormalities in the form of elevated Ig levels and of locally synthesized polyclonal or oligoclonal Ig in CNS during the acute phase and convalescence of aseptic meningitis caused by mumps virus and, for comparison, by other infectious agents. In the patients with mumps meningitis, antibody titers against mumps virus antigen were determined in parallel, and variations in CSF and serum titers were correlated to Ig alterations. The blood-brain barrier function was investigated by determination of the CSF total protein and of the CSF/serum ratios of albumin and of a2-macroglobulin. These laboratory parameters were correlated to severity of the disease. MATERIALS AND METHODS Thirty-eight patients with acute aseptic meningitis treated at the Department of Infectious Diseases were studied. Most patients were treated during the hospital stay and then followed up by one of us (A.F.). The diagnosis was based on clinical findings, leukocytes in CSF exceeding 5 x 106/liter in 37 of the 38 patients, and the course of the disease with recovery without antibiotic treatment. The patients were divided into two groups. Group
VOL. 21, 1978
CSF Ig VARIATIONS IN MENINGITIS
853
A included 19 patients 20 to 40 years old (mean, 33 Determinations of albumin, IgG, IgA, and kappa years) with serologically confirmed mumps meningitis. and lambda light chains were carried out on unconGroup B also included 19 patients, 16 to 60 years old centrated CSF and on serum by an automatic immu(mean, 32 years). In this group the etiology of menin- noprecipitation technique utilizing nephelometric gitis could be established with certainty in six patients analyses of antigen-antibody complexes in a continu(individual cases caused by herpes simplex type 1, ous flow system (Auto-analyzer II, Technicon Corp., varicella-zoster, ECHO 30, influenza A, Coxsackie B4, Inc., Tarrytown, N.Y.). a2-Macroglobulin, IgM, and and tick-borne encephalitis virus), while a probable the complement factors C3 and C4 were determined etiological agent was detected in four additional pa- by single radial immunodiffusion (20), modified as tients (Mycoplasma pneumoniae in 3 and enterovirus described (18). Antisera against albumin, IgG, IgA a2-macroglobuin 1). The etiology remained unknown in the remaining nine patients. All patients were above 15 years old lin, and kappa and lambda light chains were purchased and were informed of the purpose of the study. Only from Dakopatts (Copenhagen, Denmark), antisera those who gave full consent were included. against IgM and C3 was from Organon Teknika (Oss, CSF and serum were taken at the same time from Holland), and antiserum against C4 was from Beheach patient on three occasions, namely on the day of ringwerke (Marburg-Lahn, W. Germany). admission (specimen I), about 5 days after admission The reference values for the individual proteins in (specimen II), and 30 to 60 days after admission (spec- CSF and serum are given in Table 1. These values imen III). Because the interval between onset of men- refer to a normal material defined according to Tibingitis symptoms and admission was rather constant bling et al. (32), consisting of 56 individuals with (0 to 5 days; mean, 2 days), the day of admission was functional neurological disorders but normal findings accepted as the starting point for the investigation. at neurological examination and with normal serum Additional CSF and serum specimens (specimen IV) proteins. The CSF/serum albumin ratio and the CSF were obtained more than 3 months after onset from total protein increase with age, the upper normal those patients who displayed CSF abnormalities in values being 5.7 and 304 mg/liter, respectively, at 30 specimen III and/or persisting symptoms. years, and 9.7 and 658 mg/liter, respectively, at 60 Cell counts and determinations of total protein, IgG, years (32). The IgG level in CSF was presented by the CSFand albumin were carried out immediately after lumbar puncture on all CSF specimens. The erythrocyte IgG index equal to (CSF IgG/serum IgG):(CSF albucount in CSF did not exceed 1,000 x 106/liter and was min/serum albumin) (32). The CSF-IgG index takes 100 x 10" to 820 x 106/liter in 12 of the CSF specimens. into account the influence of fluctuations of serum IgG IgG and albumin were determined simultaneously in and of blood-brain barrier disturbances (15). An elethe CSF and in the serum, and the CSF-IgG index was vated CSF-IgG index value indicates the occurrence determined. The specimens were then stored at of IgG synthesis within the CNS (16). The IgA and -201C. A portion of the CSF was concentrated by IgM concentrations in CSF were similarity presented ultrafiltration in collodium bags (Sartorius Membran- by the CSF-IgA and CSF-IgM indexes, respectively. filter, Gottingen, W. Germany) at 40C to an IgG con- The upper normal values in our laboratory are 0.70 for centration of about 3 g/liter and then analyzed by the CSF-IgG index, and 0.62 for the CSF-IgA index. agarose gel electrophoresis in parallel with serum. All The lower limit of IgM detected with the method specimens from individual patients were then investi- used was 2 mg/liter, and IgM was therefore not degated simultaneously regarding IgA, IgM, kappa and monstrable in normal unconcentrated CSF. Although lambda light chains, a2-macroglobulin, and comple- a normal range for the CSF-IgM index has not been ment factors C3 46ic/fi, globulin) and C4 (,61E globu- defined as accurately as it has for the other Ig's, >0.6 lin). appears to be a raised value. We only evaluated >1.0 Immunochemistry. After cell counting was per- as an abnormal value. formed by phase-contrast microscopy, the CSF was The kappa/lambda ratio (17) was determined. The centrifuged. 95% confidence limits in our laboratory are 0.7 to 1.7 The total protein concentration in CSF was deter- for CSF, and 0.7 to 1.3 for serum. mined according to Lowry et al. (19). The age-dependAgarose gel (Behringwerke) electrophoresis was ent reference values calculated by Tibbling et al. (32) carried out as described previously (13). The occurwere adopted (Table 1). rence of one or more homogeneous bands in the TABLE 1. Reference values for CSF and serum proteins given as 95% confidence limits Protein
Total protein'
Albumin" IgG IgA
IgM a2-Macroglobulin
CSF (mg/liter) 304-658
Serum (g/liter)
55-400 3.8-57.8 0.15-6.7
