Archives of Physical Medicine and Rehabilitation
Volume 88, Issue 11 , Pages 1384-1393, November 2007

Clinical Correlates of Elevated Serum Concentrations of Cytokines and Autoantibodies in Patients With Spinal Cord Injury

  • Andrew L. Davies, MSc

      Affiliations

    • Graduate Program in Neuroscience, London, ON, Canada
    • University of Western Ontario, London, ON, Canada.
    • ,
  • Keith C. Hayes, PhD

      Affiliations

    • Department of Physical Medicine & Rehabilitation, London, ON, Canada
    • Lawson Health Research Institute, London, ON, Canada
    • University of Western Ontario, London, ON, Canada.
    • Corresponding Author InformationReprint requests to Keith C. Hayes, PhD, Parkwood Hospital/SJHC, 801 Commissioners Rd E, London, ON N6C 5J1, Canada
    • ,
  • Gregory A. Dekaban, PhD

      Affiliations

    • Department of Microbiology and Immunology, London, ON, Canada
    • Biotherapeutics Research Group, Robarts Research Institute, London, ON, Canada
    • University of Western Ontario, London, ON, Canada.

Article Outline

Abstract 

Davies AL, Hayes KC, Dekaban GA. Clinical correlates of elevated serum concentrations of cytokines and autoantibodies in patients with spinal cord injury.

Objective

To determine the serum cytokine profiles of patients with spinal cord injury (SCI) and varying clinical presentations relative to healthy, able-bodied, age-matched control subjects.

Design

Cross-sectional study.

Setting

Clinical research unit.

Participants

People with SCI (N=56) and different clinical presentations, and healthy, able-bodied, age-matched control subjects (N=35).

Interventions

Not applicable.

Main Outcome Measures

Serum levels of the proinflammatory cytokines interleukin (IL) 1β, IL-6, tumor necrosis factor alpha (TNF-α), the anti-inflammatory cytokines IL-4 and IL-10, the regulatory cytokine IL-2, the IL-1 receptor antagonist (IL-1RA), and autoantibodies against myelin-associated glycoprotein and GM1 ganglioside (anti-GM1) immunoglobulin (IgG and IgM), as determined by enzyme-linked immunosorbent assay. The relationship between elevated serum cytokine levels and clinical variables was also studied.

Results

SCI subjects exhibited serum concentrations of IL-6, TNF-α, IL-1RA, and anti-GM1 (IgG) that were greater (P<.05) than control group values. Elevated cytokine concentrations were not associated with high white blood cell counts, level of injury, or American Spinal Injury Association classification; they were evident in SCI subjects who were asymptomatic for medical complications, but were further elevated in subjects with pain, urinary tract infection (UTI), and pressure ulcers.

Conclusions

Elevated levels of circulating proinflammatory cytokines and autoantibodies are present in the serum of SCI subjects without medical complications, and are further elevated in SCI subjects with neuropathic pain, UTI, or pressure ulcers, relative to healthy, able-bodied control subjects. These findings may be indicative of a protective autoimmunity, simply a consequence of occult or evident infection, or evidence of cytokine dysregulation that may contribute to an immune-mediated impairment of axonal conduction.

Key Words: Autoantibodies, Cytokines, Gangliosides, Interleukin-1 receptors, Interleukin-6, Myelin-associated glycoprotein, Pressure ulcer, Rehabilitation, Spinal cord injuries, Tumor necrosis factor, Urinary tract infections

 

SPINAL CORD INJURY (SCI) initiates a sequence of major immunologic challenges, from the acute stage,1 through rehabilitation,2, 3 and in the longer-term with secondary complications such as recurrent urinary tract infection (UTI) and pressure ulcers.4, 5, 6, 7 Although appreciable insights have been gained into the immunologic processes involved in the acute state (<2wk),1, 8, 9 less is known of the immunologic status of patients in the postacute (2−52wk) or chronic (>52wk) stages.

Cruse et al10, 11 reported decreased natural and adaptive immune responsiveness in SCI patients within 2 weeks of injury. Natural killer (NK) cell function was depressed, plasma adrenocorticotrophic hormone (ACTH) and urine-free cortisol levels were increased, and T-cell function and activation were diminished. Patients with tetraplegia showed reduced NK and T-cell function compared with patients with paraplegia; rehabilitation therapy appeared to reverse many of these indicators of immunosuppression, although NK cell−induced lysis remained depressed even after 12 months.2, 3

Immunosuppression in these cases was attributed to disrupted sympathetic innervation of lymphoid tissue, stress-related neuro-endocrine responses, and interrupted afferent input to immunoregulatory neurons within the central nervous system (CNS). Iversen et al12 subsequently confirmed the first of these mechanisms by showing that lymphocyte functions were significantly depressed in SCI patients with functionally complete lesions. Long-term colony formation of all hematopoietic cell lineages, including dendritic cells, by decentralized bone marrow, was substantially reduced.

