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(Stroke. 1995;26:1393-1398.)
© 1995 American Heart Association, Inc.

Articles

Early Intrathecal Production of Interleukin-6 Predicts the Size of Brain Lesion in Stroke

Elisabeth Tarkowski, MD; Lars Rosengren, MD, PhD; Christian Blomstrand, MD, PhD; Carsten Wikkelsö, MD, PhD; Christer Jensen, MD; Sven Ekholm, MD, PhD Andrzej Tarkowski, MD, PhD

From the Departments of Clinical Immunology (E.T., A.T.), Clinical Neurosciences (section of Neurology) (E.T., L.R., C.B., C.W.), Radiology (C.J., S.E.), and Rheumatology (A.T.), University of Göteborg, Sweden.

Correspondence to Dr Elisabeth Tarkowski, Department of Clinical Immunology, Guldhedsgatan 10, S-413 46 Göteborg, Sweden.

	Abstract

Top Abstract Introduction Subjects and Methods Results Discussion References

 
Background and Purpose We have previously demonstrated that stroke influences systemic immune responses. The aim of the present study was to investigate patterns of local inflammatory response as a consequence of acute stroke.

Methods Thirty stroke patients were studied prospectively on days 0 to 3, 7 to 9, 21 to 26, and after day 90 with clinical evaluations, radiological assessments, and analysis of serum and cerebrospinal fluid cytokine levels.

Results Significantly increased levels of interleukin-6 (IL-6) in cerebrospinal fluid (P<.001) were observed in virtually all patients studied compared with healthy control subjects. This increase was observed during the whole observation period but was significantly more pronounced within the first days after stroke onset, with a peak level on days 2 and 3. This initial increase was significantly correlated (r=.65, P=.002) with the volume of infarct measured by MRI 2 to 3 months later. Serum levels of IL-6 in stroke patients were significantly lower than cerebrospinal fluid levels of IL-6 (P=.013) and did not display any significant correlation to the size of the brain lesion. Also, increase in intrathecal but not systemic production of IL-1ß was observed early during the stroke. Only minor increases of cerebrospinal fluid interferon- levels were observed in two patients.

Conclusions Our study demonstrates an intrathecal production of IL-6 and IL-1ß in patients with stroke, supporting the notion of localized inflammatory response to acute brain lesion. In addition, the significant correlation between early intrathecal production of IL-6 and the subsequent size of the brain lesion can be used as a prognostic tool, predicting the size of the brain damage before it is possible to accurately visualize it with radiological methods.

Key Words: cerebrospinal fluid • magnetic resonance imaging • interleukins • prognosis

	Introduction

Top Abstract Introduction Subjects and Methods Results Discussion References

 
The hallmark of ischemic stroke is the varying degree of brain tissue damage. However, it is difficult to assess the extent of brain damage or to visualize the lesion with radiological methods soon after stroke onset. Since new and potentially risky procedures are currently being investigated to reverse the acute effects of ischemia on brain tissue and to restore the compromised blood perfusion,¹ it is of crucial importance to identify early markers predicting the size and localization of a brain lesion for an appropriate estimation of the risk-benefit relation.

One of the consequences of an acute lesion, eg, surgical trauma, is the release of proinflammatory cytokines such as interleukin (IL)-1ß and IL-6.^{2 3} Recently, intracranial production of proinflammatory cytokines has been demonstrated after infections,^{4 5} mechanical or hypoxic injury of the brain,^{6 7} and subarachnoid hemorrhages.⁸ These cytokines are not only produced by monocytes, fibroblasts, endothelial cells, and T lymphocytes, as reviewed by Hirano,⁹ but also by microglia^{10 11} and astrocytes.¹² Once released, proinflammatory cytokines are capable of mediating cytotoxic action in surrounding tissues¹³ and inducing astrocyte proliferation¹⁴ and lymphocyte activation.¹⁵ IL-1ß and IL-6 have also been shown to exert neuroprotective¹⁶ and neurotrophic^{17 18} effects.

We have recently demonstrated that T-lymphocyte responsiveness is affected as a consequence of stroke.^{19 20 21} The aim of the present study was to investigate whether stroke induces intrathecal production of proinflammatory cytokines and, if so, to evaluate the kinetics of their release in relation to the size of the brain lesion and the clinical course of the disease.

