1 Division of Clinical Immunology and 2 Division of Infectious Diseases, Department of Molecular and Clinical Medicine, 3 Clinical Research Center, Faculty of Health Sciences and 4 Division of Pediatrics, Department of Molecular and Clinical Medicine, University of Linköping, Sweden, 5 Pediatric Clinic, University Hospital, Linköping, Sweden, 6 Pediatric Clinic, Ryhov County Hospital, Jönköping, Sweden, 7 Pediatric Clinic, Västervik Hospital, Västervik, Sweden, 8 Pediatric Clinic, Vrinnevi Hospital, Norrköping, Sweden and 9 Department of Microbiology, University of Umeå, Sweden
10 Present address: Rheumatology Research Unit, Department of Medicine, CMM L8:04, Karolinska Institutet, SE-17176 Stockholm, Sweden
Correspondence to: M. Widhe; E-mail: mona.widhe{at}cmm.ki.se
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Abstract |
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Keywords: cytokines, IL-4, IFN-, immune response, ELISPOT
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Introduction |
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The immune response elicited during infection, although aimed to eliminate disease, may contribute to the pathogenesis. A tight regulation therefore needs to take place in order not only to eradicate the pathogen, but also to prevent excessive inflammatory responses leading to tissue destruction. The type of immune response induced towards the infecting agent, in terms of type 1 or type 2 cytokine pattern, is believed to be important for clinical outcome of many infectious diseases (8, 9). The Th cell differentiation into either IFN-- and tumor necrosis factor-ß-producing Th1 cells or IL-4-, IL-5- and IL-9-producing Th2 cells is a complex process influenced by several factors, including important contributions by innate immune mechanisms. Since several cell types besides Th cells contribute to the polarization of cytokine patterns, the type 1/type 2 terminology is more appropriate and will be used in this paper. During the course of the infection, the type 1/type 2 balance is either maintained or interchanged by a cross-regulating interaction. The cytokine chiefly responsible for the pro-inflammatory effects of the type 1 response is IFN-
. It activates macrophages, stimulates phagocytosis, promotes intracellular killing of microbes, enhances antigen presentation to T cells and the secretion of pro-inflammatory cytokines and chemokines (10). The type 1 response is also involved in the production of complement activating and opsonizing antibodies (11, 12). The ultimate type 2 cytokine IL-4 down-regulates these macrophage-dependent pro-inflammatory responses. Also, IL-4 activates B-cells and induces isotype switch to IgE, which mediates activation of basophils and mast cells (13). Different animal models of Lyme borreliosis (LB) have shown contradictory beneficial roles for IL-4 (14, 15) and IFN-
(16, 17) in disease resistance and the ability to clear the infection.
The immune response in the fetus and during early life is believed to be polarized towards the type 2 profile, and to gradually proceed towards type 1 (1820). In adults with NB, showing a clinical spectrum differing substantially from that in children, a predominating IFN- response with low IL-4 has been reported in blood and cerebrospinal fluid (CSF) by us (21) and others (22). We have also shown that increased levels of IL-4 are associated with a shorter disease course and recovery of adult NB (23). In children with NB, however, the type 1/type 2 response is so far unexplored.
Since the type of immune response is believed to have an impact on the clinical course and outcome of several infections, including NB, we wanted to examine the CSF and blood Borrelia-specific and spontaneous type 1/type 2 balance in children with early-disseminated NB, relate it to the clinical outcome of these children and to compare it with the previously reported response in adults with NB.
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Methods |
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Borrelia serology
Serological testing, using IDEIATM Lyme NB capture ELISA (DAKO, Copenhagen, Denmark), was done at the Department of Clinical Microbiology, University Hospital, Linköping, as a part of the routine diagnostic procedure. In short, paired CSF and serum samples were added to microwells coated with antibody specific to human IgG (or IgM). The dilution of CSF and serum ensures that the anti-human-IgG (or IgM) antibodies are saturated with either CSF or serum IgG (or IgM). Therefore, the same amount of total IgG (or IgM) from either CSF or serum is captured in the microwells. After washing of the wells to remove excess protein, biotinylated, native Borrelia flagellum (purified from Borellia afzelii strain DK1) complexed with peroxidase-conjugated streptavidin was added to the wells. The amount of flagellum conjugate bound per well was visualized by the addition of a chromogenic substrate, and optical density was measured in a spectrophotometer.