As with other immunosuppressive disease states,13, 14, 15, 16 elevated serum concentrations of proinflammatory cytokines have been reported in SCI patients with or without secondary medical complications of infection or pressure ulcers.17, 18, 19, 20 Abnormally elevated serum titers of interleukin 2 (IL-2), tumor necrosis factor alpha (TNF-α), the soluble IL-2 receptor (IL-2R), and IL-6, as well as elevations in complement,18, 21, 22 have been reported in chronic SCI patients who were asymptomatic for bacterial or viral infection or pressure ulcers. Patients being treated for pressure ulcers or infection also have elevated serum levels of proinflammatory cytokines.20 Elevated proinflammatory cytokine levels have not been detectable in all cases; Frost et al23 reported that some chronic SCI patients with pressure ulcers or indwelling urinary catheters had elevated serum titers of the acute phase reactant, C-reactive protein, indicative of an active inflammatory process, but did not have statistically significant elevated levels of proinflammatory cytokines.

We undertook the present study to further characterize the serum proinflammatory cytokine profile of patients with SCI. In particular, we investigated the serum titers of IL-1β and its receptor antagonist (IL-1RA), IL-2, IL-6, and TNF-α in control subjects, SCI patients who were asymptomatic for pain, pressure ulcers or infection, and SCI patients being managed for these secondary complications. We also assayed serum concentrations of the anti-inflammatory cytokines IL-4 and IL-10 in a subset of SCI subjects. In addition to the cytokines, we quantified circulating levels of antibodies against the GM1 ganglioside (anti-GM1) (immunoglobulin [Ig] G, IgM) and myelin-associated glycoprotein (anti-MAG). The hypotheses we tested were that: (1) there is a subpopulation of people with SCI that has abnormally elevated levels of circulating proinflammatory cytokines in the absence of evidence of infection, and (2) that most people with SCI who are symptomatic for infection, severe neuropathic pain, or pressure ulcers have further elevated levels of serum proinflammatory cytokines.

Among the proinflammatory cytokines, IL-1β was selected for study because it is thought to play a major role in the initiation and early stages of inflammation. Its antagonist, IL-1RA, is an endogenous competitive antagonist for both IL-1β and IL-1α due to its affinity for the IL-1 receptor type I; binding of IL-1RA to the receptor does not result in signal transduction,24 although IL-1RA has also been reported to have its own agonist effects.25 We studied IL-2 because of its importance in immunoregulatory processes and its role in the development and proliferation of both T and B lymphocytes.26 IL-2 is also essential for maintenance of self-tolerance and control of autoimmune disease through induction of CD4+CD25+ T regulatory cell production.27

We chose TNF-α for the study because of its role as a mediator in inflammatory and immune function, endothelial cell function, and cytotoxicity.28 Both IL-2 and TNF-α have been implicated in axonal ion channel modulation and “channelopathy” in various neuroinflammatory diseases, and they may contribute to ongoing neurologic deficits after neurotrauma.29, 30, 31 IL-6 is a proinflammatory cytokine with several functions, including the modulation of proliferation, differentiation, and maturation of various hematopoietic cell types involved in the immune and inflammatory response to infection and injury.32 IL-6 has also been implicated in the development of neuropathic pain,33, 34 and has an important role in the development and repair of neurons and CNS tissue.35 In addition to the proinflammatory cytokines studied, IL-4 and IL-10 were assayed because of their anti-inflammatory properties, inhibition of proinflammatory cytokine production, involvement in neuroinflammatory processes, and modulation of glial cell activity.36 Several of these cytokines pass freely through the blood-brain or blood-cord barrier, and do so at an accelerated rate after SCI,37, 38 which prompted the hypothesis that immune-CNS signaling may contribute to the neurologic status of patients with SCI.17, 39 Antibodies to axolemma and myelin constituents, such as anti-GM1 and anti-MAG, have also been implicated in neurologic dysfunction.40

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Methods 

Participants 

Fifty-six people with SCI caused by trauma, and who were inpatients or outpatients of a regional SCI rehabilitation program (Parkwood Hospital and St. Joseph’s Health Care, London, ON), participated in this study. All subjects received standard treatment, including rehabilitation therapy tailored to their specific needs. Admission eligibility for the study included having an American Spinal Injury Association (ASIA) Impairment Scale classification of A, B, C, or D, a neurologic level of injury between C4 and T12, and medical stability. People with communicable diseases, a diagnosis of cancer in the last 5 years, or who were taking anti-inflammatory medications at the time of the study were excluded. The subjects’ clinical and demographic profiles are listed in table 1. People with nontraumatic etiologies, including transverse myelitis, epidural abscess, surgery, aneurysm, and arteriovenous malformation, were excluded from the study. Twenty-two of our subjects were in the postacute stage (2–52wk postinjury) and 34 had chronic SCI (>52wk).

