Modifications of immunological and neuro-endocrine parameters induced by antiorthostatic bed-rest in human healthy volunteers.

AIM: Space flight has profound effects on immunological and neuroendocrine parameters. Microgravity plays a major role in the induction of these changes. The aim of the present study was the evaluation on ground of the effects induced by antigravitary posture on immune and neuroendocrine functions. METHODS: Eight healthy male volunteers (mean age 24+/-1 years) were maintained in antigravitary posture (-10 degrees) for 72 hours. Four of them were also maintained in supine posture for 72 hours as controls. The following immunological and neuroendocrine parameters have been analysed: peripheral white blood cells count, CD11b integrin expression and H(2)O(2) production by neutrophils, lymphocyte and monocyte phenotype, intracytoplasmic cytokine (IFN-gamma, TNF-alpha and IL-4) pattern, lymphocyte proliferation to mitogens and antigens, cortisol, ACTH, catecholamines, GH, LH, prolactin and testosterone plasma levels. RESULTS: In subjects maintained in antigravitary posture, norepinephrine, dopamine, cortisol, ACTH, GH and prolactin plasma levels increased whereas H(2)O(2) production by neutrophils, lymphocyte proliferation, NK cells number and intracytoplasmic IFN-g expression decreased. No significant modifications were observed in subjects maintained in supine posture. CONCLUSION: The results of this study indicate that several neuroendocrine and immunological parameters are modulated by a prolonged antigravitary posture on ground and may negatively affect astronauts defenses against pathogens during space flights.

Soluble HLA class I and Fas ligand molecules in blood components and their role in the immunomodulatory effects of blood transfusions.

It has been known for many years that blood transfusions may have immunomodulatory effects, however an ultimate explanation of this phenomenon is lacking. In the present paper we report that the concentrations of soluble HLA class I (sHLA-I) and soluble Fas ligand (sFasL) molecules in supernatants of blood components which contain elevated numbers of residual donor leukocytes, like red blood cells and random-donor platelets, are significantly higher than in other blood components. Elevated amounts of sFasL molecules are also found in some commercial immunoglobulin preparations. sHLA-I and sFasL molecules in blood components and in immunoglobulin preparations are biologically active in vitro as they inhibit mixed lymphocyte responses and cytotoxic T cell activity in allogeneic and autologous combinations and induce apoptosis in Fas-positive cells. If these results are paralleled in vivo the amount of sHLA-I and sFasL molecules should be taken into account in clinical practice in order to select the blood component and the immunoglobulin preparation which could induce the desired immunomodulatory effect in the recipient.

Increased beta2-microglobulin-free HLA class I heavy chain serum levels in the course of immune responses to viral antigens and to mismatched HLA antigens.

Besides being present in serum in association with beta2-mu, HLA class I heavy chains are also present in serum as beta2-micro-free moieties. The increase in serum levels of beta2-micro-associated HLA class I heavy chains in conditions associated with an activation of the immune system have prompted us to measure the serum levels of beta2-mu-free HLA class I heavy chains in the course of immune responses to viral antigens and to mismatched histocompatibility antigens. The serum level of beta2-mu-free HLA class I heavy chains, like that of beta2-mu-associated HLA class I heavy chains was significantly increased in patients affected by advanced HIV-1 infection or by chronic hepatitis C (CHC). In the latter group of patients an association was found between a reduction in the beta2-mu-free HLA class I heavy chain serum level and response to therapy with interferon alpha and ribavirin. Moreover, the beta2-mu-free HLA class I heavy chain serum level was increased more than that of beta2-mu-associated HLA class I heavy chains during episodes of liver ischemia following liver transplantation and in the course of acute graft rejection and of acute graft-versus-host-disease (GVHD) after allogeneic bone marrow transplantation (BMT). These results suggest that the serum levels of beta2-mu-free and beta2-mu-associated HLA class I heavy chains are independently regulated. Furthermore, beta2-mu-free HLA class I heavy chain serum level may be a useful marker to monitor response to therapy in CHC patients and the clinical course of liver and bone marrow grafts.