Preparation of mononuclear cells from blood and CSF
Blood (heparinized) and CSF were collected from patients with suspected NB (children and adults) and control subjects. PBMCs were separated by gradient centrifugation on Lymphoprep (Medinor AB, Stockholm, Sweden) at 400 x g for 30 min in room temperature (RT) with no brake, according to Bøyum (26). The cells were washed twice with HBSS (GIBCO, UK) pH 7.2 before re-suspension in cell culture medium (tissue culture medium, TCM) consisting of Iscove's modification of Dulbecco's medium (GIBCO BRL, Paisley, UK) with the addition of L-glutamine (Flow Labs, Irvine, UK) 292 mg l1, MEM 100x, non-essential amino acids 10 ml l1 (Flow), penicillin 50 IU ml1, streptomycin 50 µg ml1 (Flow), NaHCO3 3.024 g l1 and 5% heat-inactivated FCS. Cells were counted under a phase-contrast microscope using a Bürker chamber, and the lymphocyte concentration was adjusted to 1 x 106 ml1.
CSF-MNCs were counted under a phase-contrast microscopy using a Jessen chamber before 10 min of centrifugation at 200 x g at 4°C, followed by gentle re-suspension in TCM.
Preparation of outer surface protein fraction Borrelia antigen
An outer surface protein-enriched fraction (OF), containing mainly outer surface protein (Osp) A and OspB, was prepared from the Borrelia garinii strain Ip90, as previously described (27, 28). Ip90 was cultured in BarbourStoennerKelly II medium to a concentration of 1011 cells l1 and harvested in late log phase. After washing twice with TSM buffer (10 mM Tris pH 7.4/150 mM NaCl/5 mM MgCl2) at 10 000 x g for 20 min, the cells were re-suspended in 10 ml TSM and kept on ice for 15 min. Octyl ß-D-glucopyranoside (Calbiochem, Novablochem, San Diego, CA, USA) was added at a final concentration of 2%. The cells were incubated at 37°C for 1 h, before centrifugating for 30 min, 20 000 x g at RT. The supernatant was collected and incubated for 30 min at 56°C. OF was obtained from the precipitate, which after washing once with TSEA buffer (10 mM Tris pH 7.4/150 mM NaCl/10 mM EDTA/0.05% NaN3) was solubilized with 1% sodium lauryl sarcosinate in TSEA and incubated for 1 h at 37°C and for 15 h in RT. After centrifugation, the supernatant was filtrated on a 0.45-µl nitrocellulose membrane and dialyzed for 48 h against 25% methanol in MilliQ water at RT. The precipitate was dissolved in MilliQ water and pulsed with ultrasound before freezing at 70°C. To confirm the presence of OspA and OspB in the protein fraction, the respective murine mAbs (H5332 and H6831) obtained from Alan G. Barbour and Denee D Thomas (University of Texas Health Science Center, San Antonio, TX, USA) were assayed against the OF and recombinant OspA by immunoblot analysis (29). OF stimulation of PBMC and CSF-MNC discriminates between patients with NB and patients with other neurological diseases as well as healthy controls as shown in the IFN- and IL-4 enzyme-linked immunospot (ELISPOT) assay (21, 30). The OF was used here in a previously optimized concentration of 10 µg ml1.
