J Neurol (1992) 239 : 322-326
Journal of
Neurology © Springer-Verlag1992
Specific antigen binding by activated cerebrospinal fluid B lymphocytes in acute neuroborreliosis Wolfgang Beuche, Arno Siever, and Klaus Felgenhauer Abteilung ffir Neurologie der Universitgt, Robert-Koch-Strasse 40, W-3400 G6ttingen, Federal Republic of Germany Received May 10, 1991 / Received in revised form October 25, 1991 / Accepted November 6, 1991
Summary. A recently developed immunocytochemical antigen-binding sandwich test for the identification of specific activated B lymphocytes was applied to cerebrospinal fluid cells and peripheral blood mononuclear cells in patients with acute neuroborreliosis. Discrimination of antigen-binding phagocytes was achieved by double staining with monoclonal antibodies. Specific activated B lymphocytes were much m o r e numerous in cerebrospinal fluid than in blood, showing great interindividual differences. When intrathecal immunoglobulin production was present, the n u m b e r of specific activated B lymphocytes was also high. The specificity of all activated B lymphocytes ranged f r o m 10% to 60% and was higher in the acute stage than after treatment. Key words: Cerebrospinal fluid cells - Borrelia burgdorferi - Activated B lymphocytes
Materials and methods Preparation of rat lymph node cells Male Sprague-Dawley rats weighing 350-450 g were immunized with 100 gl/ml B. burgdorferi antigen, together with 1 : 1.2 (v/v) incomplete Freund's adjuvant (Difco, Detroit) in both hind foot pads. Two to 4 weeks later the animals received one booster immunization, were sacrificed after 8 days and regional lymph nodes were removed from the knee. Lymph node tissue was digested in phosphate buffered saline (PBS) pH 7.4 with 0.2% collagenase and 0.02% DNAse (Sigma, Deisenhofen, FRG) to yield a cell suspension. Cells were washed twice in medium RPMI 1640 with 5% heat-inactivated fetal calf serum (FCS) and centrifuged over a Ficoll-Hypaque density gradient to remove dead cells and debris. Viable washed cells were counted by trypan blue exclusion and 20000-30000 cells were centrifuged on glass slides by a Shandon cytocentrifuge.
Preparation of B. burgdorferi antigens Introduction Immunoglobulin-containing, activated cerebrospinal fluid (CSF) B lymphocytes are useful diagnostic markers in inflammatory diseases of the central nervous system (CNS) [15]. Although they are reliable indicators of activity, the infectious agent remains unknown, unless the specificity of their antibodies can be defined. It has b e e n shown in patients with shingles that intracellular immunoglobulins bind to varicella zoster virus antigens [4]. This immunocytochemical sandwich technique has now b e e n applied in patients with neuroborreliosis. In CNS infections with Borrelia burgdorferi specific antibodies are intrathecally produced [22]. The bacterial antigens bind specifically to B cell antibodies and to monocytes and granulocytes via Fc receptors or other unknown mechanisms [3, 12]. Discrimination was achieved by staining these phagocytes with monoclonal antibodies prior to antigen binding. Background staining was suppressed by the addition of homologous pooled h u m a n AB serum to the antigen during the staining procedure.
Correspondence to: W. Beuche
A European isolate of B. burgdorferi was a generous gift from Dr. Scriba of the Institute of Medical Microbiology, G6ttingen, FRG. Bacteria were grown in modified Kelly II medium and prepared as described by Stiernstedt et al. [18]. Supernatants of sonicated bacteria centrifuged at 10000 g for 30 min were used as antigen.
Production of mouse antiserum to Borrelia burgdorferi Five mice, strain C57BL, were immunized with 100 ~tl/ml antigen of B. burgdorferi, together with 1:1.2 (v/v) complete Freund's adjuvant (Difco) intraperitoneally. Booster injections were made repeatedly and mice were bled from retro-ocular veins 3-4 weeks after the last booster injection. Sera were pooled and extensively absorbed to rat spleen and lymph node cells as well as with human peripheral blood lymphocytes. Antiserum was used at a final dilution of 1 : 100.
Human clinical specimen CSF cells were obtained from diagnostic lumbaI punctures. Cytospin preparations were obtained as described above for animal lymph node cells. Peripheral blood lymphocytes were isolated by density centrifugation on Ficoll-Hypaque and prepared after being washed twice in medium RPMI 1640 with 5% FCS.
323
Fig. 1. a Overview (× 285) of a cerebrospinal fluid cell cytocentrifugate from patient 2 stained for Borrelia burgdorferi antigenbinding cells. Background staining is negligible and the positive cell is easily distinguished from unstained cells. The positive cell is not double stained with monoclonal phagocyte marker, b Higher magnification ( × 700) of a different B. burgdorferi antigen-binding cerebrospinal fluid cell. Note that the cytoplasmatic content extruded near the excentric nucleus (arrowhead) is not stained. The
staining is pronounced at the cell margins, c, d B. burgdorferi antigen-binding cerebrospinal fluid cells from patient 4 that had been stored in TRIS-buffered saline for 8 days prior to immunocytochemistry. The antigen is bound at the cell surface, although the cells show plasma cell morphology (arrows). x 700. e Demonstration of immunoglobulins in the same preparation as c, d. Immunoglobulins (arrows) show the same localization as in antigen binding. x 700
The diagnosis of neuroborreliosis was suspected if a history of erythema chronicum migrans and neurological findings of Bannwarth's syndrome was confirmed by positive serology for specific IgM in serum and CSF [21]. A limited number of human specimens had been cryopreserved and stored in liquid nitrogen for 1-2 years. These cells had been frozen in medium RPMI 1640 with 20% FCS and 10% dimethylsulphoxide (Sigma) in liquid nitrogen vapour. Recovery was over 85% ; the cells were washed once after thawing and handled like freshly obtained cells. Control specimens came from patients suffering from other inflammatory CNS diseases, including viral (n = 8), bacterial infections from gram negative (n = 4) and gram positive (n = 5) organisms, multiple sclerosis (n = 6) and leukaemic infiltration of the meninges (n = 3).