Soluble human MHC class I molecules induce soluble Fas ligand secretion and trigger apoptosis in activated CD8(+) Fas (CD95)(+) T lymphocytes.

In the present study, we have evaluated the apoptotic effect of soluble human MHC class I (sHLA-I) antigens on CD8(+) T lymphocytes. sHLA-I antigens and beta(2)-microglobulin-free HLA class I heavy chains, isolated from serum, induced apoptosis on phytohemagglutinin-activated CD8(+) T lymphocytes in autologous and allogeneic combinations. The extent of CD8(+) T cell apoptosis depends on the degree of activation, time of incubation with sHLA-I antigens and amount of sHLA-I antigens added to the cultures. Apoptosis is induced by the interaction of Fas (CD95)(+) cells with soluble Fas ligand which is released following binding of sHLA-I antigens to CD8 molecules. These results suggest that sHLA-I antigens may regulate immune responses by inducing apoptosis in activated CD8(+) T cells.

Soluble HLA class I/CD8 ligation triggers apoptosis in EBV-specific CD8+ cytotoxic T lymphocytes by Fas/Fas-ligand interaction.

In the present study, we report that allogeneic soluble HLA class I (sHLA-I) molecules isolated from serum induce apoptosis on EBV-specific CD8(+) Fas(+) cytotoxic T lymphocytes (CTL). CTL apoptosis is induced by the binding of sHLA-I molecules to CD8 and its extent depends on the time of incubation with sHLA-I molecules. Apoptosis is triggered by the interaction of Fas(+) CTL with soluble Fas-ligand, which is released following the binding of sHLA-I antigens to CD8 molecules. These results suggest that sHLA-I molecules may regulate immune responses by inducing apoptosis in virus-specific CTL.

Soluble beta2-mu-associated and beta2-mu-free HLA class I heavy chain serum levels in interferon-alpha nonresponder chronic hepatitis C patients. Markers of immune activation, and response to antiviral retreatment.

The serum levels of soluble beta2-mu-associated and beta2-mu-free HLA class I heavy chains were determined in 28 interferon-alpha nonresponder chronic hepatitis C patients retreated with interferon-alpha plus ribavirin and in 70 healthy subjects. The baseline levels of beta2-mu-associated and beta2-mu-free HLA class I heavy chains were significantly higher in patients than in healthy controls (P = 0.001). The levels of beta2-mu-associated HLA class I heavy chains significantly increased in responder patients with respect to nonresponders at the third month of treatment (P = 0.03). At the sixth month of treatment and after 6 months of follow up the levels of beta2-mu-associated HLA class I heavy chains decreased in responder patients and increased in nonresponders. The levels of beta2-mu-free HLA class I heavy chains showed only minor changes during and after treatment. We suggest that the determination of hepatitis C virus RNA levels combined with soluble beta2-mu-associated HLA class I heavy chains, as a marker of immune activation, could identify interferon-alpha non responder chronic hepatitis C patients most likely to respond to a retreatment with interferon-alpha plus ribavirin.

Beta2-micro-free HLA class I heavy chain levels in sera of healthy individuals. Lack of association with beta2-micro-associated HLA class I heavy chain levels and HLA phenotype.

We have applied a double-determinant immune assay (DDIA) to measure soluble beta2-microglobulin (beta2-micro)-free HLA class I heavy chains in serum. The mean concentration of beta2-micro-free HLA class I heavy chains in serum from 120 healthy subjects was 0.21+/-0.24 microg/ml. The individual serum levels of beta2-micro-free HLA class I heavy chains had a wide distribution, did not seem to be related with HLA phenotype, were stable over time and did not change with age. The serum levels of soluble beta2-micro-free HLA class I heavy chains did not correlate with those of soluble beta2-micro-associated HLA class I heavy chains, suggesting that their release is independently regulated. Three forms of soluble beta2-micro-free HLA class I heavy chains, with apparent molecular masses of 44, 39 and 37-35 kD, respectively, circulate in human serum. These results provide a useful background to assess the serum level of soluble beta2-micro-free HLA class I heavy chains in pathological conditions and to evaluate their putative immunoregulatory function.

