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).

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.

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.

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.

Increased level of serum HLA class I antigens in HIV infection. Correlation with disease progression.

Analysis of (sHLA-I) antigens in a large number of HIV-positive subjects found a significant increase of their level, but did not detect any change in their molecular profile. Monitoring at yearly intervals for four years of the sHLA-I antigen level in 14 HIV-positive subjects with a normal sHLA-I antigen level at study entry showed a significant correlation between progressive increase of sHLA-I antigen level and disease progression. Furthermore, a Kaplan-Meier plot of the frequency of development of AIDS in 34 patients whose cases were followed for 7 years showed that sHLA-I antigen level is a strong predictor of progression to AIDS. Its predictive value is comparable to that of serum beta 2-mu level, greater than that of serum neopterin, and lower than that of CD4+ T-cell percentage. The predictive value of sHLA-I antigen level in combination with serum beta 2-mu level, neopterin level, or CD4+ T-cell percentage is greater than that of each individual variable. These results suggest that measurement of the sHLA-I antigen level may provide useful prognostic information in HIV-positive subjects.

HLA class-I-soluble antigen serum levels in liver transplantation. A predictor marker of acute rejection.

The serum levels of sHLA-I have been determined in 16 patients following liver transplantation. sHLA-I levels did not show remarkable variations in six patients without evidence of transplant-related complications. sHLA-I levels strongly increased in 10 patients undergoing acute rejection episodes. In these patients, an average 20% daily increase of sHLA-I levels was detected on the 6 days preceding and on the 2 days following the rejection episode. A fast decrease of sHLA-I levels was observed in seven patients following treatment of acute rejection with anti-CD3 mAb. The serum level of sHLA-I antigens positively correlated with ALT serum level and inversely correlated with PT. The determination of sHLA-I in serum may therefore be proposed as a useful marker in the monitoring of patients following liver transplantation. The increase of sHLA-I antigens may predict the onset of acute rejection whereas their decrease may be related to a good response of acute rejection to immunosuppressive treatment.

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.

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.

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.

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.

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.

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.

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.

Major histocompatibility gene products and human immunodeficiency virus infection.

In the present paper we analyze the role of major histocompatibility gene products, the human leukocyte antigens, in the pathophysiology of the acquired immunodeficiency syndrome. No association has been found between human leukocyte antigen (HLA) frequencies and human immunodeficiency virus type 1 (HIV 1) infection, whereas significant associations have been reported in some populations between some HLA haplotypes and the appearance of either opportunistic infections or secondary cancers. With regard to the human leukocyte class I antigens, their role as restriction elements in presenting HIV 1 to virus-specific cytotoxic T lymphocytes seems to be established. An increase in the serum levels of their soluble forms that correlate with disease stage has also been demonstrated. These circulating molecules could interfere with the immune response to HIV 1 and could contribute to the development of the immunodeficiency. Antigenic similarities have been detected between human leukocyte class II antigens and HIV 1 envelope proteins. These homologies could explain both the presence in some HIV-positive sera of anti-HLA class II antibodies that mediate the lysis of CD4(+)-HLA class II+ T cells and the false-positive reaction of some HIV-negative sera, which contain anti-HLA class II antibodies, in tests for HIV 1 antibodies. Reduced levels of some complement factors (the human leukocyte class III antigens) have been detected in HIV-infected subjects. These defects could play a role in the progression of the disease and affect both the clearance of HIV 1 and complement-mediated antibody responses. The data reported in this review suggest that HLA antigens may be involved in several steps of the immune deficiency of HIV-infected subjects and thus contribute to the pathophysiology of acquired immunodeficiency syndrome.

Use of an established cell line in the evaluation of the cytotoxic effects of various chemicals.

The HTC hepatoma cell line was used as an "in vitro" model to detect the cytotoxicity of eighteen chemicals, chosen on the basis of different biological activities and physicochemical characteristics. Two different cytotoxicity assays measuring cell lethality (CS) or inhibition of cell growth (CF) were applicated to confluent cell monolayers or to colony-forming cells, respectively. Cells were exposed to the chemicals at doses ranging from 10(-6) M to 10(-2) M for 24 h. The results indicated a wide range of IC 50 (the concentration resulting in 50% inhibition of toxicity parameters) from as low as 1 microM (Potassium dichromate) to as high as 407.5 mM (Ethanol), the sensitivity of the CF test being greater than that of the CS test. A battery of cytotoxicity tests could be established in order to offer simple, rapid and economic methods which can be complementary and, in part, alternative to the use of laboratory animals.

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.