Immunology, and Prof. Pfitzenmeier, Klinische Arbeitsgruppe der Max-Planck-Gesellschaft, G/3ttingen, FRG. They were used as cell culture supernatants without dilution. Binding of the antibodies was visualized using a peroxidase-antiperoxidase (PAP) technique with heavy metal salt amplification as described recently by Behrends [2]. In rat cells staining of phagocytes was omitted. Cells were then incubated with antigen of B. burgdorferi at 100 ~tg/ml diluted in PBS with 3% bovine serum albumin (BSA) and 0.5% Tween 20 together with 30% human AB serum pool for 45 min. After washing 5 times in PBS mouse polyclonal antiserum was applied for 30 min. Mouse antibodies were visualized by the application of alkaline phosphatase-antialkaline phosphatase (APAAP) technique with one repetition cycle [9]. Astra New fuchsin was used as the chromogen with levamisole to block endogenous alkaline phosphatase activity. Cells were counterstained with Mayer's haematoxyline. On a different glass slide immunoglobulin-containing cells (ICC) were stained using a biotinylated sheep anti-human immunoglobulin antiserum (RPN 1003, Amersham) and avidinbiotin-complex (PK4000, Vector Laboratory) with diaminobenzidine as chromogen. All antigen-binding cells (ABC) that did not stain for phagocyte markers were counted and the ratio of antigenbinding and thus antibody-forming B lymphocytes and all immunoglobulin-containing cells (ABC/ICC) was calculated as an index of specificity of B cell activation. One to three glass slides were stained for each specimen depending on the frequency of the activated B lymphocytes. The determination of blood-brain-barrier impairment and intrathecal immunoglobulin synthesis was done according to Reiber
Staining procedure Cytospin preparations were air dried and fixed with ethanolacetone (v/v)-formalin (2.6%) at room temperature for 3 min. The cells were preserved either fixed in TRIS-buffered saline, pH 7.2, for several days or unfixed for longer periods at -20°C wrapped in aluminium foil. Thawing was performed in the fixation solution. Endogenous peroxidase was inhibited with 3% H202 for 5 min. Slides were washed in PBS, pH 7.4. Monocytes and granulocytes were stained with a combination of monoclonal antibodies: M-718 (Dako, Hamburg, FRG), CD 14 (TIB 228, American Type Culture Collection, ATCC) and Mac-1 (TIB 128, ATCC). The latter two antibodies were generous gifts from Prof. Peters, Institute for
324
Fig. 2. Cerebrospinal fluid cells from patient 4 double stained for phagocytes by the combination of monoclonal antibodies M-718, CD 14 and Mac-1 (heavy metal amplificated peroxidase reaction) staining dark black and antigen binding (alkaline phosphatase reaction) staining red, counterstained with Mayer's haematoxylin, × 285. The red stained elliptic B lymphocyte (arrow) can be distinguished from the black stained monocyte (arrowhead) and Felgenhauer [13]. This intrathecal immunoglobulin production was not B. burgdorferi specific. Results
B. burgdorferi a n t i g e n is a d h e s i v e to glass a n d cell surfaces. A p p l i c a t i o n o f o t h e r p r o t e i n s such as F C S o r B S A d i d n o t r e d u c e t h e high b a c k g r o u n d staining. This b a c k Table 1. Clinical symptoms and signs and the results obtained in immunocytochemical staining and humoral CSF findings. Intrathecal immunoglobulin production that is not Borrelia burgdorferi specific was determined according to Reiber and Felgenhauer [13]. 1. indicates the first lumbar puncture prior to treatment with 2 × 10 mega penicillin G/day intravenously for 10 days; 2. indi-
g r o u n d staining c o u l d b e g r e a t l y r e d u c e d using 30% r a t serum or pooled human AB serum. An antigen concent r a t i o n o f 100 ~tg/ml gave o p t i m a l cell staining with minimal background. With lower antigen concentrations the n u m b e r of A B C d e c l i n e d . A n t i g e n b i n d i n g l y m p h o c y t e s f r o m l y m p h n o d e s c o u l d b e easily d i s t i n g u i s h e d f r o m m a c r o p h a g e s t h a t h a d p h a g o c y t o s e d a n t i g e n . T h e s e cells w e r e few in n u m b e r a n d t h e m a j o r i t y o f l y m p h n o d e m a c r o p h a g e s i d e n t i f i e d on m o r p h o l o g i c a l g r o u n d s did not bind antigen. T h e a n t i g e n b o u n d s h o w e d a c h a r a c t e r i s t i c surface d i s t r i b u t i o n in r a t l y m p h n o d e cells as well as in h u m a n cells (Fig. 1). S m a l l e r cells o f t e n s h o w e d a m o r e cytop l a s m a t i c staining. T h e A B C a p p e a r e d m o s t o f t e n as elliptic i m m u n o b l a s t s o r p l a s m a cells with e x c e n t r i c nuclei. The distribution patterns of bound antigen and immunog l o b u l i n s w e r e t h e same. T h e A B C w e r e easily d i s t i n g u i s h e d f r o m d o u b l e s t a i n e d b l a c k p h a g o c y t e s , which o c c u r r e d in m u c h g r e a t e r n u m b e r s in t h e b l o o d t h a n in t h e C S F (Fig. 2). G r a n u l o cytes