Cannabinoid influence on cytokine profile in multiple sclerosis

Abstract

Cannabinoids have been suggested as possessing immunomodulatory properties, and cannabinoid receptors are present on leucocytes. Clinically, there is some evidence that cannabinoids may be therapeutically useful in treating multiple sclerosis, which is generally believed to be an autoimmune condition. This paper reports data derived from the Cannabinoids in MS (CAMS) study, which was the largest randomized controlled trial yet conducted to evaluate the therapeutic efficacy of cannabinoids. We found no evidence for cannabinoid influence on serum levels of interferon (IFN)-γ, interleukin (IL)-10, IL-12 or C-reactive protein as measured using enzyme-linked immunosorbent assay (ELISA), in comparison to control values. Mitogenic stimulation experiments also failed to demonstrate any significant reduction in percentage of CD3⁺, IFN-γ producing cells after exposure to cannabinoids in vivo, although numbers were small. Further work is needed to establish the functional significance of cannabinoid receptors on immune cells.

Introduction

Multiple sclerosis (MS) is the most common cause of neurological disability in young adults, with a prevalence of around one in 800 in most of the United Kingdom [1]. While the precise aetiology of MS is unknown, there is much evidence implicating an autoimmune mechanism, with central nervous system white matter tracts providing the focus for inflammation and demyelination. It has been suggested that MS is predominantly a Th1-mediated disease, with good evidence that interferon (IFN)-γ makes the disease course worse [2]. Repeated inflammatory attack eventually provokes axonal loss and glial scarring, which constitute the neuropathological basis for chronic disability and progressive disease. Most MS starts with repeated clinical episodes (relapsing-remitting MS, RRMS). Unfortunately, after passing through the RRMS phase, the majority of patients will ultimately develop chronic progressive clinical decline (secondary progressive MS, SPMS, approximately 80% after 20 years). About 15% of patients may follow a progressive course from onset (primary progressive MS, PPMS).

Treatments for MS can broadly be divided into symptomatic (such as muscle relaxants and bladder treatments) and so-called disease modifying, most of which are immunomodulatory, such as beta-interferons and glatiramer acetate. The recent Cannabinoids in MS (CAMS) study [3] was designed to evaluate whether cannabinoids had any therapeutic benefit in treating MS-related symptoms, and randomized 657 patients to an oral extract of cannabis, Δ⁹-tetrahydrocannabinol (Δ⁹-THC), believed to be the major active ingredient of cannabis, or the respective placebo capsules. Although cannabinoids have been used mainly to alleviate symptoms, there is experimental evidence to suggest that they may be immunomodulatory. Cannabinoids have been reported to suppress experimental allergic encephalomyelitis (EAE) [4,5], reduce Th1 responses in mouse models of infection [6] and high concentrations (5–20 µg/ml) of Δ⁹-THC and cannabidiol (CBD) block IFN-γ release in vitro[7]. However, physiologically relevant concentrations comparable to plasma levels after smoking marijuana (10–100 ng/ml) may increase IFN-γ levels in vitro[7] and recent evidence from a small cross-over study in humans has suggested that cannabinoids may even have some proinflammatory effects [8].

We investigated the nature of potential cannabinoid immunomodulation on samples obtained from patients with MS taking part in the CAMS study, and report the results here.

Materials and methods

Patients

Patients with stable MS were recruited into a double-blind placebo-controlled trial looking at the effect of oral cannabinoids on spasticity, the CAMS study [3]. They received pure synthesized Δ⁹-THC (Marinol), natural cannabis oil extract (Cannador) and matching placebo in equally divided groups. The cannabis oil extract contained equivalent dose of Δ⁹-THC per capsule as the synthetic Δ⁹-THC, but with other components of natural cannabis, the other major cannabinoid being CBD. Doses were adjusted according to side effects, with maximal oral dose of 0·25 mg/kg/day of Δ⁹-THC. Patients from two centres (Plymouth and National Hospital, Queen Square, London) provided blood samples for further laboratory analysis. The full inclusion and exclusion criteria have been published elsewhere [3], but patients were required to have had stable MS for the 6 months and no corticosteroids for 30 days prior to study entry. Any relapses or corticosteroid medication during the study were recorded.