Soluble HLA class I, HLA class II, and Fas ligand in blood components: a possible key to explain the immunomodulatory effects of allogeneic blood transfusions.

The immunomodulatory effect of allogeneic blood transfusions (ABT) has been known for many years. However, a complete understanding of the effects of ABT on the recipient's immune system has remained elusive. Soluble HLA class I (sHLA-I), HLA class II (sHLA-II), and Fas ligand (sFasL) molecules may play immunoregulatory roles. We determined by double-determinant immunoenzymatic assay (DDIA) sHLA-I, sHLA-II, and sFasL concentrations in different blood components. sHLA-I and sFasL levels in red blood cells (RBCs) stored for up to 30 days and in random-donor platelets are significantly (P <.001) higher than in other blood components and their amount is proportionate to the number of residual donor leukocytes and to the length of storage. Blood components with high sHLA-I and sFasL levels play immunoregulatory roles in vitro as in allogeneic mixed lymphocyte responses (MLR) and antigen-specific cytotoxic T-cell (CTL) activity, and induce apoptosis in Fas-positive cells. These data suggest that soluble molecules in blood components are functional. If these results are paralleled in vivo, they should be taken into account in transfusion practice. Blood components that can cause immunosuppression should be chosen to induce transplantation tolerance, whereas blood components that lack immunosuppressive effects should be preferred to reduce the risk of postoperative complications and cancer recurrence.

Decreased surface antigen density on lymphocytes of elderly humans.

The aim of our study was to analyze some poorly investigated and controversial aspects of senescent lymphocyte phenotype and functions. We examined 100 healthy aging individuals, divided into 4 age groups, and 30 young controls, correlating lymphocyte responsiveness to mitogenic stimulation with membrane phenotypic pattern and surface molecular densities of the main functional lymphoid markers. Stability of values in the period of study was established. No age-related differences in the parameters evaluated were detected among aging subjects. Phytohemagglutinin-induced lymphocyte proliferation was found severely impaired (about halved) in all elderly individuals with respect to controls. There was no significant difference between elderly group and controls in CD2, CD3, CD4, CD8, CD19, CD25 and HLA-DR antigen distribution. CD56 positive cell percentages were slightly decreased in the elderly groups. In apparent correlation with reduced lymphocyte responsiveness, CD2, CD3, CD4 and CD8 molecular densities, to different extents, were found relevantly (about 1 to 3-fold) lower in all aging groups than in controls. We could not ascertain if those antigens were poorly synthesized, defectively transported to membrane or shed in excess. However, we suggest that decreased surface molecular densities of antigens involved in functional processes of immune responses may be responsible for an abnormal costimulatory pattern during lymphocyte activation, leading to apoptotic rather than proliferative signals in a greater proportion of cells than normal.

Immunoregulatory role of soluble HLA molecules: a new skin for an old subject?

The available evidence suggests that measurement of the level of total sHLA-1 antigens and of donor-derived and recipient-derived allospecificities as well as the characterization of their variants in recipient's serum may provide useful information to differentiate graft rejections from infections in allograft recipients. Moreover, a significant progress has been made in our understanding of the functional properties of sHLA-I antigens in serum and of their potential role in the modulation of immune responses. If these preliminary results will be confirmed, then sHLA-I antigens are likely to become important reagents to monitor and treat graft recipients.

Acetylcholine-induced, calcium-dependent norepinephrine outflow from peripheral human lymphocytes.