a v i d l y b o u n d a n t i g e n b u t w e r e d i s t i n g u i s h a b l e f r o m A B C b y t h e i r c h a r a c t e r i s t i c nuclei. T h e o p t i m i z e d staining p r o c e d u r e for r a t l y m p h n o d e cells was also o p t i m a l for h u m a n b l o o d a n d C S F cells. T h e n u m b e r o f A B C in h u m a n C S F p r e p a r a t i o n s s h o w e d g r e a t i n t e r i n d i v i d u a l d i f f e r e n c e s r e g a r d l e s s of t h e d i s e a s e stage ( T a b l e 1). T h e i r n u m b e r v a r i e d f r o m less t h a n 5 to m o r e t h a n 120 cells p e r 10000 a n d g e n e r a l ly d e c l i n e d after t r e a t m e n t . T h e r e was an o v e r a l l c o r r e l a t i o n b e t w e e n i n t r a t h e c a l i m m u n o g l o b u l i n p r o d u c t i o n a n d t h e n u m b e r of A B C . cates lumbar puncture after treatment, except patient 2 who was re-examined after 5 days. In patient 4 a third lumbar puncture was done after 20 days. ECM, Erythema chronicum migrans; F, female; M, male; ABC, antigen-binding cells; ICC, immunoglobulin-containing cells
Patient no.
Age (years)
Sex
Symptoms and signs
Cells (per ~1)
ABC % of all cells
ICC % of all cells
% ratio ABC/ICC
Blood-CSFbarrier impairment
Intrathecal Ig synthesis
1 1. 2. 2 1. 2. 3 1. 2. 4 1. 2. 3. 5 1. 2. 6 1. 2. 7a 1. 2. 8 1. 2. 9 1. 2.
77
M
Polyradiculitis
67
M
27
M
78
F
ECM, faciaInerve palsy, radicular pain ECM, facial nerve palsy, radicular pain Facial nerve palsy
F
Radicular pain
48
F
ECM, radicular pain
22
F
61
M F
0.4 5.0 0.17 3.0 0.29 0.74 6.0 6.0 10.0 0.1 0.15 1.0 3.0 0.08 0.3 0.3 n.d. 1.0 0.16
50 6 43 33 50 11 17 17 10 33 0 5 4 63 7 10
50
Tickbite, ECM, radicular pain Tickbite, ECM, radicular pain Tick bite, facial nerve palsy
0.2 0.3 0.07 1.0 0.14 0.08 1.0 1.0 1.0 0.03 0.0 0.03 0.12 0.05 0.02 0.03 0.0 0.08 0.0
+++ +++ ++ ++ ++ ++ ++ (+)
47
364 107 347 313 459 102 91 87 44 49 17 96 34 315 40 33 22 82 8
IgM/IgG/IgA IgM/IgA/IgG IgM/IgG IgM/IgG/IgA IgM/IgA/IgG IgM/IgG/IgA IgM/IgG IgM/Iga IgM IgM/IgA IgM/IgA
a Frozen specimen
8 0
-
(+) ++ (+) + (+)
325 Based on the formula of Reiber and Felgenhauer [13] approximately 40-60% of IgG, 20-40% of IgA and 6080% of IgM were produced intrathecally in these cases. CSF cells contained proportionally more ABC than blood cells. The specificity of activated B lymphocytes ranged from 10% to 60% of all ICC in the acute stage. Control specimens from patients with other inflammatory diseases showed no ABC. In patients with bacterial meningitis the granulocytes bound antigen despite staining with the monoclonal antibodies but could easily be distinguished by their characteristic morphology. Cryopreservation of CSF cells reduced the number of ICC considerably. Cytospin preparations could, however, be stored up to 4 - 6 weeks without loss of staining characteristics.
Discussion
B. burgdorferi infection was chosen since the diagnosis can be generally made on clinical grounds in an early stage of disease and a CSF pleocytosis is frequently found with high numbers of activated B lymphocytes. The staining procedure as optimized with rat lymph node cells could be applied without major changes to clinical specimens. The adherence of B. burgdorferi to mononuclear phagocytes was overcome by double staining with monoclonal antibodies. After using human AB serum in the staining procedure it was not possible to double-stain further the A B C for immunoglobulins, owing to the immunoglobulin content of the human serum that adhered to the surface of the slide. Real double-staining of closely related antigens is mainly difficult when using permanent chromogens. The morphological appearance of antigen binding and immunoglobulins in immunoblasts and plasma cells, on the other hand, is exactly the same. Detection of antibody-producing cells by antigen binding has been used for the investigation of specific immunoblasts and plasma cells in spleen and lymph nodes of mice and rabbits (for review see [20]). A B C have also been investigated by fluorescence-activated cell sorting and have been shown to contain almost the entire population of antigen-specific antibody-producing cells [6]. Other techniques for the demonstration of antibody-forming and -releasing cells are the plaque or spotforming assays that use antigen-coated erythrocytes or solid phases to demonstrate secreted antibodies of individual cells [16]. Compared with these assays the immunocytochemical detection has several advantages. The method is fast and can detect rare antibody-forming cells. Antibody-forming cells of different stages are detectable even in the absence of antibody secretion and can be easily quantified. The concentration of specific activated B lymphocytes is much higher in the CSF than in blood, confirming the results obtained with the E L I S P O T assay in neuroborreliosis [1] and in viral meningitis [5]. This concentrated immunoreactivity in the CSF is specific for neuroborreliosis. A B C were numerous in patients with intrathecal immunoglobulin synthesis.