Table 1. Demographic and Clinical Characteristics of Subjects
SubjectsN/(n)SexAge (y)ASIA ScaleTime Since Injury (mo)Neuropathic PainPressure UlcersUTI
MenWomenABCD
SCI subjects (all)56421440.6±11.91413227128±9925813
Paraplegia(25)19643.2±13.04894163±1191548
Tetraplegia(31)23839.7±11.6105133110±841045
Control35181735.1±9.8NANANANANANANANA

Abbreviation: NA, not applicable.

Our control subjects were 35 healthy able-bodied volunteers (18 men, 17 women) who ranged in age from 18 to 65 years. They were screened both to ensure that they had not had any recent infections and to determine their general health status. They were recruited from the University of Western Ontario student population, hospital staff, and community volunteers. All study subjects provided informed consent to participate and the Review Board for Health Sciences Research Involving Human Subjects at The University of Western Ontario in London, Ontario, approved the study.

Clinical Data Collection and Processing 

SCI subjects underwent a screening interview during which basic clinical and demographic information was obtained. Additional data were extracted from hospital charts. Patients with pain were screened to determine whether the pain was neuropathic or nociceptive41 and those with neuropathic pain completed the Neuropathic Pain Scale (we used an intensity rating of 1 to 10 for correlations).42 Symptomatic pressure ulcers (ie, stage 1 or greater) were quantified as present or absent in all subjects. Stage 1 was defined by “an area of non-blanchable erythema involving intact skin.”43 Symptomatic UTI evidenced by fever, spasticity, malaise, cloudy and malodorous urine, and bacterial counts greater than 100,000 organisms per milliliter of urine was also documented as present or absent in all subjects. Other medical complications or comorbidities were similarly documented, and any subjects with pre-existing disease or confounding medical conditions (eg, diabetes or any condition affecting the inflammatory response) aside from the secondary complications of neuropathic pain, pressure ulcers, and UTI were excluded. Eight SCI subjects had 2 of the 3 secondary complications.

Serum Collection 

Blood samples were drawn by standard venipuncture from all study participants between the hours of 9:00 am and 4:00 pm on the day of their first visit. No attempt was made to control for postprandial state or smoking status. Each whole blood sample was collected aseptically by a trained phlebotomist into 3 Vacutainer collection tubes,a 2 of which contained a serum separator and one that contained K3 ethylenediamine tetraacetic acid anticoagulant. The separator samples were allowed to clot for 30 minutes, and then centrifuged at 3400rpm for 10 to 15 minutes. The sera from 1 separator tube was separated into 5 plastic vials and frozen at −20°C or colder until just prior to immunoassay, per the enzyme-linked immunosorbent assay (ELISA) kit manufacturer’s specifications. The second separator tube and the anticoagulant tube were sent to a certified commercial medical laboratory for routine screening with chemistry and hematology panels.

Immunoassays 

ELISAs were used to detect cytokine levels and antibody levels in the sera using commercially available kits (IL-1β,b IL-1RA,b IL-6,b IL-2,c TNF-α,c IL-4,c IL-10,c anti-GM1 [IgG, IgM],c anti-MAGc). These assays use a quantitative sandwich assay technique to determine the concentration of the specific cytokine or autoantibody of interest. We used the manufacturers’ specified assay and analytic methods throughout. All samples were randomized and coded to ensure blinded quantification prior to assay, and completed in duplicate (multiple wells for each sample). Minimal detectable cytokine or autoantibody levels for each kit were: IL-1β (<1pg/mL), IL-1RA (<14pg/mL), IL-6 (<.70pg/mL), IL-2 (<1.10pg/mL), TNF-α (<4.8pg/mL), IL-4 (<2pg/mL), IL-10 (<1pg/mL), anti-GM1 (IgG, <210 Buhlmann titer units (BTU); IgM, <308 BTU), and anti-MAG (<89 BTU).

Duplicate readings for each standard, control, and sample were averaged and the average zero standard optical density reading was subtracted. A standard curve was created using a 4-parameter logistic curve fit. Concentration values were then determined, using this curve for control and sample average optical densities per the ELISA kit instructions. The Pearson product-moment correlation coefficient (r) was determined by comparing the known standard concentration with the curve fit; r was shown to be greater than .95 for all the assays.

Analytic Methods 

We initially used standard descriptive statistics (mean, standard deviation [SD], range) to characterize the data, and the Pearson product-moment correlations to identify association among normally distributed variables. The Bera-Jarque test of normality was used to test for normal distribution; where asymmetric distributions were evident, we used nonparametric descriptors, including the Spearman (ρ) coefficient of association among variables. We used a Kruskal-Wallis analysis of variance (ANOVA) test of ranks to compare group data in cases where assumptions of normality were violated. We used 2-tail probability (P) values to test the hypotheses that SCI subject group values exceeded control group cytokine concentrations. Multivariate analyses, including stepwise, forward solution, and multiple linear regressions, were used to model the distinguishing clinical features of SCI subjects with elevated levels of cytokines or autoantibodies (using a natural log transformation to constrain the variance and normalize the distribution).