	Subjects and Methods

Top Abstract Introduction Subjects and Methods Results Discussion References

 
Patients
Thirty patients (23 men and 7 women, 40 to 81 years old [mean±SD, 64±11 years]) at Sahlgrens' University Hospital, Department of Neurology, were consecutively incorporated into the study. All the patients experienced stroke that occurred within the first 3 days of admission into the study. None of these patients had a history of previous stroke. Patients with malignant and autoimmune diseases, with severe infections, or taking immunosuppressive drugs were excluded. All the patients were evaluated by a standardized examination of motor and sensory deficit, peripheral reflexes, muscular tone, and cranial nerve function. Occurrence of dysphasia and tactile neglect symptoms was also evaluated. Furthermore, the patients were examined according to the modified Scandinavian Stroke Scale index²² at the onset of stroke and then at 1 week, 3 weeks, and 3 months after the onset. The Scandinavian Stroke Scale index is scored between 7 (normal neurological status) and 29 (coma and paralysis). The degree of disability was evaluated with the Barthel Index²³ 3 months after onset; scoring ranges between 110 (no disability) and 60 (complete dependence for activities of daily life). All the patients were examined with CT of the brain during the first days after the onset of stroke. Twenty patients were reexamined with MRI, and the remaining 10 patients were reexamined with CT 2 to 3 months after stroke onset.

Stroke patients were stratified in minor and major stroke groups according to the criteria of Hachinski.²⁴ The definition of minor stroke requires that the patient is discharged and sent home, walks without assistance, and copes unaided with such self-care activities as eating, dressing, and toileting within 1 month after disease onset.²⁵ In contrast, patients with major stroke had stable and usually severe neurological deficit.

Cerebrospinal fluid (CSF) and serum samples were obtained for analysis of cytokine levels on days 0 to 3, 7 to 9, and 21 to 26 and 3 months after disease onset. In 10 patients, serum and CSF samples could be obtained twice within the first 3 days after stroke onset. CSF samples from 23 control individuals without any neurological disease or deficit and serum from 41 healthy blood donors were obtained to establish normal levels of IL-6 and IL-1ß.

This study was approved by the ethics committee of the University of Göteborg.

Reagents and Procedures
Cell line B13.29, which is dependent on IL-6 for growth, has been previously described.²⁶ For IL-6 determinations, the more sensitive subclone B9 was used.^{27 28} B9 cells were harvested from tissue culture flasks, seeded into microtiter plates (Nunc) at a concentration of 5000 cells per well, and cultured in Iscove's medium supplemented with 5x10^-5 mol/L 2-mercaptoethanol, 5% fetal calf serum (Seralab), penicillin (100 U/mL), and streptomycin (100 µg/mL), and CSF or serum samples were added. [³H]Thymidine was added after 68 hours of culturing, and the cells were harvested 4 hours later. The samples were tested in twofold dilutions and compared with a recombinant human IL-6 standard (Genzyme). B9 cells were previously shown not to react with several recombinant cytokines, including IL-1, IL-1ß, IL-2, IL-3, IL-5, granulocyte-macrophage colony-stimulating factor, tumor necrosis factor–, and interferon-. There was only weak reactivity with IL-4.²⁸ To assess the specificity of the bioassay, we used a highly purified monoclonal antibody specific for human IL-6 (Genzyme) in a neutralization assay. Preincubation of 10 µg/mL of this antibody with either recombinant IL-6 or CSF from stroke patients containing IL-6 (1 hour, 37°C) reduces proliferative responses of B9 indicator cells by an average of 95%.

Levels of interferon- in CSF samples were estimated by an enzyme-linked immunosorbent assay using monoclonal antibodies for coating and developing steps as previously validated²⁹ and described.²¹

Levels of IL-1ß in CSF and serum samples were estimated by an enzyme-linked immunosorbent assay (Quantikine R&D Systems). The normal levels of serum IL-1ß are below 3.5 pg/mL according to the manufacturer data.

