The significance of the inflammatory reactions for the development of clinical signs in multiple sclerosis and acute experimental autoimmune encephalomyelitis as assessed by means of the spontaneous chemiluminescence activity of peripheral blood monocytes.

We found that the course of the chemiluminescence activity (CL-A) of peripheral blood monocytes of Lewis rats with acute experimental autoimmune encephalomyelitis (EAE) correlates well with the clinical course of this autoimmune disease and that only a reduction in T helper cells caused a reduction in CL-A. However, in multiple sclerosis (MS) patients the CL-A increased when clinical improvement started. The serum of these patients contained at least two stimulatory substances. More than 50% of the stimulatory effect could be blocked by antibodies to interferon-gamma.

The inhibitory effects of prednisone, 16-methylen-prednisolone, and ACTH on con-A induced lymphokines (interferon-y) as measured by the chemiluminescence-activity of blood monocytes.

When lymphocytes are stimulated with mitogens or antigens they are enhanced via a cascade of lymphokines to produce interferon-y (IFN-y). IFN-y augments the H2O2 secretion of human monocytes which indirectly can be measured by chemiluminescence. We tested prednisone, 16-methylen-prednisolone and ACTH for their effect to inhibit the Con-A induced stimulation of the chemiluminescence-activity. All three hormones inhibited significantly the stimulation: prednisone up to 52.5% (concentration = 150 micrograms/ml, p = 0.000005), 16-methylen-prednisolone up to 22.5% (concentration = 2.5 micrograms/ml, p = 0.006) and ACTH up to 33% (concentration = 10 micrograms/ml, p = 0.0036).

The chemiluminescence activity of peripheral blood mononuclear cells during acute experimental allergic encephalomyelitis.

The spontaneous chemiluminescence activity (CL-A) of peripheral mononuclear cells (MNC) was examined in Lewis rats with acute experimental allergic encephalomyelitis (EAE), compared to rats immunized with complete adjuvant (n = 11) and healthy animals (n = 16). In rats with EAE, CL-A increased sharply 8-9 days after immunization (3420 +/- 3124 counts/10 s, n = 16) at the time of flattening of the weight curve. This CL-A peak was compared to that of animals immunized with complete adjuvant: 765 +/- 441 counts/10 s (P = 0.01) and healthy rats: 450 +/- 172 counts/10 s (P = 0.0001). After this initial peak in EAE rats, CL-A decreased almost to normal values when animals lost weight (746 +/- 251 counts, n = 19) and tail paralysis developed (557 +/- 251 counts/10 s, n = 15). CL-A increased again with the onset of paralysis of the extremities (1527 +/- 990 counts/10 s, n = 11), followed by a decrease as the clinical course deteriorated. Finally, CL-A approached normal values as the animals improved. A significant increase in the number of meningeal (P less than 0.01) and perivascular (P less than 0.01) cells in the CNS coincided with the initial CL-A peak. Gel filtration of the serum of rats with increased CL-A revealed at least one substance, with a molecular weight between 13,700 and 43,000 Da, which stimulated the CL-A of normal mononuclear cells.

The behaviour of OKT3-, OKT4- and OKT8-positive cells during phases of elevated spontaneous chemiluminescence activity (CL-A) in multiple sclerosis patients. A serial study.

The chemiluminescence activity (CL-A; synonym = burst activity, BA) and the percentage of OKT3-, OKT4- and OKT8-positive peripheral blood cells were serially examined in four control persons and in eight patients with multiple sclerosis. When the OKT values obtained in phases of increased CL-A (clinical remission) were compared with those of the control group, the percentage of OKT3-positive cells was reduced (P = 0.014), and that of OKT4-positive cells increased (P = 0.014); there were no significant changes in the percentage of OKT8-positive cells (P = 0.171). After the CL-A had returned to normal values, the OKT4-positive cells remained elevated (P = 0.029), whereas the OKT3- (P = 0.342) and OKT8-positive cells (P = 0.443) showed no significant changes. When in the same patients, phases of increased CL-A were compared with phases in which values were not elevated, a reduced percentage of OKT3-positive cells was found in phases with increased CL-A (P = 0.031); however, the OKT4-positive and OKT8-positive cells did not differ significantly (P = 0.156 and 0.281).