The reason why antigen binding and immunoglobulin staining are predominantly found on the cell surface is not clear at present. As the fixative includes acetone and ethanol the cytoplasmatic structures should be accessible for antibodies. Membrane-bound antibodies are fixed together with other membrane proteins, whereas intracellular antibodies prone to secretion that lack a transmembrane unit may not be held fast in situ by the fixatives applied and are thus eluted during washing steps. The thin surface staining of A B C from fixed specimens that had been stored in aqueous buffer further supports this view. The comparatively large proportion of activated B lymphocytes that do not bind antigen found in zoster ganglionitis and neuroborreliosis is also not explained. It may be attributable to the limits of the immunocytochemical technique but this non-specific activation seems to be a general p h e n o m e n o n [7, 8, 10, 11] and may not be due to a mitogenic effect of B. burgdorferi. While the absolute number of CSF cells had decreased after 10 days of treatment, the percentage of ICC often increased. The percentage of ABC, on the other hand, showed a minor increase or even decreased. Despite a larger proportion of immunocompetent cells among the CSF pleocytosis the specific activated B lymphocytes seem to be more closely regulated than the unspecific ones, because the specificity of B cell activation, i.e. the ABC/ICC ratio, was lower following treatment. Production of antibodies of other specificities, for example to myelin basic protein, has been reported in neuroborreliosis patients. It may account for some of the "unspecific" activated B lymphocytes and play a role in the pathogenesis of the disease [14, 17, 19].
Acknowledgements. This study was supported by a grant from the German Federal Ministry for Research and Technology (Bundesministerium fi~r Forschung und Technologie; 01 K188377). The authors accept sole responsibility for the contents of this article. Mrs. Heidi Semmler is thanked for her diligent technical assistance. References 1. Baig S, Olsson T, Link H (1989) Predominance of Borrelia burgdorferi specific B cells in cerebrospinal fluid in neuroborreliosis. Lancet I : 71-74 2. Behrends T (1988) Phosphate-coupled contrast enhancement of the DAB reaction product. Clin Neuropathol 7:145-146 3. Benach JL, Fleit HB, Habicht GS, Coleman JL, Bosler EM, Lane BP (1984) Interactions of phagocytes with the Lyme disease spirochete: role of the Fc receptor. J Infect Dis 150:497507 4. Beuche W, Thomas RS, Felgenhauer K (1989) Demonstration of zoster virus antibodies in cerebrospinal fluid cells. J Neurol 236 : 26-28 5. Forsberg P, Kam-Hansen S, Fryden A (1986) Production of specific antibodies by cerebrospinal fluid lymphocytes in patients with Herpes Zoster, Mumps meningitis and Herpes Simplex Virus encephalitis. Scand J Immunol 24 : 261-271 6. Hoven MY, De Leij L, Keij JFK, The TH (1989) Detection and isolation of antigen-specific B cells by the fluorescence activated cell sorter (FACS). J Immunol Methods 117:275-284 7. Hyypi~i T, Escola J, Laine M, Salmi A, Meurman O (1985) Polyclonal activation of B cells during Rubella infections. Scand J Immunol 21 : 615-617
326 8. Kantele AM, Takanen R, Arvillomi H (1988) Immune response to acute diarrhea seen as circulating antibody-secreting cells. J Infect Dis 158 : 1011-1016 9. Mason DY (1985) Immunocytochemical labeling of monoclonal antibodies by the APAAP immunoalkaline phosphatase technique. Tech Immunocytochem 3 : 25-42 10. Miller HRP, Ternynck T, Avrameas S (1975) Synthesis of antibody and immunoglobulinswithout detectable antibody function in cells responsing to horseradish peroxidase. J Immunol 114: 626-629 11. Minoprio P, Burlen O, Pereira P, Guilbert B, Andrade L, Hontebeyrie-Joskowicz, Coutinho A (1988) Most B cells in acute Trypanosoma cruzi infection lack parasite specifity. Scand J Immunol 28 : 553-561 12. Peterson PK, Clawson CC, Lee DA, Garlich D J, Quie PG, Johnson RC (1984) Human phagocyte interactions with the Lyme disease spirochete. Infect Immun 46:608-611 13. Reiber HO, Felgenhauer K (1987) Protein transfer at the blood cerebrospinal fluid barrier and the quantitation of the humoral immune response within the central nervous system. Clin Chim Acta 163 : 319-328 14. Ryberg B, Hindfelt B, Nilsson B, Olsson JE (1984) Antineural antibodies in Guillan-Barr6 syndrome and lymphocytic meningoradiculitis (Bannwarth's syndrome). Arch Neurol 41 : 12771281 15. Sch~idlichHJ, Felgenhauer K (1985) Diagnostic significance of IgG-synthesizing activated B cells in acute inflammatory diseases of the central nervous system. Klin Wochenschr 63 : 505510