Catecholamines (CA) were studied in peripheral human lymphocytes, as well as in the supernatants, after incubation with L-tyrosine and L-dihydroxyphenylalanine (L-Dopa) for 1 h. The effect that the addition of acetylcholine (ACh), Veratridine, lonomycin or KCI had on the outflow of norepinephrine (NE) from lymphocytes was also studied. The effect of the addition of methoxyverapamil (D600, a Ca2+ channel blocker) and cholinergic antagonists had on the ACh-induced NE outflow was assessed. CA were determined by HPLC-ECD, both in the supernatant and in the cell lysates. L-Tyrosine and L-Dopa significantly (P < 0.01) increased intracellular NE. Neither L-tyrosine, L-Dopa, nor vehicle induced a detectable outflow of NE to the supernatants. ACh [120 microM], Veratridine [100 microM], Ionomycin [10 microM] and KCl [50 mM] (with or without the simultaneous addition of L-tyrosine or L-Dopa) all induced a detectable outflow of NE to the supernatant when added 5 min before the end of incubation. NE was not detectable in the supernatant when the chemicals were added 10 to 20 min before the end of the incubation. When the chemicals were added at lower concentrations, erratic secretion or no secretion whatsoever was observed. D600 [100 microM] was able to significantly (P < 0.01) reduce the ACh-induced NE outflow. Tetraethylammonium (nicotinic antagonist), but not atropine (muscarinic antagonist), significantly (P < 0.001) decreased the ACh-induced NE outflow. The outflow of NE from peripheral human lymphocytes was seen. NE secretion seems to be ACh- and calcium-dependent since Veratridine, Ionomycin and KCl are able to induce Ca2+ entry by means of various mechanisms. The Ca2+ channel blocker employed in this study (D600) reduced the ACh-dependent NE outflow. We can conclude that both ACh (through nicotinic receptors) and calcium are involved in the outflow of NE from peripheral human lymphocytes.

Downregulation of HLA class I antigen expression in CD4+ T lymphocytes from HIV type 1-infected individuals.

The expression of HLA class I antigens is downregulated in CD4+ T cells following in vitro HIV-1 infection. We determined whether the expression of HLA class I antigens is downmodulated in peripheral blood lymphocytes (PBLs) of HIV-1-positive subjects and whether this defect correlates with disease progression. A cohort of 62 HIV-1-seropositive individuals in different stages of disease was studied. Among these, four subjects were evaluated at yearly intervals for 6 years. The expression of HLA class I, HLA class II, and CD38 antigens was analyzed in PBLs and in CD4+ and CD8+ T lymphocyte subpopulations. The percentage of HLA class I-positive cells and the membrane density of HLA class I antigens were significantly lower in PBLs from HIV-1-positive individuals than in PBLs from HIV-negative controls, proportionally decreased with disease progression, and significantly correlated with the decrease in CD4+ T lymphocytes. Furthermore, the percentage of HLA class I-positive cells and the membrane density of HLA class I antigens were significantly lower in CD4+ T lymphocytes from AIDS patients with respect to CD4+ T lymphocytes from HIV-negative controls and to CD8+ T lymphocytes from HIV-negative controls and AIDS patients. By contrast, the expression of HLA class II and CD38 antigens was upregulated in CD4+ and CD8+ T lymphocytes from HIV-1-positive subjects. The defective expression of HLA class I antigens could impair the lysis of HIV-infected CD4+ cells by virus-specific HLA class I-restricted cytotoxic T lymphocytes and contribute to the progression of disease.

L-tyrosine and nicotine induce synthesis of L-Dopa and norepinephrine in human lymphocytes.

Catecholamines (CA) were studied in peripheral human lymphocytes in basal conditions as well as after L-tyrosine and/or acetylcholine (ACh) stimulation. Nicotinic and muscarinic receptor activation and blockade were assessed. CA were determined after ultrasonic cell disruption in peripheral lymphocytes after incubation (1 h at 37 degrees C) with the chemicals employed. L-tyrosine significantly increased (P < 0.01) L-Dopa and norepinephrine (NE) content of lymphocytes. ACh in the low microM range did not modify, whereas ACh (60 microM) and (120 microM) significantly increased (P < 0.01), both L-Dopa and NE intracellular levels. L-tyrosine plus ACh (60 microM) or (120 microM) significantly increased (P < 0.01) intracellular L-Dopa and NE versus control, versus L-tyrosine alone and versus ACh alone. The increase was higher than the algebraic sum of the individual increases. Nicotine (250 microM), but not muscarine (50 microM), significantly increased L-Dopa and NE in lymphocytes. Tetraethylammonium (500 microM) (nicotinic blocker), but not atropine (100 microM) (muscarinic blocker), inhibited the ACh-mediated increase of intracellular L-Dopa and NE. These data show that lymphocyte synthesis of CA is under nicotinic control. Since intracellular L-Dopa after L-tyrosine plus ACh increased 6-fold versus basal, 2-fold versus L-tyrosine alone and 3-fold versus ACh alone, it is concluded that ACh might regulate CA synthesis in lymphocytes through an activation of the rate limiting enzyme tyrosine hydroxylase.