Determination of interferon-gamma in the peripheral blood of multiple sclerosis patients in remission, using a chemiluminescence technique.

The plasma from eight patients with multiple sclerosis (MS) whose disease was in remission, was investigated by a chemiluminescence technique for its ability to stimulate the oxidative metabolism of peripheral blood monocytes (PBM). The active fraction identified had a molecular weight between 13,700 and 43,000 Da. Its activity was reduced by incubation at pH 2, pH 4 or pH 6, or by treatment at 56 degrees C for 1-3 h. The activity was also decreased, 58-100%, by prior incubation with antibodies to human interferon-gamma (IFN-gamma). We suggest that these results indicate that the increased chemiluminescence activity (CL-A) of PBM in MS patients in remission is due mainly to the presence of circulating IFN-gamma.

The spontaneous burst activity of peripheral blood monocytes in patients with acute polyradiculoneuritis, lymphocytic meningoencephalitis, and multiple sclerosis. A comparison.

The course of the spontaneous burst activity (BA) of peripheral blood monocytes was examined in patients with acute polyradiculoneuritis (PN), lymphocytic meningoencephalitis (LE), and multiple sclerosis (MS) and the BA was compared with the clinical course. In 4 patients with postinfectious acute PN the BA was significantly increased up to values around 60 000 counts/10 s. The BA and the clinical course were closely correlated in these patients (mean of r = 0.83). In 4 patients with lymphocytic LE the BA initially was moderately increased to values between 4 000 and 5 000 counts/10 s and showed again a very close correlation with the clinical course (mean of r = 0.99). In 13 MS patients the BA values were found markedly increased showing a close correlation with clinical improvement (mean of r = -0.94) whereas in 2 patients without improvement the BA remained at low levels. The comparison of the clinical course with that of the BA in the three inflammatory diseases of the nervous system reveals that patients with PN and LE show high BA values whilst the clinical signs and symptoms are most expressed, whereas the BA is low in this phase of illness in MS patients but increases around the beginning of improvement. Possible meanings of this behaviour of the BA are discussed.

Monocytes constitute the only peripheral blood cell population showing an increased burst activity in multiple sclerosis patients.

We examined peripheral mononuclear cells (PMNC), peripheral polymorphonuclear cells (PPNC), adherent mononuclear cells, non-adherent mononuclear cells, non-phagocytic mononuclear cells and non-phagocytic Percoll-fractionated mononuclear cells of multiple sclerosis (MS) patients for their spontaneous burst activity (BA). According to previous results PMNC of MS patients showed a markedly increased BA (p = 0.0002); PPNC, however, did not show significantly elevated values (p = 0.36). Further separation of PMNC and identification of the cells obtained by FITC-conjugated antibodies, esterase and Giemsa staining revealed high mean coefficients of correlation (r) between the BA and the number of esterase-positive cells (MO; r = +0.97) and the number of surface Ig-positive cells (B cells; r = +0.97). r was negative for the correlation between the BA and OKT3-positive cells (T cells; r = -0.99) and for the correlation between the BA and the number of large granular lymphocytes (r = -0.39). As in the range below 2,000 counts/10 s r is -0.5 between the BA and the number of B lymphocytes, while r is +1.0 between the BA and MO, and there are BA values below 500 counts/10 s although around 10% B cells but no MO are present, the results suggest that MO are the only cell population responsible for the increased BA in MS patients. Based on recent findings in our laboratory the results additionally indicate that in the peripheral blood interferon-gamma stimulates the BA of MO only.

Indications of the occurrence of inflammatory reactions in the clinical improvement phase in multiple sclerosis patients.