ELISPOT for analysis of IL-4- and IFN--producing cells
The ELISPOT assay, described by Czerkinsky (31), was used for analysis of Borrelia OF-stimulated and unstimulated cytokine-secreting cells. The method was slightly modified and optimized for IL-4 and IFN- spots as described previously (21, 30). Ninety-six-well nitrocellulose-bottomed microtitre cell culture plates (Multiscreen HA; Millipore, Bedford, MA, USA) were pre-incubated with 100 µl of sterile MilliQ water for 30 min at RT before coating with 100 µl per well of mouse anti-human IL-4 mAb IL-4-I (82.4) or mouse anti-human IFN-
mAb 1-D1K (Mabtech, Stockholm, Sweden) at a concentration of 15 µg ml1 in sterile phosphate buffer (PBS) pH 7.4 overnight at 4°C or for 90 min at 37°C. The plates were either used directly after coating or frozen with the coating fluid at 20°C for a maximum of 3 months. At analysis, the plates were (when applicable) thawed at RT and emptied using a multiscreen vacuum device before washing eight times with 100 µl PBS. Unspecific binding sites on the nitrocellulose were blocked with TCM for at least 30 min in 37°C. After emptying, the PBMC suspension was added to achieve 100 000 lymphocytes per well. Due to lower cell numbers, 100097 000 (median 5000) cerebrospinal fluid lymphocytes (CSF-Ls) were added per well, depending on the number of cells available. Thereafter, 100 µl of OF diluted in TCM was added at a final concentration of 10 µg ml1. Hundred microlitres of TCM without antigen was added for the unstimulated cell secretion. As a positive control, 100 µl of PHA (Sigma Chemical Co., St Louis, MO, USA) diluted in TCM to a final concentration of 20 µg ml1 was used. As a negative control, 200 µl of TCM alone was added to wells not containing any cells. For PBMC wells, cell cultures were run in triplicates, while for CSF-MNC triplicates (n = 28), duplicates (n = 14) or single wells (n = 32) were analyzed, and in a few cases only one of the cytokines, depending on cell supply. The cells were cultured undisturbed for 48 h at 37°C, 5% CO2 with 95% humidity. The plates were then emptied and washed thoroughly 2x with 100 µl of PBS and 2x with PBS containing 0.05% Tween 20 (PBS-Tween), before 100 µl biotinylated mouse anti-human IL-4 mAb [IL-4-II (12.1)] or IFN-
mAb [7-B6-1] (Mabtech AB, Sweden) diluted in PBS-Tween to 1 µg ml1 was added. After 24 h of incubation in a moist chamber at RT, the plates were washed 4x with PBS-Tween and 100 µl of alkaline phosphatase-conjugated streptavidin diluted 1 : 1000 in PBS-Tween was added and incubated as above for 6090 min. The plates were then washed 4x with PBS and left for 510 min before emptying. The spots were developed for 15 min by adding 100 µl of BCIPNBT in development buffer (AP conjugate substrate kit, BioRad, Hercules, CA, USA). The color development was stopped by rinsing thoroughly with de-ionized water several times, and the plates were left to dry overnight at RT, in the dark. The spots were counted under a dissection microscope. One spot is equivalent to one cytokine-secreting cell.
Data handling and statistics
Mean of triplicates or duplicates was used when analyzing the cytokine results. Total Borrelia-stimulated secretion refers to the number of spots in Borrelia OF-stimulated wells, while the number of spots detected in wells with cells and medium (no antigen added) represents the unstimulated cytokine secretion. To achieve Borrelia-specific net secretion, in the following referred to as Borrelia-specific secretion, the unstimulated cytokine secretion was subtracted from the total Borrelia-stimulated secretion. The number of spots in the CSF-MNC wells was recalculated to the number of spots per 100 000 CSF-Ls, and will be referred to as cytokine secretion in CSF, whereas the number of spots in PBMC wells will be designated cytokine secretion in blood.
SPSS 10.0 for Windows was used for statistical evaluation of the results. For comparison of cytokine-secreting cell counts between the three diagnostic groups, KruskalWallis was used as a pre-test, and MannWhitney U test as post hoc. KruskalWallis was also used when comparing multiple intervals. When paired samples of IL-4 and IFN- secretion were compared within the diagnostic groups, Wilcoxon signed-ranks test was used. For correlation analyses, Pearson's correlation test was used, and Fishers exact test was used for comparison of the proportion of confirmed and possible NB in the two patient materials. Values <0.05 were considered significant.
The study was approved by the Ethical Committee of the Faculty of Health Sciences at the University of Linköping.
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Results |
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Cytokine production in children with confirmed and possible NB
Children with confirmed and possible NB had significantly higher numbers of both Borrelia-specific IL-4-secreting CSF-Ls (P < 0.05 and P < 0.01, respectively) and Borrelia-specific IFN--secreting CSF-Ls (P < 0.01), compared with non-NB (Fig. 2). In blood, the cytokine response showed a similar pattern, but was not as pronounced as in CSF, and the differences between the diagnostic groups were not statistically significant (Fig. 3). When comparing IL-4 and IFN-
within the diagnostic groups, there were no significant differences in CSF or blood for possible NB, showing that there was no predominance of either cytokine in this group. However, in confirmed NB, Borrelia-specific IFN-
was significantly higher than IL-4 in both CSF (P < 0.05, Fig. 2) and blood (P < 0.001, Fig. 3).