16. Sedgwick JD, Holt PG (1983) A solid-phase immunoenzymatic technique for the enumeration of specific antibody-secreting cells. J Immunol Methods 57 : 301-309 17. Sigal LH, Steere AC, Dwyer JM (1988) In vivo and in vitro evidence of B cell hyperactivity during Lyme disease. J Rheumatol 15 : 648-654 18. Stiernstedt GT, Granstr6m M, Hederstedt B, Sk61denberg B (1985) Diagnosis of spirochetal meningitis by enzyme-linked immunosorbent assay and indirect immunofluorescence assay in serum and cerebrospinal fluid. J Clin Microbio121 : 819-825 19. Suchanek G, Kristoferitsch W, Stanek G, Bernheim H (1986) Anti-myelin antibodies in cerebrospinal fluid and serum of patients with meningopolyneuritis Garin Bujadoux-Bannwarth and other neurological diseases. Zentralbl Bakteriol Hyg A 263 : 160-168 20. Van Rooijen N, Claassen E (1986) Recent advances in the detection and characterization of specific antibody-forming cells in tissue sections. Histochem J 18:465-471 21. Wassmann K, Borg-von Zepelin M, Zimmermann O, Stadler M, Eifert H, Thomssen R (1989) Determination of immunoglobulin M antibody to Borrelia burgdorferi to differentiate between acute and past infections. Lyme Borreliosis II. Zentralbl Bakteriol 18 [Suppl] : 282-286 22. Wilske B, Schierz G, Preac-Mursic V, Busch K yon, Kfihbeck R, Pfister HW, Einh~upl K (1986) Intrathecal production of specific antibodies against Borrelia burgdorferi in patients with meningoradiculitis (Bannwarth's syndrome). J Infect Dis 153 : 304-314
Journal of
Neurology © Springer-Verlag1992
Specific antigen binding by activated cerebrospinal fluid B lymphocytes in acute neuroborreliosis Wolfgang Beuche, Arno Siever, and Klaus Felgenhauer Abteilung ffir Neurologie der Universitgt, Robert-Koch-Strasse 40, W-3400 G6ttingen, Federal Republic of Germany Received May 10, 1991 / Received in revised form October 25, 1991 / Accepted November 6, 1991
Summary. A recently developed immunocytochemical antigen-binding sandwich test for the identification of specific activated B lymphocytes was applied to cerebrospinal fluid cells and peripheral blood mononuclear cells in patients with acute neuroborreliosis. Discrimination of antigen-binding phagocytes was achieved by double staining with monoclonal antibodies. Specific activated B lymphocytes were much m o r e numerous in cerebrospinal fluid than in blood, showing great interindividual differences. When intrathecal immunoglobulin production was present, the n u m b e r of specific activated B lymphocytes was also high. The specificity of all activated B lymphocytes ranged f r o m 10% to 60% and was higher in the acute stage than after treatment. Key words: Cerebrospinal fluid cells - Borrelia burgdorferi - Activated B lymphocytes
Materials and methods Preparation of rat lymph node cells Male Sprague-Dawley rats weighing 350-450 g were immunized with 100 gl/ml B. burgdorferi antigen, together with 1 : 1.2 (v/v) incomplete Freund's adjuvant (Difco, Detroit) in both hind foot pads. Two to 4 weeks later the animals received one booster immunization, were sacrificed after 8 days and regional lymph nodes were removed from the knee. Lymph node tissue was digested in phosphate buffered saline (PBS) pH 7.4 with 0.2% collagenase and 0.02% DNAse (Sigma, Deisenhofen, FRG) to yield a cell suspension. Cells were washed twice in medium RPMI 1640 with 5% heat-inactivated fetal calf serum (FCS) and centrifuged over a Ficoll-Hypaque density gradient to remove dead cells and debris. Viable washed cells were counted by trypan blue exclusion and 20000-30000 cells were centrifuged on glass slides by a Shandon cytocentrifuge.
Preparation of B. burgdorferi antigens Introduction Immunoglobulin-containing, activated cerebrospinal fluid (CSF) B lymphocytes are useful diagnostic markers in inflammatory diseases of the central nervous system (CNS) [15]. Although they are reliable indicators of activity, the infectious agent remains unknown, unless the specificity of their antibodies can be defined. It has b e e n shown in patients with shingles that intracellular immunoglobulins bind to varicella zoster virus antigens [4]. This immunocytochemical sandwich technique has now b e e n applied in patients with neuroborreliosis. In CNS infections with Borrelia burgdorferi specific antibodies are intrathecally produced [22]. The bacterial antigens bind specifically to B cell antibodies and to monocytes and granulocytes via Fc receptors or other unknown mechanisms [3, 12]. Discrimination was achieved by staining these phagocytes with monoclonal antibodies prior to antigen binding. Background staining was suppressed by the addition of homologous pooled h u m a n AB serum to the antigen during the staining procedure.
Correspondence to: W. Beuche
A European isolate of B. burgdorferi was a generous gift from Dr. Scriba of the Institute of Medical Microbiology, G6ttingen, FRG. Bacteria were grown in modified Kelly II medium and prepared as described by Stiernstedt et al. [18]. Supernatants of sonicated bacteria centrifuged at 10000 g for 30 min were used as antigen.