MRI and CT Analyses
To evaluate the extent and localization of brain lesions, neuroimaging was performed 2 months or later after onset of stroke. The delay in imaging was chosen to get a better delineation of the permanent damage. The neuroimaging techniques used were CT as well as multiplanar MRI. The CT scans were routinely performed parallel to the canthomeatal plane (ie, a gantry tilt about +10° from Reid's baseline with 5-mm [posterior fossa] and 10-mm [supratentorial] slice thickness). The MRI examinations (Philips Gyroscan T5-II) were performed with axial proton density and T₂-weighted images of the brain. If a lesion was identified, a 3-D volume sampling was also performed. All scans were evaluated to correlate each lesion to its anatomic location. The evaluation of the scans was done by two experienced neuroradiologists without knowledge of clinical data. Sixty-six important anatomic brain structures were defined in the scans according to the criteria of Kretschmann and Weinrich,³⁰ and all the lesions were related to these structures. Volume measurements of the lesions were done by means of a 3-D reconstruction program in the work-station environment (Philips Gyroview). This technique entails the use of a proper segmentation and subsequent seeding within the lesion. The volume of the lesion thus created is automatically given when reconstruction is finished.

Statistics
Statistical analysis was carried out by Student's two-tailed t test. The ² test was used to analyze categorical data. Spearman's rank-order correlation method was used to calculate the correlation and the level of significance between the IL-6 levels and the volume or size of a brain lesion. A value of P<.05 was considered statistically significant.

	Results

Top Abstract Introduction Subjects and Methods Results Discussion References

 
Clinical Findings
Twenty-three patients included in the study displayed varying degrees of hemiparesis when examined at the onset of stroke. Eighteen of the hemiparetic patients were able to perform some voluntary movements, whereas 5 had a complete paralysis. Seven displayed no motor deficit but showed other stroke-related symptoms such as isolated hemisensory deficit or aphasia.

Twelve patients displayed impaired sensory function. Four patients displayed neglect symptoms. Eight patients had dysphasia. Sixteen had neurological signs on the right side of the body, 11 on the left side, and 3 patients had no symptom lateralization. Twenty-four of the patients were classified as having a minor and 6 a major stroke.

Use of the Scandinavian Stroke Scale index showed that the average neurological deficit was limited already during the first days after the disease onset (mean±SD, 10.8±4.7) and diminished with time (days 7 to 9, 10.5±5.0; days 21 to 26, 9.3±4.2; and after 3 months, 9.2±4.5). The degree of disability, measured 3 months after the onset of stroke by the Barthel Index, was also low (103.1±14.4), indicating that the majority of patients could perform daily life activities without any help.

Radiological Findings
Fourteen patients exhibited a single infarct, 13 multiple infarcts, and 3 no pathological changes in support of infarct. Seventeen patients displayed white matter lesions in the nonaffected brain hemisphere, whereas in 13 patients such changes were not found. Fourteen patients had a large infarct (ie, the sum of the largest transversal and sagittal diameters divided by 2, if greater than 1.5 cm), 13 a small infarct (ie, the sum of the largest transversal and sagittal diameters divided by 2, if less than 1.5 cm). The 3 patients with no radiologically visible infarct changes were assumed to have a small infarct. These two groups of patients differed significantly in relation to several other radiological and clinical aspects (Table 1). MRI analysis of brain infarct in 20 stroke patients revealed that the average volume of lesion was 21.7±7.5 mL (mean±SEM).

View this table: [in this window] [in a new window]   Table 1. Radiological and Clinical Features of Stroke Patients With Large Versus Small Infarcts

Cytokine Levels in Cerebrospinal Fluid and Serum
CSF IL-6 Levels in Stroke Patients
Twenty-eight of 30 patients displayed elevated levels of IL-6 in the CSF within the first days after disease onset. Two patients with minor stroke had no early production of IL-6. One of those had radiologically verified small infarct, whereas the other one did not display any infarct. The latter patient started to produce IL-6 after 3 weeks, whereas the other one had no IL-6 production during the observation period. As shown in Fig 1, top, levels of IL-6 were already significantly higher than in control subjects on the day of the stroke onset, increased rapidly to reach a peak on days 2 and 3, and then decreased successively until day 90. However, even on day 90, the IL-6 levels in CSF remained significantly elevated compared with the control subjects (Fig 1, top).

View larger version (22K): [in this window] [in a new window]   Figure 1. Top, Bar graph shows kinetics of interleukin-6 (IL-6) production in cerebrospinal fluid (CSF) and serum in patients with acute stroke. Bottom, Bar graph shows relationship between levels of IL-6 in CSF and in serum at different intervals after stroke.