Blood samples

Serum was available from patients taken before treatment and at the end of treatment, 13 weeks later for cytokine and C-reactive protein (CRP) measurements. Because timing of clinic attendance varied between patients, it was not possible to obtain samples at the same point after medication in all patients, although wherever possible each patient attended at the same time of day at each clinic visit. The relatively long half-life of cannabinoids and pharmacokinetic modelling suggested that after a number of weeks patients would achieve a steady state of serum cannabinoid levels, so we would not expect short-term confounding of any longer-term effects. Lithium heparin blood samples were taken for cell culture at visits 2 and 7. Controls were obtained from random anonymized patient samples sent to the immunology laboratory for routine immunological analyses. Peripheral blood mononuclear cells (PBMC) were isolated within 12 h of blood sampling (see below).

Measurement of serum cytokines [IFN-γ, interleukin (IL)-10 and IL-12]

Serum cytokine levels (IFN-γ, IL-10 and IL-12) were measured using commercially available, highly sensitive enzyme-linked immunosorbent assays (ELISAs) (Insight Biotechnology Limited, Wembley, UK). Cytokine levels were measured in pg/ml.

Measurement of CRP

CRP levels were measured as a marker of concurrent infection and a surrogate marker of IL-6. CRP was measured on serum samples remaining after cytokine assays on a MIRAS analyser using a routine immunoturbidimetric assay capable of measuring levels as low as 2 mg/ml [9].

Quantification of numbers of cytokine-secreting cells after stimulation in vitro

As described previously [10], PBMC were isolated by density gradient centrifugation, washed and resuspended at a concentration of 1 × 10⁶ cells/ml in culture medium in duplicate in 24-well culture plates. Phorbol myristate acetate (PMA), ionomycin and monensin were added to the wells to give final concentrations of 20 ng/ml, 1 µ M/ml and 3 µg/ml, respectively. Control wells contained monensin only. The plates were placed in a humidified incubator at 37°C, 5% CO₂ for 7 h. The cells were subsequently harvested, washed, fixed and permeabilized using a commercial kit (Dako Intrastain; Becton-Dickinson, Oxford, UK). Cells were labelled with surface FITC CD3 and cytoplasmic phycoerythrin (PE) IFN-γ. Murine surface IgG1 fluorescein isothiocyanate (FITC) and murine cytoplasmic IgG1 PE monoclonal antibodies were used as controls. Numbers of IFN-γ-expressing T cells were quantified by two-colour flow cytometry. Measurements were taken in duplicate using samples from each of the cell culture wells. The proportion of cells expressing surface CD3 that also expressed cytoplasmic IFN-γ was calculated.

Addition of THC to cell cultures

Initial experiments using THC produced by Sigma (Gillingham, Dorset, UK), dissolved in ethanol and added in increasing concentrations, showed that only concentrations above 40 µg/ml caused a significant fall in the percentage of IFN-γ-producing T cells. This dose of Δ⁹-THC was then used to see whether any reduction of IFN-γ positive cells could be achieved in vitro using patient cells from the end of the treatment period.

Statistical analysis

All experiments and data collection were completed prior to unblinding. Analysis was performed using SPSS (Chicago, IL, USA) with appropriate parametric and non-parametric tests.

The study was approved by the South-West multi-centre research ethics committee and was undertaken under licence from the UK Home Office. All participants provided written informed consent.

Results

A total of 130 patients were recruited to the main CAMS study from the two centres (Plymouth, 79; Queen Square, 51) after the initial pilot phase of 20 patients had been recruited in Plymouth. Samples were available from 102 patients, two of whom did not commence medication. The characteristics of the 100 patient cohort were broadly similar to the whole CAMS cohort [3], so that mean age was 52 (SD 7·7), 51% were unable to walk, 75% were female, 25% had PPMS and 75% had SPMS. The disposition of patients according to treatment group and disease type is shown in Table 1. Only two patients in this cohort had a relapse treated with 1 g methylprednisolone for 3 days during the study period, one in each of the cannabis extract and placebo groups.