In patients with multiple sclerosis (MS) the spontaneous burst activity (BA) of peripheral blood mononuclear cells was related to the clinical course of the disease. In five patients clinical improvement was found while the BA was significantly increased (more than 300% of the controls). During the appearance of new or deteriorating signs and in the period without clinical changes, the BA was not at all or not markedly increased. In two patients without clinical improvement the BA did not reach levels above 300% of the controls. Our findings suggest that inflammatory reactions represented by the BA occur in the phase of clinical improvement. Since burst-stimulating activity was found in the serum of MS patients, cytokines produced by activated immuno-competent cells are assumed to cause the increased BA. A possible role of prednisone and ACTH on the BA of peripheral mononuclear leucocytes is discussed.

Correlation of the chemiluminescence-activity of peripheral blood monocytes with CSF parameters of inflammation and the clinical course of patients with lymphocytic meningoencephalitis.

The chemiluminescence-activity (CL-A) of peripheral blood monocytes (MO) was measured in eight patients with lymphocytic meningitis or meningoencephalitis and compared to CSF parameters and the clinical course. The initial maximum CL-A was around four times above the control and decreased to normal values within approximately 20 days. Poor correlations were found when the CL-A was compared to CSF parameters in the total group of patients. With regard to the CSF parameters in individual patients the CL-A was closely related to the cell count, to a lesser degree to the protein content, but not to the IgG content. Finally, a very good correlation of the CL-A was found with the clinical course in individual patients. This preliminary data suggests the clinical usefulness of CL-measurements and supports the conception that the CL-A predominantly reflects the specific cellular phase of inflammation, which is dominated by T cells.

Hemichorea associated with varicella-zoster reinfection and endocarditis. A case report.

A 20-year-old woman developed transient right-sided hemichoreatic movements after household exposure to varicella-zoster. Some days before the appearance of involuntary movements a vesicular rash had occurred. About 6 months later an elevated IgG serum titer against varicella virus was found and two-dimensional echocardiography showed signs of an endocarditis. During the following 2 months the IgG value returned to within the normal range and the choreatic movements disappeared almost totally. The possibility is discussed that endocarditis had been caused and maintained by serum antibodies to varicella-zoster virus which cross-reacted with valvular tissue. Embolization to the region of the left striatum and/or postinfectious encephalitis in this region are assumed to be the most plausible causes of the transient hemichorea.

Changes in the number of complement-binding leucocytes in the peripheral blood of multiple sclerosis (MS) patients: indication to a reduction of EAC3bi-binding T cells in the early acute phase of MS.

Peripheral blood leucocytes (PBL) of multiple sclerosis (MS) patients and healthy controls were examined for their reactivity with EAC3b, EAC3b-beta 1H, and EAC3bi intermediates and C3b- or beta 1H-coated microspheres (ms). A marked decrease was found in the number of EAC3b-beta 1H (P = 0.04) and EAC3bi (P = 0.03) rosettes accompanied with a decrease in the number of total T cells (P = 0.0015), when MS patients were examined within the first 14 days after occurrence of disturbances. On the other hand, when MS patients were examined later than 14 days after onset of disturbances or those with a progressive course were examined, EAC3b-beta 1H (P = 0.0002) and EAC3bi (P = 0.0002) rosettes were markedly increased. In studies where fluorescence-activated cell sorter (FACS) analysis was applied on C3b- and beta 1H-coated ms, similar results were obtained as found with EAC intermediates. Based on previous findings these results strongly suggest that the decrease in EAC3b-beta 1H, EAC3bi, and beta 1H-ms-binding PBL was predominantly due to a loss of beta 1H-binding T cells. A possible relevance of this finding to the demyelinating mechanism(s) is discussed.

Measurement of the respiratory burst in human monocytes and polymorphonuclear leukocytes by nitro blue tetrazolium reduction and chemiluminescence.