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Results 

Hematology and Chemistry 

Select parameters from the hematology and chemistry panels are summarized in table 2. ANOVA revealed significantly (P<.01) reduced serum protein and albumin levels in subjects with tetraplegia and significantly (P<.05) reduced serum albumin levels in subjects with paraplegia, compared with control group values. Hemoglobin levels were not significantly reduced in subjects with paraplegia or tetraplegia compared with controls. SCI subjects with UTI or pain had protein levels significantly (P<.05) less than the control group values. Contrasts between SCI subjects with or without complications and between subjects with different ASIA classifications all yielded nonsignificant differences. Only 2 SCI subjects had abnormally low white blood cell (WBC) values (ie, below the standard laboratory reference value of <4×109/L) that are suggestive of immunosuppression. Both subjects were paraplegic; 1 had symptomatic UTI and the other was without medical complications. ACTH levels were above the laboratory reference range in only 3 of 40 SCI subjects, which indicates that the majority of subjects were not experiencing undue stress.

Table 2. Hematology and Blood Chemistry of Subjects
SubjectsN/(n)Red Blood Cells (1012/L)WBC (109/L)Neutrophils (109/L)Lymphocytes (109/L)Monocytes (109/L)Eosinophils (109/L)Basophils (109/L)Protein (g/L)Albumin (g/L)Hemoglobin (g/L)
SCI subjects (all)564.8±0.66.8±1.74.2±1.32.0±0.7.43±.17.23±.15.08±.0472.0±5.840.7±4.0⁎145.0±17.4†
Paraplegia(25)4.8±0.56.7±1.94.1±1.11.9±0.6.43±.20.23±.18.09±.0675.7±5.441.6±4.2⁎141.2±22.5
Complications(15)4.7±0.67.1±1.84.3±1.42.2±0.7.46±.26.32±.24.10±.0776.0±6.840.7±4.5139.2±25.8
No complications(10)4.8±0.56.2±1.93.8±0.71.7±0.6.41±.16.16±.08.08±.0575.5±4.743.0±3.5142.8±21.8
Tetraplegia(31)4.8±0.66.9±1.64.2±1.32.0±0.7.43±.16.22±.13.08±.0470.1±5.3†39.9±3.7†146.5±15.4
Complications(9)4.8±0.77.1±1.44.4±0.92.0±0.6.42±.14.22±.11.08±.0369.8±6.040.7±5.5146.1±17.8
No complications(22)4.8±0.56.9±1.64.1±1.52.0±0.8.44±.17.22±.14.08±.0470.3±5.239.6±2.8146.6±14.8
Control354.8±0.56.8±2.53.9±1.92.0±0.6.49±.20.15±.12.08±.0474.8±3.743.5±3.1143.7±13.6
Reference valuesMale4.2–5.74.0–11.01.8–7.01.0–3.2.00–.80.00–.40.00–.2064.0–81.034.0–48.0135.0–170.0
Female3.6–5.0115.0–155.0

Abbreviation: WBC, White blood cells.

⁎P<.05;

†P<.01.

Cytokine Assays 

The principal descriptive data for the serum cytokine concentrations of the control and SCI subject groups are summarized in table 3 and illustrated in figure 1. Table 3 reports the means and SDs. Because many of the cytokine concentrations were not normally distributed, nonparametric statistics (median and range) are also reported. Nonparametric statistics were also used for inferential analysis. No detectable levels of IL-1β or IL-10 were obtained from either the controls or the SCI subjects. The serum concentrations for IL-2, IL-6, TNF-α, and IL-1RA of control subjects were comparable to previously reported values for healthy adults.13, 44 Abnormal cytokine concentrations (> mean +3 SDs of control values) were evident in 14 (25%) of the 56 SCI subjects.

Table 3. Serum Cytokine Levels of Subjects
Subjects IL-2 (pg/mL)IL-6 (pg/mL)TNF-α (pg/mL)IL-1RA (pg/mL)IL-4 (pg/mL)IL-10 (pg/mL)
SCI subjects (all) (N=56)Mean16.8±72.86.8±13.5⁎51.1±129.1⁎608.7±849.2⁎1.3±2.00.0±0.0
Median0.03.80.0321.40.30.0
Range0.0−409.50.0−89.20.0−728.658.1−3948.40.0−7.40.0−0.0
Paraplegia (n=25)Mean1.5±3.18.9±17.8⁎40.0±51.9⁎607.5±781.52.9±3.00.0±0.0
Median0.04.117.3346.21.70.0
Range0.0−8.30.0−89.20.0−143.9169.1−3668.40.8−7.40.0−0.0
Tetraplegia (n=31)Mean21.4±82.85.1±8.1⁎54.5±145.0⁎609.8±917.50.9±1.60.0±0.0
Median0.00.00.0273.10.00.0
Range0.0−409.50.0−32.60.0−728.658.1−3948.40.0−4.40.0−0.0
Control (N=35)Mean1.1±4.72.0±2.36.4±17.1331.2±262.00.6±0.80.0±0.0
Median0.00.80.0193.10.10.0
Range0.0−25.90.0−6.00.0−81.3114.0−906.80.0−2.50.0−0.0

⁎P<.05.