Soluble HLA class I and class II molecule levels in serum and cerebrospinal fluid of multiple sclerosis patients.

Increased concentrations of soluble HLA class I and class II molecules (sHLA-I and sHLA-II) have been observed in infectious, inflammatory, and autoimmune diseases. Because autoimmune mechanisms are considered to play a role in the pathogenesis of multiple sclerosis (MS), we decided to dose sHLA-I and sHLA-II in serum and cerebrospinal fluid (CSF) of MS patients comparing their concentrations with those observed in serum and CSF of patients with other neurologic diseases (OND) without evidence of neuroradiologic involvement of central nervous system (CNS) and in serum of healthy donors. The serum concentrations of sHLA-I were higher in both MS and OND patients than in healthy donors (P < 0.05) whereas sHLA-II serum concentrations were lower in MS patients than in both OND patients and healthy donors (P < 0.01). Detectable amounts of sHLA-II were observed in the CSF of 45% of MS patients and in CSF of only 6% of OND patients (P < 0.001). In MS patients a significant correlation between sHLA-I serum and CSF concentrations was observed (P < 0.01), whereas sHLA-II serum and CSF levels did not correlate. In conclusion, alterations of sHLA-I and sHLA-II serum and CSF concentrations are present in MS patients and could be involved in the induction of enhanced susceptibility to develop MS or in MS pathogenesis.

Catecholamine content and in vitro catecholamine synthesis in peripheral human lymphocytes.

We studied the catecholamine (CA) content in peripheral human lymphocytes and the ability of these cells to synthesize CA in vitro. CA were separated by high performance liquid chromatography (HPLC) and determined in the supernatant by electrochemical detection as well as being determined after ultrasonic cell disruption in mononuclear leukocytes, adherent cells (monocytes/macrophages), total lymphocytes, and B- and T-cell enriched fractions. T lymphocytes contained L-Dopa and norepinephrine (NE), whereas B lymphocytes contained only L-Dopa. Lymphocytes seem to be able to synthesize NE from both L-tyrosine and L-Dopa added to the incubation medium in concentrations similar to the peripheral venous plasma (i.e. 5 x 10(-5) m and 10(-8) m, respectively). The addition of D-Dopa did not increase intracellular NE. alpha-methyl-p-L-tyrosine, benserazide, disulfiram, and fusaric acid (which are inhibitors of the enzymatic pathway) all decreased the synthesis of NE. After the addition of [3H]-L-Dopa (10(-8) m and 10(-7) m) to the incubation medium, [3H]-NE and [3H]-dopamine appeared. By increasing the concentration of L-Dopa in the medium (< 10(-6) m), CA were detected in the supernatant as well. These data show that peripheral human T lymphocytes contain and are able to synthesize CA from normal precursors in physiologic concentrations, i.e. a CA synthetic pathway is shown in nonneural cells. These data seem to support the hypothesis of autocrine and paracrine loops in the regulation of lymphocyte activity in lymphocytes taken from human cerebrospinal fluid (as suggested by other authors).

Serum HLA class I antigen levels in allogeneic bone marrow transplantation: a possible marker of acute GVHD.