We compared measurements of chemiluminescence (CL) assessing the rate of production of oxygen intermediates at a given instance, and of nitro blue tetrazolium (NBT) reduction detecting total superoxide produced during the assay period, for the assessment of the respiratory burst of both human monocytes (M phi) and polymorphonuclear leukocytes (PMN). In the CL experiments, opsonized and non-opsonized zymosan was used to stimulate peripheral blood M phi and PMN. Opsonized zymosan yielded an earlier and 2-fold higher peak response than non-opsonized zymosan. The stimulatory action of zymosan in CL varied with the time of incubation and depended on the concentration of cells and zymosan. No light generation was observed in the absence of viable cells. In contrast to NBT reduction, incubation of PMN with dextran sulfate did not result in increased light generation but rather in a quenching of the response in CL. Opsonized zymosan also yielded a higher level of NBT reduction than non-opsonized zymosan. Comparing NBT reduction with CL in 21 healthy individuals, with 2 X 10(5) M phi and 2.5 X 10(5) PMN for NBT and 5 X 10(5) PMN for CL tests, we found that NBT brings about spontaneous oxidative metabolism, possibly reflecting the intracellular compartment, the chemiluminescent response of resting cells being only marginal. The data suggest higher sensitivity of the CL method. These results provide useful comparative data for 2 established methods used to document the respiratory burst in phagocytic cells.

Multiple sclerosis patients show an increased spontaneous activity of their peripheral blood monocytes as measured by chemiluminescence.

I has been reported that myelin basic protein (BP) reacts extremely sensitively to peroxide, which is formed when monocytes/macrophages are stimulated to produce a "respiratory burst" (RB). We measured the RB activity by means of chemiluminescence in peripheral blood monocytes/macrophages (MO) of 17 MS patients, 5 patients with a viral infection of the CNS, and 14 control persons. The median of the spontaneous RB activity of MS patients compared with the median of our control group showed a highly significant increase (P = 0.0002). All MS patients examined possessed a clearly increased MO activity. The highest values, however, were found in MS patients in a bout (means = 315%, means = 296%). Since a viral infection is discussed as being involved in the pathogenesis of MS, we also examined patients with a viral infection of the CNS, but this group of patients did not show a significant increase (P = 0.34). Our data indicate that MO in MS patients generate and/or secrete an increased amount of unspecific mediators of inflammation. The possibility is discussed that this altered MO function might be caused by an altered reaction of MO in MS patients to a viral infection or superinfection.

Conditions for the enhancing effect of protease inhibitors on the concanavalin A induced thymidine response of murine lymphocytes.

Incorporation of [3H]-thymidine - [3H]-TdR - into concanavalin A (Con A) stimulated murine splenocytes and thymocytes was found to be enhanced by addition of certain concentrations of phenyl-methylsulfonylfluoride (PMSF), di-isopropylfluorophosphate (DFP), N-alpha-tosyl-L-lysyl-L-chloromethylketone (TLCK), and soybean trypsin inhibitor (SBTI). No enhancement could be observed when mononuclear cells of the peripheral blood were used, and a medium enhancement when thymocytes were applied. Furthermore, no enhancing effect of the protease inhibitors (PI) on the Con A response of murine splenocytes could be observed within the first 24 h of the culturing period. DFP, PMSF, and TLCK enhanced the Con A response to a similar degree, whereas SBTI was less effective. DFP and SBTI proved to be also effective when they were added after 15-24 h to the Con A cultures, if the cultures were harvested 48 h later. Removal of adherent and phagocytic spleen cells or reduction of the concentration of spleen cells shifted the effective DFP concentration to lower concentrations, whereas addition of adherent spleen cells caused a shift of the enhancing DFP amounts to higher concentrations. The data presented suggest that the enhancing effect of PI on the T cell response depends on the concentration of PI, the time of culturing and incubation, the PI used, the origin of the stimulated cells, and especially on the number of adherent and phagocytic cells. These findings might explain - at least in part - the different results on the effect of PI on the T cell response obtained in the past.

Differential effect of low molecular weight alcohols on the Con A stimulation of mouse spleen cells.

Incorporation of [3h]thymidine ([3H]TdR) into concanavalin A (Con A)-stimulated murine splenocytes was found to be enhanced by addition of certain concentrations of ethanol, 2-propanol and acetone. The alcohol/acetone-induced enhancement of the Con A response was found to be accompanied by an increase of the percentage of living cells as assessed by trypan blue exclusion. Concentrations of ethanol and 2-propanol which caused maximum [3H]TdR uptake in Con A cultures were also found to lead to higher percentages of aggregated cells than in comparison to Con A cultures without alcohol. The data suggest that alcohols in certain concentrations are capable to achieve optimum Con A stimulation.