Serum IL-6 Levels in Stroke Patients
The IL-6 level in serum was significantly higher in the stroke patients compared with control subjects during the whole observation period (Fig 1). However, in contrast to IL-6 levels in the CSF, the stroke patients did not display any distinct time-related variation of the IL-6 levels in serum (Fig 1). Notably, the level of IL-6 in serum was significantly lower compared with that of CSF on days 1 to 3 (mean±SD, 73±42 versus 231±313 pg/mL; P=.03), indicating intrathecal production and secretion of this cytokine. With time, CSF IL-6 levels became successively equal to those of serum (day 7, 83±102 versus 59±48 pg/mL; NS) and then decreased significantly compared with serum (day 21, 21±30 versus 54±35 pg/mL, P<.001; day 90, 31±38 versus 60±44 pg/mL, P<.001). IL-6 levels in serum and CSF did not show any significant correlation initially. However, there was a significant correlation on days 21 to 26 and 90 (r=.42, P=.044; and r=.79, P<.001; respectively). The ratios between IL-6 levels in CSF and in serum in relation to progression of stroke are presented in Fig 1, bottom.

IL-6 Levels in Relation to Ratios of Albumin CSF to Serum
Ratios of CSF albumin to serum albumin and IgG index have been investigated for every patient and at each time point. During the first week after stroke onset, 16 of the 30 patients showed no increases in CSF to serum albumin ratio (days 1 to 3 [mean±SEM], 0.0061±0.0003; day 7, 0.0058±0.0003), in favor of an intact blood-brain barrier, whereas the 14 remaining patients exhibited a small increase of CSF to serum albumin ratio (days 1 to 3, 0.0099±0.0005; day 7, 0.0104±0.0006), in favor of a modest blood-brain barrier damage. However, when analyzed separately, these two groups of patients displayed similar levels of IL-6 both in CSF and in serum. Thus, IL-6 levels in CSF within the first 3 days after stroke were 233±69 pg/mL in the group of patients without any signs of blood-brain damage, whereas corresponding values for patients with blood-brain damage were 229±97 pg/mL (NS). At day 7 after the onset of stroke, patients with an intact blood-brain barrier displayed somewhat higher CSF IL-6 levels compared with patients with blood-brain barrier damage (102±32 versus 55±15 pg/mL, NS). IgG index remained unaltered throughout the course of stroke (data not shown).

IL-6 Level in CSF Predicts the Size of Subsequent Brain Lesion
The initial levels of IL-6 in CSF were significantly correlated to the volume of the brain infarct measured 2 to 3 months later (Table 2). No similar correlation was found between the IL-6 levels in serum and the brain lesion volume (Table 2). Moreover, when analyzed separately, the patients with a large brain lesion displayed significantly increased (P=.001) IL-6 levels in CSF early in the course of stroke (Fig 2) but not in serum (data not shown), compared with patients with small brain lesions.

View this table: [in this window] [in a new window]   Table 2. Correlation Between the Levels of Interleukin-6 in Cerebrospinal Fluid and Subsequent Volume of Brain Lesion as a Consequence of Stroke

View larger version (20K): [in this window] [in a new window]   Figure 2. Graph shows kinetics of interleukin-6 (IL-6) production in cerebrospinal fluid with respect to the size of the subsequent brain lesion (14 stroke patients with large infarcts and 16 stroke patients with small infarcts).

Patients with a brain lesion affecting mainly (>50%) the gray matter displayed significantly higher IL-6 levels in CSF on days 1 and 7 (days 1 to 3, 433±365 versus 42±21 pg/mL; P=.003; days 7 to 9, 154±129 versus 31±43 pg/mL; P=.015; respectively) compared with stroke patients with mainly white matter lesions. Moreover, only the former group displayed kinetics similar to the whole stroke group with significantly elevated levels of IL-6 on days 1 to 3 compared with the subsequent period (Fig 3). No significant difference was seen in stroke patients with mainly gray matter lesions compared with patients with white matter lesions with respect to serum IL-6 levels (days 1 to 3, 69±41 versus 79±48 pg/mL; NS; days 7 to 9, 50±34 versus 60±55 pg/mL; NS).

View larger version (20K): [in this window] [in a new window]   Figure 3. Graph shows kinetics of interleukin-6 (IL-6) production in cerebrospinal fluid with respect to gray versus white matter lesions (14 stroke patients with mainly gray matter lesions and 10 patients with mainly white matter lesions).