  • View full-size image.
  • Fig 1. 

    Serum cytokine concentrations for control subjects and SCI patients (all). (A) IL-2, (B) IL-6, (C) TNF-α, and (D) IL-1RA. Note that all serum cytokine concentrations have been transformed to their natural logarithm (In) values. *P<.05 (Kruskal-Wallis ANOVA).

Interleukin-2 

Kruskal-Wallis ANOVA did not show a significant difference between all SCI subjects and controls, and there were no evident differences among controls, subjects with tetraplegia, and subjects with paraplegia (see fig 1A). Analyses comparing the control group, SCI group without complications, and SCI subjects with pain, UTI, or pressure ulcers (treated as separate variables), did not detect any significant differences.

Interleukin-6 

The Kruskal-Wallis ANOVA yielded significant (P<.05) differences among all study subjects for IL-6 concentrations. Subjects with paraplegia had the highest mean concentration, with subjects with tetraplegia having intermediate concentrations; collectively, the SCI patients’ values were higher than the control group values (P<.05). This is evident in figure 1B, where the data are illustrated using a natural log transformation to constrain the variance. Kruskal-Wallis ANOVA also showed that SCI subjects with complications had higher concentrations (P<.01) than did the controls and the subjects without complications. More specific analysis showed that SCI subjects with either pain, UTI, or pressure ulcers had significantly (P<.01) higher concentrations than did the subjects without complications and the control subjects. These relationships are evident in figure 2A; again, the data have been transformed to their natural log values.

  • View full-size image.
  • Fig 2. 

    Serum cytokine concentrations of (A) IL-6 and (B) IL-1RA for control subjects and SCI subjects categorized according to the presence or absence of the medical complication of UTI, pressure ulcers, or pain. Note that all serum cytokine concentrations have been transformed to their natural logarithm (In) values. *P<.05 (Kruskal-Wallis ANOVA).

Tumor Necrosis Factor Alpha 

There was a significant (P<.05) difference between control subjects and all SCI subjects in TNF-α levels (fig 1C), and among the control, paraplegia, and tetraplegia groups (P<.05). Kruskal-Wallis ANOVA showed the subjects with tetraplegia had the highest levels, followed by the paraplegic subjects, and then the controls. SCI subjects with complications tended to have higher levels than the other subjects and the controls, although this difference was not significant (P=.11).

IL-1 Receptor Antagonist 

There was a significant difference (P<.05) in IL-1RA concentrations between control subjects and all SCI subjects (fig 1D). There was no significant difference between the paraplegic and tetraplegic subjects. Kruskal-Wallis ANOVA also revealed significantly (P<.01) higher concentrations in SCI subjects with pain or UTI compared with the other patients and the control group (fig 2B).

Anti-GM1 

Kruskal-Wallis ANOVA revealed that anti-GM1 IgG levels were greater in all SCI subjects than in the controls (P<.05) (fig 3). The patients with complications had higher serum levels than did those without complications and the controls (P<.05). Within this group, SCI patients with UTI (P<.05), and pain (P<.05), but not pressure ulcers (P>.05), had greater serum levels than either the patients without complications or the controls.

Anti-MAG 

There were no significant differences in serum anti-MAG levels among controls and all SCI subjects, or among control subjects, subjects with tetraplegia, and subjects with paraplegia. There were no differences among SCI subjects with or without complications and the control subjects. Table 4 summarizes the anti-MAG and anti-GM1 results.

Table 4. Serum Autoantibody Levels of Subjects
Subjects Anti-GM1 IgG (BTU)Anti-GM1 IgM (BTU)Anti-MAG (BTU)
SCI subjects (all) (N=56)Mean1735.7±2109.2⁎1092.1±2137.1602.4±277.5
Median887.4538.1566.4
Range0.0−6725.10.0−12724.6120.9−1838.0
Paraplegia (n=25)Mean1786.9±2324.3715.4±578.1713.1±476.9
Median758.9533.1623.5
Range0.0−6694.20.0−1777.1120.9−1838.0
Tetraplegia (n=31)Mean1722.5±2097.41189.8±2381.5569.2±182.2
Median888.9541.1555.7
Range0.0−6725.10.0−12724.6228.2−1126.6
Control (N=35)Mean1025.3±1095.9710.8±273.1610.4±146.2
Median702.6634.3555.0
Range0.0−4513.50.0−1028.7478.2−1299.4

Abbreviation: BTU, Buhlmann titer units.

⁎P<.05.

