GM-CSF production by CD4+ T cells in MS patients: regulation by regulatory T cells and vitamin D.

BACKGROUND/OBJECTIVE: Data from animal models of MS suggest that GM-CSF(+)CD4(+)T cells are pathogenic cells. Therefore, GM-CSF production by CD4(+)T cells of MS patients and their susceptibility to regulatory mechanisms were investigated. METHODS: Intracellular flowcytometry was performed to determine the GM-CSF(+)CD4(+)T cell fraction in PBMC and CSF of MS patients and controls. The effect of regulatory T cells (Tregs) on GM-CSF production by CD4(+)T cells was studied in MS patients using a proliferation-suppression assay. Finally, GM-CSF(+)CD4(+)T cell fraction and GM-CSF protein levels in supernatant were assessed in anti-CD3-stimulated CD4(+)T cell cultures derived from healthy controls and MS patients, in the presence or absence of the active vitamin D metabolite calcitriol. RESULTS: The GM-CSF(+)CD4(+)T cell fraction in the peripheral blood did not differ between controls and MS patients. This T cell population could also be detected in the CSF of both subjects with MS as well as subjects with another diagnosis. In the CSF, it comprised a significant fraction of the T cell population. Upon in vitro stimulation of PBMC with anti-CD3 antibody, no differences were observed in GM-CSF(+)CD4(+)T cell frequencies. GM-CSF secretion was susceptible to regulation by Treg and vitamin D. Suppression of GM-CSF secretion by vitamin D was reduced in MS patients. CONCLUSIONS: Our study showed no elevation in GM-CSF(+)CD4(+)T cell fractions in MS patients compared to controls. Furthermore, GM-CSF secretion was prone to regulation by Treg and vitamin D, the latter being less effective in MS patients.

Increased inflammasome related gene expression profile in PBMC may facilitate T helper 17 cell induction in multiple sclerosis.

The NLRP3 inflammasome is a macromolecular complex importantly involved in IL-1ß processing. A role for this has been described in multiple sclerosis (MS). One mechanism by which IL-1ß might be involved in MS is by inducing pathogenic Th17 cells, i.e. GM-CSF+ Th17 cells. In the present study, we show that expression of the inflammasome related genes, NLRP3, caspase-1, IL-1ß and the IL-1ß/IL-1Ra ratio, was increased in PBMC from MS patients compared to healthy controls (HC). However, in an in vitro inflammasome activity assay with PBMC, IL-1ß protein secretion and the IL-1ß/IL-1Ra protein ratio were similar in MS patients and HC. Th cells cultured in the presence of supernatant derived from LPS/ATP inflammasome activated PBMC showed increased Th17 and GM-CSF+ Th17 cell frequencies in HC and MS patients and decreased anti-inflammatory IL-10+Th cell frequency in HC compared to Th cells cultured in the presence of control supernatant. Moreover, addition of the immune modulator calcitriol to the former condition resulted in reduced frequencies of Th17 and GM-CSF+Th17 cells, and also of IL-10+ Th cells. Evidently, our data indicate that inflammasome activity can skew the Th cell population toward a more pro-inflammatory composition, an effect that might be inhibited by vitamin D, and that might be importantly involved in inflammation within the central nervous system.

Monocytes, but not T cells, respond to insulin with Akt(S473) phosphorylation independent of the donor glucometabolic state.

BACKGROUND: Obesity is associated with insulin resistance and chronic low-grade inflammation. Insulin has been described to have anti-inflammatory effects in immune cells. Therefore, insulin resistance in immune cells can be expected to have important consequences for immune function. Here, we investigate whether freshly isolated monocytes and T cells, isolated from study subjects with a normal or disturbed glucometabolic state, respond to insulin with phosphorylation of Akt, a key molecule in the insulin signalling pathway. METHODS: A total of 25 study subjects were enrolled in the study. An oral glucose tolerance test (OGTT) was performed, and from fasting insulin and glucose, the homeostasis model assessment of insulin resistance (HOMA-IR) index was calculated. Peripheral blood mononuclear cells were isolated from heparinized blood and phenotypically characterized by flow cytometry. Basal and insulin-induced fractions of pAkt(S473)-positive monocytes and T cells were determined by Phosflow. RESULTS: On the basis of the OGTT, 11 subjects were classified as normal glucose tolerant (NGT), 9 had an impaired glucose metabolism (IGM) and 5 had type 2 diabetes (T2DM). The fraction of pAkt(S473)positive-T cells and monocytes, in the absence of insulin, was low in all subjects. Incubation with insulin did not induce Akt phosphorylation in CD4⁺ and CD8⁺ T cells in normal subjects. However, in the monocyte fraction, an insulin-dose-dependent increase of the pAkt(S473)positive-cell fraction was observed. This response did not differ between NGT, IGM and T2DM and was not correlated with HOMA-IR. CONCLUSIONS: In this study, we show that freshly isolated monocytes, but not T cells, are insulin-sensitive cells and that this insulin sensitivity of monocytes is not negatively affected by the glucometabolic state of the donor.