Table 1.

Cross-tabulation of treatment (CE = cannabis extract) against disease type (PPMS = primary progressive MS, SPMS = secondary progressive MS).

	Disease
	PPMS	SPMS	Total
CE	10	23	33
Δ9THC	4	28	32
Placebo	12	23	35
Total	26	74	100

Effect of THC and cannabis extract on serum cytokines and CRP

Using Kruskal–Wallis non-parametric evaluation, there was no significant detectable difference between pretreatment and treatment levels of serum IFN-γ, IL-10, IL-12, or CRP levels (Table 2). Grouping the two active treatment groups together gave similar results and there were no significant treatment effects on measured cytokines when the cohort was analysed according to disease type (PPMS or SPMS). Results of cytokine change according to disease group are shown in Figs 1 and 2. Serum IFN-γ, IL-10 and IL-12 levels correlated with each other with Kendall and Spearman correlation coefficients > 0·35 with all P-values < 0·001.

Table 2.

Number of pairs for Kruskal–Wallis analysis and P-values.

	Placebo	Cannabis extract	THC	P-values
IFN-γ	22	22	22	0·692
IL-10	21	21	20	0·608
IL-12	18	21	19	0·816
CRP	17	19	19	0·271

Fig. 1.

Box-plot presentation of data from patients with primary progressive MS for change in interferon (IFN)-γ (a), interleukin (IL)-10 (b), IL-12 (c) and CRP (d). The asterisk represents an extreme value more than three interquartial ranges from the box hinges.

Fig. 2.

Box-plot presentation of data from secondary progressive patients for change in interferon (IFN)-γ (a), interleukin (IL)-10 (b), IL-12 (c) and CRP (d). Asterisks represent extreme values more than three interquartial ranges from the box hinges; open circles represent outliers more than 15 interquartial ranges from the box hinges.

Comparison between control and MS serum cytokine levels

Using the Mann–Whitney U-test and Wilcoxon matched pairs, there was no evidence of a significant difference in serum cytokine levels between controls and MS patients (Table 3).

Table 3.

Mann–Whitney U-test comparing serum cytokine results in vivo between MS patients and controls.

	Mean rank (total number of patients)
	Serum IFN-γ	Serum IL-10	Serum IL-12
MS patients	69 (102)	52 (75)	51 (74)
Controls	55 (29)	64 (34)	45 (34)
Sig. (two tailed)	0·083	0·066	0·301

Effect of in vivo THC and Marinol on in vitro IFN-γ expression in CD3⁺ T cells

Data from 20 patients was available before and during treatment for analysis of the effect of IFN-γ secretion following co-incubation of patients cells with Δ⁹-THC (placebo n = 6, cannabis extract n = 7, Δ⁹-THC n = 7) and results are shown in Fig. 3. Although five of seven patients treated with cannabis extract and six of seven patients treated with Δ⁹-THC showed a decrease in IFN-γ before and after treatment, compared to only two of six patients in the placebo group, these differences were not statistically significant. Addition of Δ⁹-THC to cells in vitro following treatment at a concentration of 40 µg/ml achieved a further small reduction of IFN-γ in all cases across all three groups. A paired-samples test showed a mean reduction of 2·31% (95% CI, 0·14–4·49), P = 0·04. A representative FACS plot is included (Fig. 4).

Fig. 3.

Percentage of CD3 positive cells expressing interferon (IFN)-γ either pretreatment (pre) or during treatment (post) with 95% CI. CE = cannabis extract, THC = Δ⁹-THC, Plac = placebo).

Fig. 4.

Flow cytometric profile of frequencies of CD3⁺ T cell producing interferon (IFN)-γ. Peripheral blood mononuclear cells (PBMC) were cultured with progressive multiple sclerosis (PMS), ionomycin and monensin for 7 h, fixed and permeabilized.