The levels of serum HLA class I antigens were determined at weekly intervals up to 5 weeks in 46 patients who had undergone allogeneic BMT. In patients with GVHD grade I or with GVHD grade I and fever of unknown origin (FUO), serum HLA class I antigen levels did not change during the observation period. In patients with GVHD grade II-IV serum HLA class I antigen level significantly increased in the week before the onset of GVHD, was maximal at the onset of GVHD and then persisted unchanged in the following 2 weeks. In patients with GVHD grade I or GVHD grade II-IV and infections whose onset coincided with that of acute GVHD a significant increase of serum HLA class I antigen level was found 2 weeks after the onset of the infectious episode. An increase of serum HLA class I antigen level was also found before the onset of repetitive GVHD grade II-IV episodes as well as during and after infectious episodes whose onset occurred after the onset of acute GVHD. The mean +/- s.d. concentrations of serum HLA class I antigens during GVHD grade II-IV episodes (9.4 +/- 3.4 micrograms/ml) and 2 weeks after the onset of infectious episodes (7.1 +/- 1.6 micrograms/ml) are significantly (P < 0.01 and P < 0.05, respectively) higher than that found 2 weeks before the onset of GVHD (3.0 +/- 0.5 micrograms/ml). The results of the present investigation suggest that measurement of serum HLA class I antigen level may be a possible marker to detect an acute GVHD following BMT.

Increased serum concentration of soluble HLA-DR antigens in HIV infection and following transplantation.

HLA class I and class II antigens circulate in serum as soluble molecules. Increased concentrations of soluble HLA class I molecules have been demonstrated in viral diseases, in rejection episodes following organ transplantation and in graft versus host disease. To explore the possibility of a variation of the serum concentrations of soluble HLA class II molecules in the same pathologic conditions we developed a double determinant immune assay that detects whole soluble HLA-DR molecules (sHLA-DR). The mean level of sHLA-DR antigens in sera from 23 healthy individuals was 0.64 +/- 0.72 microgram/ml. Elevated serum concentrations of sHLA-DR molecules were detected in sera from HIV infected patients in CDC2/3 and in CDC4 C1 stages (2.0 +/- 1.7 micrograms/ml and 4.6 +/- 1.7 micrograms/ml, respectively), in sera from patients affected by acute rejection after liver transplantation (5.3 +/- 3.7 micrograms/ml) and in sera from patients affected by severe acute graft versus host disease following bone marrow transplantation (8.8 +/- 3.1 micrograms/ml). The increase of sHLA-DR molecules in these sera significantly correlated with the elevation of soluble HLA class I antigens (P = 0.0004). The reported data suggest that both soluble HLA class I and class II molecules serum levels increase during viral infections and strong immune reactions and could suggest the involvement of these molecules in immunoregulation.

Behavior of soluble HLA class I antigens in patients with chronic hepatitis C during interferon therapy: an early predictor marker of response?

Soluble HLA class I antigens (sHLA-I), beta 2-microglobulin (beta 2-mu) and alanine aminotransferase (ALT) serum levels have been evaluated in 16 patients affected by chronic hepatitis C treated for six months with recombinant interferon-alpha (rIFN-alpha, 3 MU three times a week). The predictor role of sHLA-I and ALT modifications with respect to the response to rIFN-alpha therapy was also evaluated. Six patients responded (group 1), five patients relapsed followed in initial responses (group 2), and five did not respond to rIFN-alpha treatment (group 3). The baseline serum levels of sHLA-I and beta 2-mu were significantly higher in all three groups of HCV-positive patients with respect to HCV-negative controls (P < 0.05). A significant increase of sHLA-I serum level with respect to baseline value (P < 0.001) was observed in group 1 patients after two weeks of rIFN-alpha treatment. sHLA-I serum level then decreased, although remaining steadily and significantly increased with respect to baseline (P values ranging from 0.05 to 0.01) in the following five months and then returned to baseline one month after the end of rIFN-alpha administration. No significant variations of beta 2-mu serum levels were detected throughout the observation period. In group 1 patients ALT serum levels significantly decreased after two weeks of rIFN-alpha treatment (P < 0.001) and then remained in the normal range throughout the observation period. In the other two groups of patients no relevant variations of sHLA-I and beta 2-mu serum levels were found during and after rIFN-alpha therapy.(ABSTRACT TRUNCATED AT 250 WORDS)