Stressor-induced alteration of cytokine production in multiple sclerosis patients and controls.

OBJECTIVE: We administered an acute psychological stressor to multiple sclerosis (MS) patients and normal controls to determine whether differences in subjective and physiological responses to stress may underlie the susceptibility of MS patients to stress-related exacerbations. METHOD: Twenty-five MS patients (18 female, 7 male) and 25 age- and gender-matched controls participated in the study. They were asked to give a 5-minute videotaped speech defending themselves in a hypothetical scenario in which they were wrongly accused of stealing. Subjective and autonomic responses were monitored, and blood was sampled at baseline, 5, 20, and 60 minutes after the stressor to assess mitogen-stimulated production of interleukin-1beta(IL-1beta), interleukin-4 (IL-4), tumor necrosis factor-alpha (TNF-alpha), and interferon-gamma (IFN-gamma). RESULTS: MS patients and controls demonstrated similar subjective and physiological responses to the stressor that were independent of gender, mood, and disability status. The macrophage-derived cytokines IL-1beta and TNF-alpha were increased during the stressor, and remained elevated through 60 minutes. Th1 lymphocyte-derived IFN-gamma production also was increased at 5 and 60 minutes relative to baseline; however, there was no change in the Th2 lymphocyte-derived cytokine IL-4. CONCLUSIONS: These results favor the hypothesis that MS patients do not differ in stress response from normal controls; however, psychological stress may enhance cellular immune responses that would be potentially harmful to MS patients.

Immunologic response to acute psychological stress in MS patients and controls.

To determine whether MS patients differ from healthy subjects in stress-related immune changes, we examined immunologic alterations following a public speaking task in 25 MS patients and 25 healthy controls. Both groups demonstrated similar autonomic, neuroendocrine and immunologic responses to acute stress. Neutrophils, monocytes, CD8+ suppressor/cytotoxic T-lymphocytes and NK-cells transiently increased, with parallel changes in NK-cell activity. T-cell proliferation declined at 20 min, followed by increased reactivity at 60 min relative to baseline. This data suggests that stress-induced immune alterations remain intact in MS patients, and may contribute to immune changes associated with disease exacerbation.

Neonatal sympathetic denervation alters development of natural killer (NK) cell activity in F344 rats.

The development of natural killer (NK) cell activity was assessed in Fischer 344 (F344) rats sympathectomized as neonates with the neurotoxin, 6-hydroxydopamine (6-OHDA). No NK cell activity was detected in sympathectomized or vehicle-injected control animals at 7 days of age. At 10 and 14 days of age, NK cell activity was reduced in spleens from sympathectomized male and female rats. At 21 days of age, a reduction in NK cell activity was detected only in sympathectomized male rats. Sympathectomy did not alter NK cell activity at 28 and 42 days of age in either gender. At 56 days of age, NK cell activity was increased in neonatally sympathectomized females; rats of both genders acutely sympathectomized at 56 days of age also showed enhanced NK cell activity. No differences were observed at 90 days of age in neonatally or acutely sympathectomized males of females. Prior treatment with desipramine, which blocks uptake of 6-OHDA into nerve terminals and prevents the destruction of sympathetic nerve terminals, prevented these 6-OHDA-induced effects, suggesting that sympathectomy, and not direct toxic effects of 6-OHDA treatment of NK cells, accounted for the alterations in NK cell activity. Together, these results indicate that the sympathetic nervous system is an integral component of the developing lymphoid and hematopoietic microenvironment.

A longitudinal study of age-related loss of noradrenergic nerves and lymphoid cells in the rat spleen.