Production of mouse antiserum to Borrelia burgdorferi Five mice, strain C57BL, were immunized with 100 ~tl/ml antigen of B. burgdorferi, together with 1:1.2 (v/v) complete Freund's adjuvant (Difco) intraperitoneally. Booster injections were made repeatedly and mice were bled from retro-ocular veins 3-4 weeks after the last booster injection. Sera were pooled and extensively absorbed to rat spleen and lymph node cells as well as with human peripheral blood lymphocytes. Antiserum was used at a final dilution of 1 : 100.
Human clinical specimen CSF cells were obtained from diagnostic lumbaI punctures. Cytospin preparations were obtained as described above for animal lymph node cells. Peripheral blood lymphocytes were isolated by density centrifugation on Ficoll-Hypaque and prepared after being washed twice in medium RPMI 1640 with 5% FCS.
323
Fig. 1. a Overview (× 285) of a cerebrospinal fluid cell cytocentrifugate from patient 2 stained for Borrelia burgdorferi antigenbinding cells. Background staining is negligible and the positive cell is easily distinguished from unstained cells. The positive cell is not double stained with monoclonal phagocyte marker, b Higher magnification ( × 700) of a different B. burgdorferi antigen-binding cerebrospinal fluid cell. Note that the cytoplasmatic content extruded near the excentric nucleus (arrowhead) is not stained. The
staining is pronounced at the cell margins, c, d B. burgdorferi antigen-binding cerebrospinal fluid cells from patient 4 that had been stored in TRIS-buffered saline for 8 days prior to immunocytochemistry. The antigen is bound at the cell surface, although the cells show plasma cell morphology (arrows). x 700. e Demonstration of immunoglobulins in the same preparation as c, d. Immunoglobulins (arrows) show the same localization as in antigen binding. x 700
The diagnosis of neuroborreliosis was suspected if a history of erythema chronicum migrans and neurological findings of Bannwarth's syndrome was confirmed by positive serology for specific IgM in serum and CSF [21]. A limited number of human specimens had been cryopreserved and stored in liquid nitrogen for 1-2 years. These cells had been frozen in medium RPMI 1640 with 20% FCS and 10% dimethylsulphoxide (Sigma) in liquid nitrogen vapour. Recovery was over 85% ; the cells were washed once after thawing and handled like freshly obtained cells. Control specimens came from patients suffering from other inflammatory CNS diseases, including viral (n = 8), bacterial infections from gram negative (n = 4) and gram positive (n = 5) organisms, multiple sclerosis (n = 6) and leukaemic infiltration of the meninges (n = 3).
Immunology, and Prof. Pfitzenmeier, Klinische Arbeitsgruppe der Max-Planck-Gesellschaft, G/3ttingen, FRG. They were used as cell culture supernatants without dilution. Binding of the antibodies was visualized using a peroxidase-antiperoxidase (PAP) technique with heavy metal salt amplification as described recently by Behrends [2]. In rat cells staining of phagocytes was omitted. Cells were then incubated with antigen of B. burgdorferi at 100 ~tg/ml diluted in PBS with 3% bovine serum albumin (BSA) and 0.5% Tween 20 together with 30% human AB serum pool for 45 min. After washing 5 times in PBS mouse polyclonal antiserum was applied for 30 min. Mouse antibodies were visualized by the application of alkaline phosphatase-antialkaline phosphatase (APAAP) technique with one repetition cycle [9]. Astra New fuchsin was used as the chromogen with levamisole to block endogenous alkaline phosphatase activity. Cells were counterstained with Mayer's haematoxyline. On a different glass slide immunoglobulin-containing cells (ICC) were stained using a biotinylated sheep anti-human immunoglobulin antiserum (RPN 1003, Amersham) and avidinbiotin-complex (PK4000, Vector Laboratory) with diaminobenzidine as chromogen. All antigen-binding cells (ABC) that did not stain for phagocyte markers were counted and the ratio of antigenbinding and thus antibody-forming B lymphocytes and all immunoglobulin-containing cells (ABC/ICC) was calculated as an index of specificity of B cell activation. One to three glass slides were stained for each specimen depending on the frequency of the activated B lymphocytes. The determination of blood-brain-barrier impairment and intrathecal immunoglobulin synthesis was done according to Reiber
Staining procedure Cytospin preparations were air dried and fixed with ethanolacetone (v/v)-formalin (2.6%) at room temperature for 3 min. The cells were preserved either fixed in TRIS-buffered saline, pH 7.2, for several days or unfixed for longer periods at -20°C wrapped in aluminium foil. Thawing was performed in the fixation solution. Endogenous peroxidase was inhibited with 3% H202 for 5 min. Slides were washed in PBS, pH 7.4. Monocytes and granulocytes were stained with a combination of monoclonal antibodies: M-718 (Dako, Hamburg, FRG), CD 14 (TIB 228, American Type Culture Collection, ATCC) and Mac-1 (TIB 128, ATCC). The latter two antibodies were generous gifts from Prof. Peters, Institute for
324