Correlational Analysis 

Table 5 shows the Spearman ρ correlation coefficients for hematologic and cytokine data. There were significant (P<.02) correlations between IL-6 concentration and WBC counts (ρ=.30) and IL-6 and neutrophil counts (ρ=.29). IL-1RA was significantly (P<.01) associated with WBC (ρ=.40), neutrophil counts (ρ=.38), and monocyte counts (ρ=.30), and negatively associated with albumin levels (ρ=−.23). IL-2 and IL-4 concentrations did not correlate significantly with any of the hematologic or chemistry values. Albumin levels correlated positively with hemoglobin levels (ρ=.28), and protein (ρ=.73) and negatively with eosinophil counts (ρ=−.29). There was no significant correlation between time since injury (in months) and any of the cytokine or autoantibody levels.

Table 5. Cytokine and Clinical Data Correlations
AgeWBCNeutrophilsLymphocytesMonocytes
IL-2.08.08.07.18–.21
IL-4–.08–.04–.13.11–.19
IL-6.15.30⁎.29⁎.11–.05
TNF-α.06–.14–.07–.12–.22
IL-1RA.37†.40†.38†.08.30†

⁎P<.02;

†P<.01.

Multivariate Models of Cytokine Concentrations 

A series of forward and backward solution, stepwise, linear regression models was developed to characterize the individual cytokine concentrations, based on combinations of clinical neurologic variables. The intent was to identify the clinical characteristics of those people most likely to have elevated levels of circulating proinflammatory cytokines. The variables included time since injury, presence of complications, paraplegia versus tetraplegia, and ASIA classification. Only the IL-6 concentration was successfully modeled, that is, greater than a univariate regression, and yielded the function:

where Y is the natural logarithm transform of IL-6 concentration; x1 is the presence or absence of secondary complications (0,1); and x2 is time since injury (in months). (Note that the regression [β] coefficients were not standardized.) The variance explained by the model (R2) was 48.5%. The model expands on the nature of the regression implicit in figure 2A, showing differences in IL-6 concentrations among controls, SCI subjects without complications, and those with pain, UTI, or pressure ulcers.

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Discussion 

We undertook this study to characterize the serum cytokine and autoantibody profiles of people with SCI and control subjects. In particular, we sought to confirm and extend previous reports of abnormally elevated levels of circulating proinflammatory cytokines in SCI subjects with or without medical complications. We attempted to determine the clinical (neurologic and medical complication), hematologic, and blood chemistry correlates of elevated levels of circulating cytokines. In addition, we sought to obtain additional information relevant to the hypothesis that proinflammatory cytokines and autoantibodies contribute to neurologic deficits in some SCI subjects.17

The results showed significantly elevated levels of IL-6 and TNF-α in SCI subjects compared with control group levels. A quarter of the SCI subjects had elevated levels in 1 or more of the proinflammatory cytokines. In contrast, only 4 of 23 SCI subjects had elevated levels of the anti-inflammatory cytokine IL-4 (there were no subjects, control or SCI, with detectable levels of IL-1β or IL-10). Circulating IL-1RA levels of SCI subjects were also elevated (6/52) over those of the control subjects, and concentrations of anti-GM1 (IgG) were elevated in 5 of 40 SCI subjects. Several SCI subjects had low levels of protein, albumin, and/or hemoglobin, although only 2 had WBC counts below the laboratory reference values for the normative range; none of these subjects had elevated serum levels of ACTH.

The fact that appreciable numbers of SCI subjects have abnormally high levels of proinflammatory cytokines in the absence of IL-4 and IL-10, whether or not they are symptomatic for infection, may be indicative of a T helper cell 1 (Th1) biased immune status.36 The Th1:Th2 paradigm is emerging as a helpful concept in understanding disease states,45 however, it can only properly be evaluated by detection of cytokine-producing cells using flow cytometry.46 Chronic Th1-like low level immune activation has been indicated in other disease states in which subjects have been exposed to prolonged and/or extreme stress and who, like SCI subjects, have multisystem illness.46 A possible link between chronic immune activation, cytokine production, and the multiorgan pathologic sequelae of SCI has been theorized previously.47, 48

There was little evidence of immunosuppression in our sample of SCI subjects, based on the hematologic and serologic markers studied. This may be because of the small proportion (14/56) of subjects with high-level lesions, that is, tetraplegia and complete functional loss (ASIA class A). Previous reports of immunosuppression in SCI subjects have been largely based on ASIA class A subjects with tetraplegia, a presumed complete disruption of sympathetic outflow to lymphoid tissue, and deafferentation of immunoregulatory neurons in the CNS.12

SCI is not the only chronic condition typically associated with immunosuppression12 in which there are elevated levels of circulating proinflammatory cytokines (eg, IL-6, TNF-α).13, 14, 15, 16 During the course of infection with human immunodeficiency virus, secretion of the Th1 cytokines IL-2 (critical for activation-induced cell death in T cells49 and protective functions of regulatory CD4+CD25+ T cells50) and antiviral interferon-gamma is decreased, whereas production of the proinflammatory cytokines IL-1, IL-6, IL-8, and TNF-α is increased.51 This abnormal cytokine profile most likely leads to a disruption of the normal cytokine-mediated control of homeostasis of the immune system, that is, the continuous reciprocal balance between cell-mediated immunity and humoral immunity.52 Disruption of this balance leads to immune dysfunction and immunopathogenesis. In the case of SCI, this may contribute to reported reductions in NK cell function,3, 11 various indications of low grade chronic inflammatory processes, and some of the evident abnormal autoimmunity reported in this study and elsewhere.17, 53