A comparative evaluation of receptor reactivities for C3b, iC3b, and C3d on Raji lymphoblastoid cells.

Raji cells were described to carry receptors for iC3b, C3d, C3b-beta 1 H and beta 1 H. Controversial opinions, however, exist whether or not these cells carry also receptors for C3b. Using highly purified C3, definitely devoid of beta 1 H and C5, for preparation of C3b intermediates, it could be shown that Raji cells bound to C3b cells. Furthermore, Raji cells reacted with monoclonal antibodies that interfered with binding of C3b to human erythrocytes, lymphocytes and renal cells. The receptor for C3b on Raji cell, however, exhibited some special properties and, therefore, required some distinct experimental conditions for its detection: (1) The origin of the erythrocytes used for preparation of the C3b intermediates seemed to be important; this was not the case when iC3b and C3d receptor reactivity was assessed. (2) Rosettes already formed between Raji cells and EAC1423b showed the tendency to disintegrate within the first 30 min after the rosette formation assay. Again, this effect could not be observed with iC3b- and C3d-dependent rosette formation. (3) Incubation of the Raji cells at 37 degrees C as well as 4 degrees C before rosette formation resulted in a rhythmic loss and reappearance of C3b receptor reactivity. At room temperature (19-22 degrees C) this effect was much less expressed. There was no influence of preincubation at 4 and 37 degrees C, respectively, on the iC3b and C3d receptor reactivity of Raji cells. (4) Diisopropylfluorophosphate (DFP) present during rosette formation enhanced, within a certain range of concentration, the percentage of C3b-dependent rosette formation. iC3b and C3d receptor reactivity was not influenced. A similar reaction pattern was observed with pokeweed mitogen (PWM)-stimulated tonsil lymphocytes. In the concentrations tested, DFP showed no effect on the rosette formation between C3b, iC3b, and C3d cells, respectively, and unstimulated tonsil lymphocytes. The data presented suggest that C3b receptors on Raji cells undergo some special metabolism, possibly controlled by fluid phase or cell-bound proteases. This might be a common property of C3b receptors on blast-like and transformed cells, differing from that of unstimulated small lymphocytes.

Role of beta 1H for the binding of C3b-coated particles to human lymphoid and phagocytic cells.

Coating of EAC14oxy23b with highly purified human serum beta 1H globulin (beta 1H) led to acceleration of rosette formation with human peripheral blood lymphocytes (PBL), tonsil lymphocytes, B lymphoblastoid (Raji) cells, granulocytes and monocytes. This reaction was discernible from C3bi-dependent rosette formation. Enhancement of rosette formation of C3b cells by beta 1H was most effective at limiting amounts of C3 per EAC14oxy23b. The beta 1H effect was not due to trace contamination with C3b inactivator. beta 1H-dependent rosette formation with the various lymphoid and phagocytic cells could be suppressed by the F(ab')2 fragment of anti-beta 1H suggesting beta 1H-mediated binding of beta 1H-coated particles to complement receptor-positive (CR+) cells. In turn, binding of fluid-phase beta 1H to lymphoid and phagocytic cells could be demonstrated by fluorescence and by 14C-labeled beta 1H. In addition, the functional status of these cells with respect to their receptor reactivity was altered. Treatment of normal lymphocytes (PBL, tonsil lymphocytes) and of granulocytes with beta 1H improved their rosette formation with both EAC14oxy23b and EAC14oxy23b-beta 1H. The reaction of monocytes was hardly affected. The beta 1H effect on Raji cells resulted in reduced rosette formation with EAC14oxy23b-beta 1H, while binding of EAC14oxy23b remained unchanged. These results suggest the presence of sites on CR+ cells, to which soluble and particle-bound beta 1H can bind, leading to alteration of the functional status of the cells. In all likelihood, EAC14oxy23bi can attach to the beta 1H-binding sites on CR+ cells.