Fig. 2. Cerebrospinal fluid cells from patient 4 double stained for phagocytes by the combination of monoclonal antibodies M-718, CD 14 and Mac-1 (heavy metal amplificated peroxidase reaction) staining dark black and antigen binding (alkaline phosphatase reaction) staining red, counterstained with Mayer's haematoxylin, × 285. The red stained elliptic B lymphocyte (arrow) can be distinguished from the black stained monocyte (arrowhead) and Felgenhauer [13]. This intrathecal immunoglobulin production was not B. burgdorferi specific. Results
B. burgdorferi a n t i g e n is a d h e s i v e to glass a n d cell surfaces. A p p l i c a t i o n o f o t h e r p r o t e i n s such as F C S o r B S A d i d n o t r e d u c e t h e high b a c k g r o u n d staining. This b a c k Table 1. Clinical symptoms and signs and the results obtained in immunocytochemical staining and humoral CSF findings. Intrathecal immunoglobulin production that is not Borrelia burgdorferi specific was determined according to Reiber and Felgenhauer [13]. 1. indicates the first lumbar puncture prior to treatment with 2 × 10 mega penicillin G/day intravenously for 10 days; 2. indi-
g r o u n d staining c o u l d b e g r e a t l y r e d u c e d using 30% r a t serum or pooled human AB serum. An antigen concent r a t i o n o f 100 ~tg/ml gave o p t i m a l cell staining with minimal background. With lower antigen concentrations the n u m b e r of A B C d e c l i n e d . A n t i g e n b i n d i n g l y m p h o c y t e s f r o m l y m p h n o d e s c o u l d b e easily d i s t i n g u i s h e d f r o m m a c r o p h a g e s t h a t h a d p h a g o c y t o s e d a n t i g e n . T h e s e cells w e r e few in n u m b e r a n d t h e m a j o r i t y o f l y m p h n o d e m a c r o p h a g e s i d e n t i f i e d on m o r p h o l o g i c a l g r o u n d s did not bind antigen. T h e a n t i g e n b o u n d s h o w e d a c h a r a c t e r i s t i c surface d i s t r i b u t i o n in r a t l y m p h n o d e cells as well as in h u m a n cells (Fig. 1). S m a l l e r cells o f t e n s h o w e d a m o r e cytop l a s m a t i c staining. T h e A B C a p p e a r e d m o s t o f t e n as elliptic i m m u n o b l a s t s o r p l a s m a cells with e x c e n t r i c nuclei. The distribution patterns of bound antigen and immunog l o b u l i n s w e r e t h e same. T h e A B C w e r e easily d i s t i n g u i s h e d f r o m d o u b l e s t a i n e d b l a c k p h a g o c y t e s , which o c c u r r e d in m u c h g r e a t e r n u m b e r s in t h e b l o o d t h a n in t h e C S F (Fig. 2). G r a n u l o cytes a v i d l y b o u n d a n t i g e n b u t w e r e d i s t i n g u i s h a b l e f r o m A B C b y t h e i r c h a r a c t e r i s t i c nuclei. T h e o p t i m i z e d staining p r o c e d u r e for r a t l y m p h n o d e cells was also o p t i m a l for h u m a n b l o o d a n d C S F cells. T h e n u m b e r o f A B C in h u m a n C S F p r e p a r a t i o n s s h o w e d g r e a t i n t e r i n d i v i d u a l d i f f e r e n c e s r e g a r d l e s s of t h e d i s e a s e stage ( T a b l e 1). T h e i r n u m b e r v a r i e d f r o m less t h a n 5 to m o r e t h a n 120 cells p e r 10000 a n d g e n e r a l ly d e c l i n e d after t r e a t m e n t . T h e r e was an o v e r a l l c o r r e l a t i o n b e t w e e n i n t r a t h e c a l i m m u n o g l o b u l i n p r o d u c t i o n a n d t h e n u m b e r of A B C . cates lumbar puncture after treatment, except patient 2 who was re-examined after 5 days. In patient 4 a third lumbar puncture was done after 20 days. ECM, Erythema chronicum migrans; F, female; M, male; ABC, antigen-binding cells; ICC, immunoglobulin-containing cells
Patient no.
Age (years)
Sex
Symptoms and signs
Cells (per ~1)
ABC % of all cells
ICC % of all cells
% ratio ABC/ICC
Blood-CSFbarrier impairment
Intrathecal Ig synthesis
1 1. 2. 2 1. 2. 3 1. 2. 4 1. 2. 3. 5 1. 2. 6 1. 2. 7a 1. 2. 8 1. 2. 9 1. 2.
77
M
Polyradiculitis
67
M
27
M
78
F
ECM, faciaInerve palsy, radicular pain ECM, facial nerve palsy, radicular pain Facial nerve palsy
F
Radicular pain
48
F
ECM, radicular pain
22
F
61
M F
0.4 5.0 0.17 3.0 0.29 0.74 6.0 6.0 10.0 0.1 0.15 1.0 3.0 0.08 0.3 0.3 n.d. 1.0 0.16
50 6 43 33 50 11 17 17 10 33 0 5 4 63 7 10
50
Tickbite, ECM, radicular pain Tickbite, ECM, radicular pain Tick bite, facial nerve palsy
0.2 0.3 0.07 1.0 0.14 0.08 1.0 1.0 1.0 0.03 0.0 0.03 0.12 0.05 0.02 0.03 0.0 0.08 0.0
+++ +++ ++ ++ ++ ++ ++ (+)
47
364 107 347 313 459 102 91 87 44 49 17 96 34 315 40 33 22 82 8
IgM/IgG/IgA IgM/IgA/IgG IgM/IgG IgM/IgG/IgA IgM/IgA/IgG IgM/IgG/IgA IgM/IgG IgM/Iga IgM IgM/IgA IgM/IgA
a Frozen specimen
8 0
-
(+) ++ (+) + (+)
325 Based on the formula of Reiber and Felgenhauer [13] approximately 40-60% of IgG, 20-40% of IgA and 6080% of IgM were produced intrathecally in these cases. CSF cells contained proportionally more ABC than blood cells. The specificity of activated B lymphocytes ranged from 10% to 60% of all ICC in the acute stage. Control specimens from patients with other inflammatory diseases showed no ABC. In patients with bacterial meningitis the granulocytes bound antigen despite staining with the monoclonal antibodies but could easily be distinguished by their characteristic morphology. Cryopreservation of CSF cells reduced the number of ICC considerably. Cytospin preparations could, however, be stored up to 4 - 6 weeks without loss of staining characteristics.