Regulatory B cells in ANCA-associated vasculitis.

OBJECTIVES: B cells have immunoregulatory function acting as antigen-presenting cells. A separate subset of interleukin (IL)-10 producing B cells (Breg) regulating T cell mediated immunity has been identified. In the present study, we investigated the role of Breg in antineutrophil cytoplasmic antibodies-associated vasculitis (AAV). METHODS: 17 healthy controls (HCs) and 41 patients with AAV were enrolled. 30 patients with AAV were in remission. Furthermore, 11 patients with AAV with active disease were studied. Breg were defined as IL-10(+)CD19(+) B cells upon culture with cytosine-phosphate-guanosine oligodeoxynucleotide (CpG ODN) 2006. Next to Breg, CD4(+)CD127(low)CD25(hi)CD39(neg)/CD39(+) regulatory T-cells (Treg), interferon (IFN)γ(+), IL-4(+) and Il-17A(+)T helper cell subsets were determined via flow cytometry. RESULTS: Patients with active or quiescent disease showed a diminished fraction of Breg as compared with HCs. The frequency of IFNγ(+) T helper cells was negatively associated with Breg in untreated AAV in remission but not in active vasculitis or in HCs. Interestingly, the total Treg population and the CD39(+) Treg subpopulation correlated positively with Breg in inactive patients with AAV. CONCLUSIONS: IL-10 producing B cells are diminished in AAV. Furthermore, Breg might regulate Th1 cells and are associated with Treg in quiescent AAV. Suppression of Th1 cells by Breg may be insufficient in active AAV.

Fraction of IL-10+ and IL-17+ CD8 T cells is increased in MS patients in remission and during a relapse, but is not influenced by immune modulators.

In the present study, circulating proportions of CD8(+) T (Tc) cell subsets, including IL-17 (Tc17) and IL-10 (Tc10) producing cells, were assessed in relapsing-remitting MS (RRMS) patients and a possible effect of beta interferon (IFN-ß), glatiramer acetate (GA), and vitamin D (VitD) on these cell subsets was investigated. We show that both Tc17 and Tc10 cell fractions are elevated in the circulation of RRMS patients in remission compared to healthy subjects and that these Tc subsets remain unaffected by current immune modulating regimens.

Influences of pelvic floor structures and sacral innervation on the response to distension of the cat rectum.

The contributions to the rectal response to distension of the pelvic floor structures surrounding the rectum and of the sacral spinal innervation have never been studied. Using paralysed intercollicularly decerebrate, anaesthesia-free cats, we studied pressure-volume relationships during slow ramp distensions of the rectum. Results obtained from animals with intact pelvic cavities were compared with those following mobilization of the rectum from the pelvic floor musculature. To assess the influences of spinal outflow and afferent input, rectal pressure-volume relationships were measured in the mobilized rectum following bilateral sequential transection of the spinal roots S1 to S3, first dorsal, then ventral. Isolation of the rectum from the pelvic floor structures resulted in a decrease in balloon volume in the lower range of distension pressure but did not affect volumes at higher pressures. The only afferent effect was seen after sectioning of dorsal roots S1, which resulted in a decrease in balloon volume. The only efferent effect was seen after sectioning of ventral roots S3, which decreased balloon volume further. In conclusion, the rectal response to distension depends on the properties of the rectal wall. It may be influenced by somatic inputs, inputs from the myenteric nervous plexus, and from the parasympathetic and sympathetic nervous systems. Afferent inputs and spinal autonomic reflexes may decrease the tone of the rectal musculature during distension.

On the adequate stimulus for rectal mechanoreception and perception: a study in cat and humans.

The adequate stimulus that is specific for both rectal mechanoreceptor excitation and rectal perception is still undefined. Using a visual analogue scale, healthy male volunteer subjects rated the intensity of the non-noxious 'pressure sensation evoked by slow balloon-induced distensions of the rectum. In a parallel study, the responses of spinal afferents originating from intramural mechanoreceptors of the rectum to the same stimulus were recorded in decerebrate cats. Both receptor activity and sensation intensity were linearly related to the diameter of the rectum, which is in turn a linear function of the tangential length of the rectal wall. In contrast, both saturated when expressed as a function of intrarectal pressure or rectal wall tension. It is concluded that the perception associated with rectal dis tensions in the non-noxious range is mediated by intramural mechanoreceptors that linearly encode tangential wall length, and that the underlying information is linearly transmitted throughout the CNS.