A longitudinal study of sympathetic noradrenergic (NA) innervation of the spleen was carried out in 3-, 8-, 12-, 17-, 21-, and 27-month-old Fischer 344 (F344) rats using (i) fluorescence histochemistry for localization of norepinephrine (NE); (ii) immunocytochemistry (ICC) for localization of tyrosine hydroxylase (TH)-positive nerve fibers alone, and in combination with specific markers for T and B lymphocytes (OX19 and anti-mu respectively), and macrophages (ED3); and (iii) high-performance liquid chromatography with electrochemical detection for quantitation of NE. Fluorescence histochemistry revealed extensive loss of NA nerve fibers in all compartments of the spleen in 21- and 27-month-old rats. With single-label ICC, a decline in TH+ nerve fibers in all compartments of the spleen was observed by 17 months of age and became more severe with advancing age; these findings suggest that both the rate-limiting enzyme and the transmitter itself (NE) are depleted from sympathetic nerves in aged rat spleen. Double-label ICC demonstrated the loss of TH+ nerve fibers in spleen from 17-, 21-, and 27-month-old rats, and a parallel loss of OX19+ T lymphocytes and ED3+ macrophages in these cellular compartments. Neurochemical measurement of NE demonstrated a decline in NE per wet weight at 27 months of age. The age-related decline in NA innervation of spleen and in the density of specific populations of cells of the immune system (T lymphocytes and antigen-presenting ED3+ macrophages), that follow remarkably similar time courses, supports functional evidence for dynamic interactions between the immune system and NA sympathetic nerves in the spleen, and further suggests a causal relationship between these age-related phenomena, i.e., that age-related immunosenescence promotes sympathetic denervation of the spleen which further compromises immune function. This hypothesis, however, requires further testing.

Neonatal sympathetic denervation alters the development of in vitro spleen cell proliferation and differentiation.

The ontogeny of spleen cell proliferation to T and B cell mitogens and immunoglobulin secretion, measured in vitro, was examined in neonatally sympathectomized Fischer 344 (F344) rats, administered the neurotoxic drug 6-hydroxydopamine (6-OHDA) from 1 to 3 days of age. Compared to cells from age-matched controls, spleen cells from neonatally sympathectomized animals, aged 7-14 days, exhibited a shift in the proliferative response to the T cell mitogen, concanavalin A (Con A), with reduced proliferation in the presence of low doses of Con A, but increased proliferation with higher doses. During the same period, from 7 to 14 days, the B cell mitogen STM/DxS inhibited proliferation by spleen cells from all rats, and no effect of sympathectomy was observed. As adult-like patterns of mitogen responsiveness emerged from 21 to 42 days of age, neonatally sympathectomized rats showed reduced proliferative responses of both T and B cells. This effect dissipated by 56 days of age. Polyclonal immunoglobulin (Ig) production by B cells was assessed in vitro in the presence or absence of STM/DxS. Neonatal sympathectomy resulted in reduced spontaneous IgM production throughout development. From 28 to 42 days of age, when mitogen-triggered IgM secretion first developed, neonatal sympathectomy decreased the magnitude of the response. By 56 days of age, mitogen-induced IgM secretion was no longer affected by sympathectomy, similar to the proliferative response. Gender influenced the time course of sympathectomy-induced changes in spleen cell proliferation and differentiation; however, the magnitude and direction of these changes were similar in both males and females. Desipramine, administered prior to 6-OHDA, prevented both sympathetic denervation and the 6-OHDA-induced changes in spleen cell responsiveness. This indicates that the alterations in immune function were dependent on NA nerve fiber destruction and were not simply the result of direct 6-OHDA action on other cells. The results of this study suggest that sympathetic innervation may play an important potentiating role in the development of the lymphoid system, through effects on lymphocyte proliferation and differentiation.

Parallel development of noradrenergic innervation and cellular compartmentation in the rat spleen.

By combining neurochemical measurement of norepinephrine (NE) with double-label immunocytochemistry for tyrosine hydroxylase-positive (TH+) noradrenergic nerves and specific lymphoid markers, we have examined the developmental compartmentation of noradrenergic nerves in the rat spleen. TH+ nerve fibers were present in the white pulp of the spleen at birth, among surface IgM-positive (sIgM+) B lymphocytes at the outer border of the periarteriolar lymphatic sheath (PALS), distant from the central artery. During the first 7 days, noradrenergic innervation developed rapidly, forming plexuses of nerve fibers along the central artery and its branches, among T and B lymphocytes of the PALS, and along the developing marginal sinus where ED3+ macrophages accumulate. The splenic concentration of NE (per mg wet wt.) and 3-methoxy-4-hydroxy-phenetheleneglycol (MHPG), a NE metabolite, increased rapidly during this period, suggesting that NE is available and released from these nerves. From 7-14 days, the white pulp expanded to include an inner PALS, outer PALS, marginal sinus, and marginal zone; during this period, TH+ fibers arborized principally among T lymphocytes of the inner PALS and adjacent to macrophages along the marginal sinus. By 14 days of age, NE concentration reached adult levels, although the MHPG/NE ratio (an index of NE turnover) remained higher throughout development than in adulthood. Finally, from 14-28 days, the outer PALS expanded to include follicles containing sIgM+ B lymphocytes. At the earliest stages of follicular development, a parafollicular rim of noradrenergic fibers was present, providing occasional branches which arborized within the follicle. No further changes were observed in either noradrenergic innervation or cellular compartmentation after 28 days of age. These findings suggest that noradrenergic fibers are present in developing compartments of the spleen at the earliest stages of their development, providing norepinephrine for interaction with a variety of adrenoceptor-bearing lymphoid and nonlymphoid cells.