Discussion

Results from the present experiments do not demonstrate any significant effects of cannabinoids on the cytokine profiles examined. However, numbers are small and standard deviations large, so that relatively small but possibly clinically useful effects cannot be excluded from these results. The small numbers also make it very difficult to distinguish any differences between cytokine profiles in primary and secondary progressive disease patterns. Although we did demonstrate a small reduction of IFN-γ expression in CD3⁺ T-cells in vitro at very high concentrations of THC (several orders of magnitude higher than measured serum levels), this suggests that there is unlikely to be any significant effect on IFN-γ at therapeutic doses.

The majority of cannabinoid actions are thought to be mediated via cannabinoid receptors, two types of which have been identified and cloned. CB₁ receptors [11] are found predominantly in the central nervous system whereas CB₂ receptors [12] are principally located peripherally, especially on leucocytes. The majority of experimental evidence suggests that cannabinoids are anti-inflammatory and may be associated with a Th1 to Th2 shift. For example, in vitroΔ⁹THC suppresses T cell proliferation, inhibits the production of IFN-γ and shifts Th1 to Th2 [13]. This may be mediated via a reduction in IL-12 [14]. TNF-α release from microglia has also been shown to reduce after cannabinoid exposure in vitro[15]. Both in rat EAE [4,5] and Theiler's murine encephalomyelitis virus affected mouse models [16], cannabinoids appear to ameliorate the effects of the disease by reducing Th1 effector functions and inhibiting central nervous system (CNS) expression of mRNA coding for proinflammatory cytokines TNF-α, IL-1β and IL-6. It should be noted, however, that doses of Δ⁹-THC used in animal experiments have tended to be between 8 and 40 mg/kg, compared to human doses of around 0·25 mg/kg. Similarly, levels used in vitro have been 1–40 µg/ml (3–127 µ M) [4,13], whereas serum levels in humans following oral dosing is rarely higher than 5 ng/ml (16 n M, unpublished data). At a receptor level, the K_i of Δ⁹-THC is around 40 nM at both CB₁ and CB₂ receptors [17], so doses used in vitro would seem to be excessive. The correlation between serum level and symptomatic or immunological effect is the focus of ongoing work. The data are difficult to interpret as high lipid solubility of this group of drugs means that serum levels may not necessarily reflect therapeutic doses, due to concentration within particular compartments such as the central nervous system.

These differences in doses may explain partly why the situation in humans appears to be more complicated than experimental data would suggest. In people smoking cannabis, levels of both CB₁ and CB₂ receptor mRNA increase on PBMC, with an apparently preserved ratio of 1CB₁ : 3CB₂[18]. Cannabis use has also been associated with a decrease in IL-2 levels and Th1-type cytokine profile and an increase in IL-10 and Th2 cytokines [19]. However, in the only other study examining cytokines in people with MS treated with cannabinoids [8], although no change was seen in plasma levels of TNF-α, IL-10 and IL-12, after LPS stimulation of whole blood the authors reported a slight increase in TNF-α production in patients taking whole cannabis extract and not THC. It is difficult to assess the significance of these results, as numbers were small and no adjustments were made for multiple comparisons. The fact that anti-TNF therapy has been shown to worsen the course of MS [20] suggests that classical concepts of proinflammatory cytokines being detrimental to the disease course may be an over-simplification of disease pathogenesis. Overall, the bulk of evidence would suggest that cannabinoids are anti-inflammatory. Interestingly, there has been a recent report of a new diagnosis of MS occurring following treatment with a cannabinoid receptor-1 antagonist for obesity [21], and longer-term follow-up results from the CAMS study suggest that cannabinoids may have more than just a symptomatic role in the treatment of MS [22]. More work is now needed both to determine the functional significance of cannabinoid receptors on cells of the immune system, and to investigate whether cannabinoids have an immunomodulatory role at doses likely to be used therapeutically.