A concept of “protective autoimmunity” has been developed in the context of SCI54 and is relevant to our observations of autoreactivity (elevated anti-GM1 IgG) in SCI patients. Controlled autoimmune responsiveness is considered to represent an endogenous and beneficial response to CNS insult.54, 55, 56, 57, 58 T cell−dependent increases in neuronal survival are observed after transfer of myelin basic protein (MBP)−reactive T cells in animal models of SCI,58 and were reduced in animals immunized with myelin proteins as neonates.59 Activation of MBP-reactive T cells is evident in chronic SCI patients.60 CD4+CD25+ regulatory T cells regulate these processes by maintaining a balance between beneficial and destructive autoimmunity, although this equilibrium may be precluded by a genetic predisposition to autoimmune disease.61 Such a predisposition may account for the acute, disseminated, encephalomyelitis that followed cord trauma that was evident in our SCI subject who presented with the highest circulating levels of IL-6 and elevated levels of anti-MAG and anti-GM1 (IgM). Increased effector T-cell activity, coupled with decreased regulatory T-cell activity, would be expected to enhance neuronal survival after an SCI, and be reflected as increases in Th1 and proinflammatory cytokine activity in the absence of anti-inflammatory (Th2) cytokines. The presence of persisting inflammatory processes may, however, prevent functional restoration60 due to structural62, 63 or channelopathic17, 29, 30 contributions to axonal dysfunction in surviving tissue.

Our results indicate that symptomatic UTI is associated with reduced protein levels and elevated serum concentrations of IL-6 and IL-1RA. Their circulating levels were elevated compared with the values of the SCI subjects without complications and the control subjects. These results are consistent with previous reports of elevated levels of serum IL-6 in bacteremic patients with febrile UTI who were compared with nonbacteremic patients.64 Serum IL-6 levels have been shown to be further elevated in patients with clinical signs of pyelonephritis.64 It is likely that IL-6 production in the urinary tract triggers the systemic host response and this may occur even in the absence of bacteremia. IL-1RA serum levels (as well as IL-10 and soluble TNF receptor types I and II) have also been reported in patients with culture-proven UTI. These patients did not have elevated levels of IL-1RA in urine, thus prompting the suggestion that during urosepsis the anti-inflammatory cytokine response is principally systemic. Our observation that UTI is associated with elevated serum IL-1RA levels in SCI patients is consistent with previous reports of a systemic host response to localized UTI.

In our study, the reporting of neuropathic-type pain was associated with elevated levels of circulating IL-6 and IL-1RA. SCI subjects with pain had higher serum levels of IL-6 and IL-1RA than either asymptomatic SCI subjects or the controls. IL-6 has been implicated in the pathogenesis of neuropathic pain, although its role has yet to be fully elucidated;33, 34, 65 there are several possible neuromodulatory mechanisms in the CNS and the peripheral nervous system. Circulating levels of IL-6 are elevated in patients who have undergone diskectomy but have persistent sciatic pain66 and in patients with peripheral nerve injury,67 which indicates systemic responses. DeLeo et al68 have shown in rats that IL-1, IL-6, and TNF-α are also expressed by cells in the spinal cord as a result of sciatic nerve injury. Exogenous recombinant IL-6, administered intrathecally, produced touch-evoked allodynia (increased sensitivity to a non-noxious stimulus) as well as thermal hyperalgesia. Exogenous IL-6 administered to the exposed cord produced a dose-related inhibition of electrically evoked C-fiber activity and other measures of neuronal hyperexcitability, as well as mechanical allodynia. Collectively, these results point to differential actions of IL-6 alone, or in association with a cascade of other cytokine activity, at various locations such as the actual site of tissue damage or within afferent neuronal pathways and second-order afferent nociceptive signal processing pathways.

In our study, serum IL-6 levels were also elevated in SCI subjects with pressure ulcers when compared with the SCI subjects who were asymptomatic for secondary complications and the controls. Protein and albumin concentrations were reduced in this group and in asymptomatic SCI subjects, relative to those concentrations in the control subjects. These findings are consistent with previous reports of elevated serum levels of IL-6 of a localized origin, but not IL-1 or TNF-α, in elderly, non-SCI people with pressure ulcers.69 Within the SCI population, the soluble IL-2R has also been shown to be elevated in the serum of patients with slow-healing pressure ulcers, and numeric increases in IL-6 were also reported but did not achieve significance.20 IL-6, as well as IL-1α, IL-1β, TNF-α, and several growth factors are present in extremely high concentrations within fluid extracted from chronic, persistent wounds; decreases in these concentrations have been associated with wound healing.70, 71 It appears that high levels of proinflammatory cytokines, such as IL-6, are associated with the inflammatory and infectious processes of pressure ulcers, and may be inhibitory to tissue restoration per se.