Discharge patterns of intramural mechanoreceptive afferents during selective distension of the cat's rectum.

The afferent input from the rectum to the central nervous system (CNS) has yet to be thoroughly characterized. The characteristics of mechanoreceptive rectal afferents have been studied in unanaesthetized decerebrate cats. Following lumbo-sacral laminectomy, single-unit activity (occasionally multi-unit activity) was recorded from centrally cut filaments of the sacral dorsal roots (predominantly S2), while a balloon was inflated in the rectum. Starting from their background activities (mean 15.1 imp sec-1, SD 7.6 imp sec-1), afferent discharge rate increased with increasing balloon pressure (mean threshold 6.3 mmHg, SD 3.6 mmHg). The dependence of firing rate on intrarectal pressure began to flatten out at 25 mmHg (mean; SD 10 mmHg). For 22 out of 29 units (76%) complete saturation occurred at 35 mmHg (mean; SD 15 mmHg) with a maximum discharge rate of 31 imp sec-1 (mean; SD 12.6 imp sec-1). In a number of recording sessions, cyclical rectal contractions were observed. In these cases, changes in firing of the units were closely related to changes in intrarectal pressure. Pressure-related afferent activity could be enhanced by parasympathomimetic drugs which augmented rectal contractions. We conclude that sacral dorsal roots contain afferents from low-threshold mechanoreceptors located in the rectal wall, and that these afferents monitor the filling state and contraction level of the rectum.

Dopaminergic influence on the excitability of antidromically activated Renshaw cells in the lumbar spinal cord of the rat.

The interaction between dopaminergic and cholinergic systems in the mammalian central nervous system, which is thought to have important implications in the pathophysiology of major extrapyramidal disorders, has never been adequately demonstrated in vivo. Renshaw cell burst responses to single electrical shocks to lumbar ventral roots in spinalized and decerebrated rats were studied. In this monosynaptic cholinergic pathway, apomorphine, a dopaminergic receptor agonist, inhibited whereas the D2-antagonist sulpiride facilitated the burst responses. The mutual antagonism of the two drugs and the depression coupled with the faster decay of post-tetanic potentiation of Renshaw cells by apomorphine demonstrate the involvement of presynaptic D2-receptors through which dopamine can modulate acetylcholine-mediated central synaptic transmission in vivo. The study also provides further evidence for the involvement of the spinal cord in extrapyramidal disorders.

Presynaptic dopaminergic inhibition of the spinal reflex in rats.

Dopaminergic influence on spinal monosynaptic transmission was examined in rats. Monosynaptic mass reflex (MMR) was recorded from the ventral root L6 following supramaximal stimulation (0.2 Hz; 0.1 ms) to the ipsilateral dorsal root L6 in spinalized rat under pentobarbitone sodium (40 mg/kg, i.p.) anaesthesia. MMR was inhibited by intravenous administration of the dopaminergic agonist, apomorphine (50-200 ug/kg) in a dose-dependent manner. The attenuatory effect of apomorphine (200 ug/kg i.v.) on the reflex could be reversed by the dopaminergic antagonist haloperidol (0.5 mg/kg, i.v.). Under tetanic stimulation (200 Hz; 15s), the pretetanic relative inhibition induced by apomorphine (200 ug/kg, i.v.) was increased only for a short period immediately after the cessation of tetanic stimulation. The results indicate existence of presynaptic dopamine receptors on the afferent terminals converging on the motoneurone which may functionally modulate the spinal motor output.

Inhibitory connections of ipsilateral semicircular canal afferents onto Renshaw cells in the lumbar spinal cord of the cat.

1. In intercollicularly decerebrate cats, the excitability of lumbar spinal Renshaw cells (tested by single shocks to ventral roots and deafferented muscle nerves) decreased for 600-1000 ms after conditioning electrical stimulation of ipsilateral semicircular canal nerves. 2. Conditioning stimulation of posterior canal afferents and combined stimulation of anterior and lateral canal afferents were equally effective in causing inhibition of Renshaw cells. No significant differences were observed for Renshaw cells excitable from hind-limb flexor or extensor nerves. 3. Inhibition appeared when one to five stimuli were applied to the canal afferents and arrived at the spinal segmental level 11-15 ms after the onset of conditioning stimulation. 4. Evidence is adduced to suggest that the inhibitory effects on Renshaw cells following stimulation of semicircular canal afferents were mediated directly, i.e. they were not caused by alterations of motoneurone activity. 5. Excitation of Renshaw cells due to stimulation of the canal afferents was rarely observed; it could not be excluded that it was secondary to motoneurone discharges. 6. It is suggested that vestibular inhibition of Renshaw cells ensure a high gain of hind-limb alpha-motoneurones during postural adjustments following a massive disturbance of body equilibrium.