Noradrenergic sympathetic innervation of the spleen: III. Development of innervation in the rat spleen.

The ontogeny of noradrenergic innervation and its compartmental development were studied in the rat spleen using glyoxylic acid histofluorescence and high-performance liquid chromatography (HPLC). Noradrenergic nerves were present at birth in bundles adjacent to the splenic artery and vein. On days 1-3, fluorescent profiles largely were associated with the vasculature and with the perivascular zone. By day 6, these fibers formed increasingly elaborate and tortuous plexuses around the central arteries and their branches. By day 10, fibers were present along the marginal sinus and extended into the developing marginal zone. Between day 10 and day 13 the largest increase in norepinephrine (NE) levels (per mg protein) were noted, and the periarteriolar lymphatic sheath (PALS) achieved its adult form, with increased innervation of the parenchyma. In contrast, the venous/trabecular system developed relatively late. The first trabecular fibers were evident at day 10, and the capsule was not innervated until day 13. From 13 days to adulthood, there was a gradual refinement and extension of existing patterns with no change in NE levels as measured by HPLC (per mg protein), suggesting that the innervation was keeping pace with rapid increases in spleen growth. The pattern of growth and development for noradrenergic nerves in the PALS remarkably parallels changes in T cell compartmentation during this period. We propose that norepinephrine is available for interaction with T cells at the earliest stages of development and could play a role in such processes as lymphocyte packing and the onset of immunocompetence.

Noradrenergic sympathetic innervation of the spleen: I. Nerve fibers associate with lymphocytes and macrophages in specific compartments of the splenic white pulp.

Sympathetic noradrenergic nerve fibers, stained with antiserum for tyrosine hydroxylase (TH), richly innervate the splenic white pulp. These fibers distribute with the vascular and trabecular systems, and associate mainly with the central artery and its branches, the periarteriolar lymphatic sheath (PALS), the marginal sinus, and the parafollicular zone, with occasional delicate fibers also present in the follicles. Simultaneous staining of TH-positive nerve fibers and markers for specific lymphoid cells has shown several regions of contact between nerves and lymphocytes or macrophages. The TH-positive nerve fibers in the plexuses around the central arterial system and in the PALS are present among T lymphocytes (OX-19-positive cells) including both T helper and T suppressor cells, and interdigitating cells. At the marginal sinus, TH-positive fibers run adjacent to macrophages (ED3-positive cells), B lymphocytes (IgM-positive), and intensely fluorescent IgM-positive cells. Along the parafollicular zone, TH-positive nerve fibers run adjacent to T lymphocytes, peripheral follicular B lymphocytes, and intensely fluorescent IgM-positive cells. Within some follicles, delicate fibers end adjacent to both T and B lymphocytes. These relationships suggest a direct interaction between norepinephrine release from the TH-positive nerve terminals and the lymphocytes and macrophages closely associated with them, and focuses attention on the potential neural modulation of related functions such as T and B lymphocyte entry into the spleen and antigen capture (marginal zone), antigen presentation and T cell activation (PALS), B cell activation (parafollicular zone and marginal zone), and lymphocyte egress (outer marginal zone).

Dynamics of REM sleep in narcolepsy.

The discovery of sleep onset REM periods (SOREMPs) in narcolepsy first suggested the important role of REM sleep in the disorder. We have conducted a series of studies exploring factors that affect the onset and termination of REM sleep in narcolepsy. Following a preliminary study of REM sleep deprivation, we compared the sleep onset response of narcoleptic and normal subjects to awakenings at REM sleep onsets and awakenings during NREM sleep. In addition, we have investigated the relationship of these awakenings to daytime sleepiness. We have demonstrated that an index of the REM sleep process predicts the sleepiness of both normal and narcoleptic subjects. The finding of increased frequency of SOREMPs following both REM and NREM sleep awakenings in the narcoleptic patient suggests that accelerated triggering and inertia of the REM sleep process are pathophysiological mechanisms of the disorder.