Patients with cortical lesions and patients with subcortical lesions displayed similar patterns of IL-6 levels in the CSF (data not shown). Patients with a single brain lesion did not show any significant difference in CSF IL-6 levels compared with patients with multiple brain lesions (data not shown).

IL-6 Levels in CSF in Relation to Clinical Findings
When analyzed separately, patients with a minor versus major stroke showed a similar pattern regarding the kinetics of IL-6 production in CSF (data not shown). However, patients with a major stroke had somewhat higher absolute levels of CSF IL-6 (338±315 versus 202±315 pg/mL; NS) during the first 3 days. Morever, patients with a major stroke had significantly more elevated levels of IL-6 on days 7 to 9 compared with patients with a minor stroke (145±138 versus 57±52 pg/mL, P=.023). This indicates that a long-lasting IL-6 production in patients will relate to major stroke features. There was no significant correlation between the levels of IL-6 in CSF or serum and the Scandinavian Stroke Scale index or Barthel Index, respectively.

Patients with right-sided neurological deficit had more elevated levels of IL-6 early after stroke compared with patients with left-sided deficit (269±355 versus 93±135 pg/mL; NS).

IL-1ß Levels in CSF and the Serum of Stroke Patients
All 6 patients with a major stroke, but only 18 of 23 patients with a minor stroke, displayed IL-1ß production in the CSF. IL-1ß levels were low initially but increased significantly compared with the healthy control subjects (P=.008) on day 2, to drop successively during the following days to levels that did not differ significantly from those of the control subjects (Fig 4). Moreover, the number of stroke patients producing IL-1ß in the CSF was significantly higher on day 2 compared with control subjects (8/10 versus 4/20; P=.01). The levels of IL-1ß in serum were within the normal range at all time points and below the levels of CSF IL-1ß (Fig 4), indicating intrathecal IL-1ß production early during the stroke.

View larger version (19K): [in this window] [in a new window]   Figure 4. Bar graph shows kinetics of interleukin (IL)-1ß production in cerebrospinal fluid (CSF) and serum in patients with acute stroke.

Levels of IL-1ß in CSF or serum did not display any significant correlation to either the size of infarct or its location. In addition, we did not find any significant correlation between the levels of IL-6 and IL-1ß in CSF or serum at any time. Only 2 patients with minor stroke exhibited a low level of interferon- in CSF early during the stroke.

	Discussion

Top Abstract Introduction Subjects and Methods Results Discussion References

 
We have recently demonstrated that stroke gives rise to aberrant T-cell responses both early after the onset²¹ and in the chronic phase of the disease.^{19 20} The present study demonstrated an initial and transient increase of IL-1ß in CSF and an early but also persisting IL-6 presence in CSF and serum, with the highest levels in the CSF within the first days after the onset of stroke.

One of the major questions is whether the cytokines observed in the CSF were produced locally by cells within the nervous system or rather originated from the systemic compartment. A systemically produced IL-1ß and IL-6 could potentially be able to passively enter into the cerebrospinal compartment through the damaged blood-brain barrier. Such a hypothesis would require high levels of IL-1ß and IL-6 in serum of stroke patients early after disease onset. We found, however, no increase of IL-1ß in serum and significantly lower levels of IL-6 in serum compared with CSF during the first week after the onset of stroke. This clear-cut difference between the levels of the cytokines in CSF and serum within the first days after the onset of stroke argues strongly against the hypothesis of a primary systemic production of IL-6 and IL-1ß with subsequent passage to CSF. Instead, it favors the local production of these two cytokines. The finding of IL-6 in serum of stroke patients is in accordance with a recent study by Fassbender et al,³¹ who demonstrated increased serum IL-6 levels in an early stage of the disease.

The intrathecal production of the IL-6 and IL-1ß could be a result of an inflammatory process secondary to brain tissue damage. This is in accordance with a previous study by Woodroofe et al,³² who has demonstrated a rapid production (peak at 48 hours) of IL-6 and IL-1ß, after mechanical injury of the adult rat brain, using intracerebral tissue microdialysis technique. The cellular origin of IL-6 production in the brain remains unknown, but both macrophages and endothelial cells, as well as brain-derived microglia and astrocytes, have the capacity to synthesize this cytokine.^{9 10 11 12} In this respect, histological studies of transient brain ischemia in animal models of stroke have demonstrated an initial activation of astrocytes within the first 48 hours after the onset of stroke, whereas macrophage activation was detected a few days later.³³ Thus, the kinetics of IL-1ß and IL-6 production in CSF with a peak on days 2 to 3 in the stroke patients, as observed in the present study, support the astrocytes as the main source of these cytokines.