Certain circulating cytokines (eg, TNF-α) pass through the normal blood-brain and blood-cord barrier via active transport, and do so at an accelerated rate after SCI.72 Cytokines also enter the CNS in regions where the barrier is absent or less restrictive, such as in the choroid plexus or circumventricular organs. The presence of elevated cytokines in the circulation and the periphery has also been associated with vagal nerve stimulation and subsequent activation of neural networks, as well as glial cell activation and cytokine production within the CNS. All of these pathways represent putative routes of immune-to-CNS signaling and neuromodulation,73 yet the specific mechanisms by which they influence neuronal activity remain unclear.

An emergent concept is that cytokines within the CNS have normal physiologic, neuromodulatory functions,74 but when their concentrations exceed a certain level they contribute to conduction deficits (axonal channelopathy) and eventually cytotoxicity (eg, oligodendrogliopathy).63 Although these concepts have developed largely around neuroinflammatory diseases, they may also be applicable to SCI.75, 76 Thus, elevated levels of circulating cytokines associated with protective immunity, or with medical complications, may actually induce additional cytokine responses intrinsic to the cord,77 and cause axonal dysfunction that compounds the neurologic deficit resulting from trauma-induced axonopathy and myelinopathy.29 Such cytokine-mediated alteration in axonal conduction in the mammalian spinal cord that has recently been demonstrated supports this possibility.29 We have also reported preliminary evidence that certain proinflammatory cytokines (IL-6, TNF-α) are present in the cerebrospinal fluid (CSF) of people with long-standing SCI who undergo surgery for syringomyelia or detethering at elevated levels comparable to those seen in neuroinflammatory disease.78

Within the rehabilitation context, management of pressure ulcers, neuropathic pain, and UTI continues to be a considerable challenge. It is unclear from the present cross-sectional analysis whether the elevated serum cytokine levels are contributing to the complications, or are a consequence of infection and the accompanying inflammatory response. It is notable that elevated proinflammatory cytokine concentrations appear to be reduced as wounds heal. Many of the asymptomatic subjects in our study had a history of pressure ulcers that had resolved; this group had lower cytokine levels than symptomatic patients. It does seem likely that effective management of the secondary complications will result in lowered serum cytokine concentrations and thereby mitigate any possible contribution to immune-mediated neurologic deficits.

The relatively short half life of cytokines in vivo79 suggest that the evident immunoactivity is associated with current inflammatory events. It may also indicate that the measured concentrations actually underestimate the levels closer to the site of the inflammation. Clearly, longitudinal studies of serum (and CSF) cytokine concentrations during the clinical course of infections and pressure ulcers will help elucidate these issues.

In summary, our results showed elevated circulating levels of IL-6, TNF-α, IL-1RA, and anti-GM1 (IgG) in SCI subjects in comparison with healthy able-bodied controls. They provide evidence of a chronic low level of immunoactivity and autoreactivity in SCI subjects with paraplegia and tetraplegia of various ASIA classifications and who are asymptomatic for pain, UTI, or pressure ulcers. The immunoactivity is further elevated in SCI subjects who are being treated for these secondary complications. The functional and clinical significance of these results remains uncertain; they may be reflective of a beneficial “protective autoimmunity,” or conversely, indicative of a process in which peripherally circulating cytokines penetrate the blood-cord barrier and, in association with locally produced cytokines, modify ion channel conductance in glia or axons, thereby contributing to axonal dysfunction.

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Conclusions 

Proinflammatory cytokines and autoantibodies are elevated in the serum of subjects with SCI who are asymptomatic for secondary complications, and are further elevated in people with symptomatic neuropathic pain, pressure ulcers, or UTI, in comparison with healthy able-bodied control subjects. Serum protein and albumin were also reduced in subjects with SCI, again in comparison with controls.

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Acknowledgments 

We gratefully acknowledge the assistance of Cindy Moniz, RN, George McCullagh, Heather Askes, BSc, Bonita Stevenson, and Patrick Potter, MD, FRCPC.

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  • a BD Canada, 2771 Bristol Cir, Oakville, ON I6H 6R5, Canada.
  • b R&D Systems Inc, 614 McKinley Place NE, Minneapolis, MN 55413.
  • c ALPCO Diagnostics, 26-G Keewaydin Dr, Salem, NH 03079.

 Supported by the Parkwood Hospital Foundation and the Ontario Neurotrauma Foundation.

 No commercial party having a direct financial interest in the results of the research supporting this article has or will confer a benefit upon the author(s) or upon any organization with which the author(s) is/are associated.

PII: S0003-9993(07)01342-1

doi:10.1016/j.apmr.2007.08.004

Archives of Physical Medicine and Rehabilitation
Volume 88, Issue 11 , Pages 1384-1393, November 2007

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