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Cytokine production in relation to clinical recovery of NB
The time required to obtain clinical recovery after the start of treatment varied between 1 day to >2 months in our patients with confirmed and possible NB. The distribution of children according to the multiple-choice questionnaire is shown in Fig. 1(b). When comparing the cytokine secretion in these intervals, no significant differences were found in CSF or in blood (data not shown). Fifty-three children were fully recovered at follow-up after 68 weeks, reported in questionnaires or in medical files, while 11 children still reported ongoing neurological symptoms. The children not recovering within this 2-month follow-up period showed similar patterns of cytokine secretion as the children that did recover.
Cytokine production in children with NB as compared with adults with NB
To be able to compare the Borrelia-specific cytokine pattern in children with NB reported here with adults having NB, a reference material of adult patients with confirmed or possible NB was used (see Methods). Children with NB had both significantly higher Borrelia-specific secretion and unstimulated secretion of IL-4 in the CSF, as compared with adults (P < 0.05 for both comparisons, Fig. 4), while no such differences were seen in blood (data not shown). The Borrelia-specific IFN- secretion was increased in blood (P < 0.05) but not in CSF in children as compared with adults with NB (data not shown).
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Discussion |
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The type 1 immune response with high production of IFN- has been suggested to be the optimal response to all infections caused by intracellular or phagocytosable microbes (32). By clearing these pathogens, the type 1 response diminishes further antigenic stimulation, allowing a switch to a type 2 response, resulting in reestablishment of homeostasis. However, if the type 1 response fails to completely clear the infection, persistent antigenic stimulation may induce chronic type 1 responses, leading to host damage. The relative lack of type 2 responses may predispose for development of chronic disease in adults. However, in addition to active infection and persistent antigenic stimulation, possibly mediated, e.g. by antigen containing blebs or inactive cystic forms of Borrelia burgdorferi (33), other mechanisms have been suggested for the development of chronic symptoms after acute NB. These include mimicry-mediated (autoimmune-like) pathologic immune responses (34, 35) or post-Lyme syndrome (36) where patients experience neurological symptoms despite the lack of objective findings of ongoing disease (37).
The immune system in young children is prone to respond with a type 2-like response to antigens (18, 38), a characteristic thought to be established during pregnancy, when the mother's immune system is believed to be type 2-deviated, aiming to prevent rejection of the fetus (20, 39). Such type 2-like responses in children have been reported in viral infections, e.g. respiratory syncytial virus (4042), but never in NB. It is likely that this propensity to respond with type 2 may influence the recovery process and clinical outcome of NB, since IFN- and IL-4 seem to be involved in the ability to clear the Borrelia infection. Resistance or susceptibility to experimental LB has been correlated with the production of IL-4 or IFN-
, respectively, as suggested by early animal studies (15, 43). Furthermore, increased IL-4 : IFN-
ratio was reported in disease-resistant mice (44). Treatment of Borrelia-infected mice with recombinant IL-4 (45) as well as recombinant IFN-
(46) resulted in suppression of the disease. However, when closely followed, an early IFN-
response was demonstrated to precede the IL-4 response seen in disease-resistant mice, while in susceptible mice, the IFN-
response appeared only later (16). This finding suggests that IFN-
plays a role in early eradication of Borrelia burgdorferi, while IL-4 is important for resolution of symptoms. We have recently demonstrated a similar relationship in adults with NB, where an early IFN-
response with a subsequent up-regulation of IL-4 was associated with a benign disease course, whereas patients with chronic NB showed increasing levels of IFN-
and lacked IL-4 (23). Since children displayed both IL-4 and IFN-
responses in the target organ, this is a prerequisite for the eradication of the disease and avoidance of harmful effects, which might be connected to the beneficial course of NB in children.