What is the function of locally produced cytokines as a consequence of stroke? In this respect, IL-1ß has been demonstrated to enhance the expression of adhesion molecules on endothelial cells^{34 35} and hence support the initial invasion of polymorphonuclear leukocytes and monocytes to the focus of the lesion. The influx of inflammatory cells might then aggravate brain damage by the production of toxic oxygen radicals³⁶ and edema formation³⁶ as well as increasing the tendency to develop thrombosis in the surrounding blood vessels.^{33 36} IL-1ß also has the capacity to trigger the synthesis of IL-6,³⁴ a cytokine that on one hand gives rise to the production of acute-phase proteins but on the other suppresses IL-1ß and tumor necrosis factor production,^{37 38} as well as the delayed-type hypersensitivity reaction,³⁹ hence acting as an anti-inflammatory agent. However, IL-1ß and IL-6 may also synergize to induce production of adrenocorticotropic hormone,⁴⁰ which presumably acts beneficially in acute stroke by controlling several stages of the inflammatory process. Thus, the cytokine cascade has the potential of both triggering and controlling the inflammatory responses after brain damage.

Another interesting aspect of the early intrathecal production of IL-1ß is its neuroprotective role in ischemia-induced neuronal damage.¹⁶ Thus, a localized brain lesion might trigger IL-1ß production to protect nearby neurons from further damage. Moreover, IL-6 has been demonstrated to act as nerve growth factor,^{4 17} being able to induce neuronal synthesis and differentiation of neurites and to increase the number of sodium-dependent channels.¹⁸ In this respect, sprouting and activation of nearby neurons has been demonstrated to be a part of recovery after brain damage.⁴¹ Since IL-6 is not only present in the CSF during the acute phase of the stroke, when the inflammatory changes have been observed,³³ but also during the resolution and the recovery phase, it is tempting to ascribe this cytokine a role in the healing process.

Lastly, we have demonstrated that the early production of IL-6 in CSF is significantly correlated to the size of brain lesion visualized 2 to 3 months after stroke onset. This relation indicates that intrathecal production of IL-6 can, within the first 24 hours of the stroke onset, predict the definitive extent of the brain damage. This is earlier than it is possible to visualize lesions with any accuracy with radiological methods. Thus, determination of CSF IL-6 levels in the acute phase of stroke may enable an early selection of patients for different therapeutic strategies. Our finding with respect to differential IL-6 CSF levels in patients with white matter lesions compared with gray matter lesions (Fig 3) could simply relate to the size of the brain infarction. Thus, it is known that in general small infarcts involve mainly the white matter, whereas large infarcts are likely to extend to the cortex. However, we believe that the differences with respect to CSF IL-6 production between white and gray matter lesions might not only reflect the size of the lesion but also involvement of different types of brain cells (eg, glia cells versus neurons). For example, within our proband group, there was a stroke patient who, despite having a large infarct in white matter, displayed only a minute increase of CSF IL-6 (maximum, 65 pg/mL). In contrast, one of the patients with a small infarct engaging gray matter displayed high CSF IL-6 levels (maximum, 450 pg/mL).

In conclusion, this study showed an intrathecal synthesis of IL-1ß and IL-6 as a consequence of ischemic stroke, suggesting cytokine-mediated inflammatory reaction in the central nervous system in the early stage of stroke. Moreover, the initial IL-6 production predicts the size of brain damage and can be a useful tool to select patients for hyperacute treatments. Studies are continuing in our laboratory to further characterize the pattern of local cytokine production in stroke.

	Acknowledgments

 
This study was supported by grants from the Göteborg Medical Society, University of Göteborg, The Swedish Association Against Rheumatism, the King Gustaf V:s 80-year Foundation, Stroke Foundation, Gamla Tjänarinnor Foundation, John and Brit Wennerströms Foundation, and the Swedish Medical Research Council. We thank Margareta Verdrengh for excellent technical assistance.

Received February 6, 1995; revision received April 13, 1995; accepted May 2, 1995.

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