Discussion
B. burgdorferi infection was chosen since the diagnosis can be generally made on clinical grounds in an early stage of disease and a CSF pleocytosis is frequently found with high numbers of activated B lymphocytes. The staining procedure as optimized with rat lymph node cells could be applied without major changes to clinical specimens. The adherence of B. burgdorferi to mononuclear phagocytes was overcome by double staining with monoclonal antibodies. After using human AB serum in the staining procedure it was not possible to double-stain further the A B C for immunoglobulins, owing to the immunoglobulin content of the human serum that adhered to the surface of the slide. Real double-staining of closely related antigens is mainly difficult when using permanent chromogens. The morphological appearance of antigen binding and immunoglobulins in immunoblasts and plasma cells, on the other hand, is exactly the same. Detection of antibody-producing cells by antigen binding has been used for the investigation of specific immunoblasts and plasma cells in spleen and lymph nodes of mice and rabbits (for review see [20]). A B C have also been investigated by fluorescence-activated cell sorting and have been shown to contain almost the entire population of antigen-specific antibody-producing cells [6]. Other techniques for the demonstration of antibody-forming and -releasing cells are the plaque or spotforming assays that use antigen-coated erythrocytes or solid phases to demonstrate secreted antibodies of individual cells [16]. Compared with these assays the immunocytochemical detection has several advantages. The method is fast and can detect rare antibody-forming cells. Antibody-forming cells of different stages are detectable even in the absence of antibody secretion and can be easily quantified. The concentration of specific activated B lymphocytes is much higher in the CSF than in blood, confirming the results obtained with the E L I S P O T assay in neuroborreliosis [1] and in viral meningitis [5]. This concentrated immunoreactivity in the CSF is specific for neuroborreliosis. A B C were numerous in patients with intrathecal immunoglobulin synthesis.
The reason why antigen binding and immunoglobulin staining are predominantly found on the cell surface is not clear at present. As the fixative includes acetone and ethanol the cytoplasmatic structures should be accessible for antibodies. Membrane-bound antibodies are fixed together with other membrane proteins, whereas intracellular antibodies prone to secretion that lack a transmembrane unit may not be held fast in situ by the fixatives applied and are thus eluted during washing steps. The thin surface staining of A B C from fixed specimens that had been stored in aqueous buffer further supports this view. The comparatively large proportion of activated B lymphocytes that do not bind antigen found in zoster ganglionitis and neuroborreliosis is also not explained. It may be attributable to the limits of the immunocytochemical technique but this non-specific activation seems to be a general p h e n o m e n o n [7, 8, 10, 11] and may not be due to a mitogenic effect of B. burgdorferi. While the absolute number of CSF cells had decreased after 10 days of treatment, the percentage of ICC often increased. The percentage of ABC, on the other hand, showed a minor increase or even decreased. Despite a larger proportion of immunocompetent cells among the CSF pleocytosis the specific activated B lymphocytes seem to be more closely regulated than the unspecific ones, because the specificity of B cell activation, i.e. the ABC/ICC ratio, was lower following treatment. Production of antibodies of other specificities, for example to myelin basic protein, has been reported in neuroborreliosis patients. It may account for some of the "unspecific" activated B lymphocytes and play a role in the pathogenesis of the disease [14, 17, 19].
Acknowledgements. This study was supported by a grant from the German Federal Ministry for Research and Technology (Bundesministerium fi~r Forschung und Technologie; 01 K188377). The authors accept sole responsibility for the contents of this article. Mrs. Heidi Semmler is thanked for her diligent technical assistance. References 1. Baig S, Olsson T, Link H (1989) Predominance of Borrelia burgdorferi specific B cells in cerebrospinal fluid in neuroborreliosis. Lancet I : 71-74 2. Behrends T (1988) Phosphate-coupled contrast enhancement of the DAB reaction product. Clin Neuropathol 7:145-146 3. Benach JL, Fleit HB, Habicht GS, Coleman JL, Bosler EM, Lane BP (1984) Interactions of phagocytes with the Lyme disease spirochete: role of the Fc receptor. J Infect Dis 150:497507 4. Beuche W, Thomas RS, Felgenhauer K (1989) Demonstration of zoster virus antibodies in cerebrospinal fluid cells. J Neurol 236 : 26-28 5. Forsberg P, Kam-Hansen S, Fryden A (1986) Production of specific antibodies by cerebrospinal fluid lymphocytes in patients with Herpes Zoster, Mumps meningitis and Herpes Simplex Virus encephalitis. Scand J Immunol 24 : 261-271 6. Hoven MY, De Leij L, Keij JFK, The TH (1989) Detection and isolation of antigen-specific B cells by the fluorescence activated cell sorter (FACS). J Immunol Methods 117:275-284 7. Hyypi~i T, Escola J, Laine M, Salmi A, Meurman O (1985) Polyclonal activation of B cells during Rubella infections. Scand J Immunol 21 : 615-617
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