NB in children is an illness with an acute course and rather good prognosis. The clinical spectrum is clearly dominated by facial palsy and/or subacute meningitis (1), while manifestations like radiculitis, involvement of other cranial nerves and recurrent headache are less frequent. The total disease duration is most often rather short (1), and chronic NB in children is seldom reported (2). According to previous studies (1) and our own observations, the time interval between the onset of illness and admission to hospital for children with NB is short (daysweeks). This means that sample collection occurs during the very early-disseminated infection, a fact which should be kept in mind when interpreting the cytokine patterns, especially when comparing with adults, who in general seek medical attendance at a later stage, weeks (months) (47, 48). However, when comparing the cytokine responses in relation to duration of symptoms on admission, which in our material varied between 1 day to >2 months (median interval: 12 weeks), we could not find any obvious changes either for IL-4 or IFN-. Furthermore, the very early samples from adults showed low IL-4, whereas the late samples of our children still had increased IL-4. There is also a possibility that the early admission of children has implications on other laboratory findings, e.g. the intrathecal IgG antibody response, an important parameter for the diagnosis of NB. Intrathecal antibodies usually occur only after
6 weeks post-infection, which would result in lack of antibodies in early-admitting children with NB. Indeed, our children with possible NB, i.e. with negative intrathecal Borrelia-specific antibody production, had significantly shorter disease duration at admission than confirmed NB (Fig. 1a). Another consequence is that treatment usually begins earlier in children than in adults, which of course may be one important reason for the differences in recovery and occurrence of chronic neurological symptoms between children and adults with NB. On the other hand, facial palsy might improve or heal even when left untreated (3). The process behind this spontaneous remission is unknown and contribution of the type 2 immune response in NB children to the frequent remission of neurological symptoms and the fast recovery cannot be excluded. However, we did not find any significant differences between children recovering within 2 months after admission and children with remaining neurological symptoms. This may, in part, be explained by the small number of NB children with persistent symptoms (n = 11), or the fact that the follow-up period should have been longer in order to find differences. Obviously other cytokines, not analyzed here, might contribute to the recovery process, and would be interesting to study further. It is noteworthy that regulatory T cells have been suggested to play an important role in immunity to infectious diseases (49), and it would be of certain interest to study the immunosuppressing cytokines IL-10 and transforming growth factor ß in relation to clinical recovery of NB.
Six of the adult patients were under antibiotic treatment (three cefotaxime and three doxycycline) at the time of sample collection. In vitro studies indicate an immunostimulatory effect of cefotaxime, such as increased production of pro-inflammatory cytokines and activation of macrophages and lymphocytes, whereas doxycycline seems to have down-regulatory effects on these immune functions [reviewed in (50, 51)]. However, little is known about the effects of these drugs on the IFN- or the IL-4 response. Despite the ongoing treatment in these six adult patients, they did show the same cytokine pattern as the adult patients who had not yet started their treatment.
The notable high Borrelia-specific secretion of cytokines detected in our children with possible NB (CSF pleocytosis but no intrathecal Borrelia-specific antibody production) suggests that these children in fact might have a Borrelia infection in the central nervous system compartment. Our findings support the Swedish treatment traditions of NB, where antibiotics are prescribed on the basis of typical neurological symptoms and mononuclear pleocytosis in CSF. Even if intrathecal antibody production is required for confirmed diagnosis, absence of antibodies does not exclude CNS involvement due to Borrelia infection, especially when lumbar puncture is performed early in disease (24, 52), which was the case in our children with possible NB (Fig. 1a). Also, the fact that the children with possible NB did respond to antibiotic treatment to the same degree as the children with confirmed NB does support an ongoing Borrelia infection within the CNS of these children (Fig. 1b). The antibody-based diagnostic procedure is known to have certain limitations, and the Borrelia-specific cytokine secretion, as measured by ELISPOT, could supplement the existing diagnostic tools in certain cases. Our findings of higher Borrelia-specific secretion in CSF compared with blood also corroborates our previous findings of compartmentalization of the immune response to the CNS in NB (21, 53).
In conclusion, children with NB produce significantly increased Borrelia-specific IL-4 in combination with IFN- in their CSF, measured as increased numbers of Borrelia-specific cytokine-secreting cells, in contrast to the low IL-4 and high IFN-
reported in adults with NB. Since IL-4 is known to down-regulate the aggressive and possibly harmful effects of a long-standing IFN-
response, and has been suggested to play a role in resolution of symptoms in Borrelia infection, the observed prominent IL-4 response in the CNS might contribute to the more benign disease course seen in children with Lyme NB.
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Acknowledgements |
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Abbreviations |
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CSF | cerebrospinal fluid |
CSF-L | cerebrospinal fluid lymphocyte |
CSF-MNC | cerebrospinal fluid mononuclear cell |
ELISPOT | enzyme-linked immunospot |
i.v. | intravenously |
LB | Lyme borreliosis |
NB | neuroborreliosis |
OF | outer surface protein-enriched fraction |
Osp | outer surface protein |
PBL | peripheral blood lymphocytes |
PBMC | peripheral blood mononuclear cells |
RT | room temperature |
TCM | tissue culture medium |
VP | ventriculoperitoneal |
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Notes |
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Received 25 June 2003, accepted 1 July 2005.
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References |
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