Intrinsic capacity of monocytes to produce cytokines ex vivo in patients with acute lymphoblastic leukaemia.

Monocytic cytokine profiles of fifteen children with acute lymphoblastic leukaemia (ALL) were included to determine whether malignancy per se contributes to impaired cytokine profiles in vivo and ex vivo. The ex vivo tumour necrosis factor-alpha (TNF-alpha) and interleukin 1beta (IL-1beta) production was positively correlated with the monocyte number and with the number of intracellular TNF-alpha or IL-1beta positive cells in lipopolysaccharide (LPS)-stimulated MNC cultures. The mean ex vivo TNF-alpha and IL-1beta production per 1x10(4)monocytes in these cultures was not significantly different in children at diagnosis of ALL, at remission or in controls. High IL-10 plasma levels at diagnosis of ALL had no effect on the ex vivo TNF-alpha and IL-1beta production of monocytes in LPS stimulated MNC cultures. These results show that monocytes of ALL patients have a normal intrinsic capacity to produce cytokines ex vivo. However, the decreased monocyte number is responsible for the lower TNF-alpha and IL-1beta concentrations ex vivo upon LPS stimulation.

Low HLA-DR expression on peripheral blood monocytes predicts bacterial sepsis after liver transplantation: relation with prednisolone intake.

Bacterial sepsis remains a frequent complication after liver transplantation. We previously reported the results of a pilot study that suggested that low expression of HLA-DR on monocytes is a predictive marker for the occurrence of sepsis. We have studied the value of this marker in an additional cohort of patients, and have analyzed the relation of HLA-DR expression with the use of immunosuppressive agents. 20 adult liver transplantation patients were prospectively monitored during the first 4 weeks after transplantation. All were treated according to standard protocols. The percentage of monocytes expressing HLA-DR was measured by flow cytometry. In addition, the effects of incubation of monocytes with prednisolone in vitro on the expression of HLA-DR was determined in 7 healthy volunteers. Seven patients developed bacterial sepsis after a median 15 (range 10-20) days after transplantation. HLA-DR expression was significantly lower in these patients on days 7, 14, 21, and 28 after transplantation compared with non-septic patients. The percentage of HLA-DR positive monocytes was 30% or less, 3 (1-8) days before onset of sepsis. On day 7 after transplantation, HLA-DR expression on 50% or less of monocytes had a positive predictive value for sepsis of 71%, whereas the negative predictive value was 85%. Patients who developed sepsis received significantly more prednisolone. Incubation with prednisolone in vitro lowered the expression of HLA-DR in a dose-dependent manner. We conclude that low HLA-DR expression on monocytes is a marker for a high risk of subsequent sepsis in liver transplantation patients. This high risk may be (at least partly) related to the dose of prednisolone.

Donor-specific immunological non-responsiveness after liver transplantation.

Since the rate of immunological losses of liver allograft after the immediate posttransplant period is much lower than in other organs, we studied the immune responses against donor HLA antigens in 18 patients with a good long-term outcome to determine whether the development of a state of immunological non-responsiveness to donor antigens might account for this favorable outcome. The reactivity against donor spleen cells was measured before and 2 years after transplantation. The reactivity in mixed lymphocyte culture (MLC) and the frequencies of cytotoxic T cell precursors (CTLp) were determined. Responses against third-party spleen cells were determined concurrently to exclude a generalized reduction of immunocompetence due to chronic immunosuppressive treatment. Before orthotopic liver transplantation, the majority of patients had normal T cell responses against donor antigens that were comparable to those against third-party antigens. Two years after transplantation, donor-specific MLC non-reactivity had developed in 10 of the 18 (56%) patients. In addition, 15 of 18 (83%) patients had developed donor-specific cytotoxic T cell (CTL) non-responsiveness; 2 had reduced numbers of CTLp against both donor and third-party cells, while the remaining patient had maintained reactivity against donor antigens. In conclusion, donor-specific non-responsiveness is present in the majority of patients 2 years after successful liver transplantation, but occurs predominantly at the CTL level.

Quantification of immunosuppression by flow cytometric measurement of intracellular cytokine synthesis.

The availability of a method to measure the effects of drugs on immune reactivity should be helpful in optimizing treatment after organ transplantation. Since cyclosporine A (CSA) interferes with activation of T cells and cytokine synthesis, production of IL-2 and IFN-gamma might constitute a marker of this drug's effects. We measured the capacity for mitogen-stimulated production of these cytokines in whole blood by using immunostaining of intracellular and membrane antigens, followed by flow cytometry. The percentage of CD4+ T cells producing IL-2 or IFN-gamma was strongly reduced in 20 transplant patients compared with 24 healthy controls. The capacity for IL-2 production of CD4+ and CD8+ cells correlated inversely with CSA blood levels (P values 0.0087 and 0.0396, respectively). IFN-gamma production by CD4+ T cells showed a negative correlation with the prednisolone dose (P = 0.0175) and, for the CD8+ subset, with CSA trough levels (P = 0.0023). These data show that inhibition of T cell cytokine synthesis by CSA and prednisolone can be quantified.

Quantitation of immunosuppression by flow cytometric measurement of the capacity of T cells for interleukin-2 production.

BACKGROUND: Methods to quantitate the effects of immunosuppressive drugs on immune reactivity might be helpful for monitoring immunosuppressive treatment. Cyclosporine (CsA) inhibits the induction of cytokine synthesis in T cells, and measurement of interleukin (IL)-2 production might constitute a parameter of this drug's effect. METHODS: We determined the percentages of CD4+ and CD8+ lymphocytes producing IL-2 upon stimulation by phorbol myristate acetate and calcium ionophore in whole blood culture, using immunostaining of intracytoplasmatic and membrane markers, followed by multiparameter flow cytometry. A total of 38 clinically stable transplant patients on various immunosuppressive protocols were studied. RESULTS: The percentage of CD4+ T cells producing IL-2 was strongly reduced in patients compared with healthy controls (23% [range, 3-68%] vs. 59.0% [range, 41-70%]; P=0.000035). The percentage of CD4+ T cells producing IL-2 was negatively correlated with the CsA level (Rc=-0.0821, P=0.00002297) but not with prednisolone or azathioprine doses. Fewer CD8+ T cells produced IL-2 in transplant patients compared with controls, but the difference failed to reach statistical significance. The percentage of CD8+ T cells capable of producing IL-2 was inversely correlated to CsA levels (Rc=-0.0375, P=0.0011). CONCLUSIONS: These data suggest that the functional effects of CsA in transplant recipients can be quantitatively determined and that the capacity of CD4+ T cells to produce IL-2 upon stimulation constitutes a functional parameter of CsA effects on the immune system. Prospective studies are required to determine whether this method is useful for clinical monitoring.

Lipopolysaccharide-induced cytokine production in peripheral blood mononuclear cells: intracellular localization of tumor necrosis factor alpha and interleukin 1 beta detected with a three-color immunofluorescence technique.

Lipopolysaccharide (LPS) can induce monocytes to produce various cytokines such as tumor necrosis factor alpha (TNF alpha) and interleukin 1 beta (IL-1 beta). In the present study, the kinetics of both intracellular and extracellular accumulation of TNF alpha and IL-1 beta in LPS stimulated mononuclear cell (MNC) cultures has been determined. A three-color-immunofluorescence technique was used to detect intracellular accumulation of cytokines. Intracellular accumulation of TNF alpha in monocytes starts shortly after initiation of the culture; i.e., TNF alpha is detectable after 1 h, reaching a peak level after 3-4 hours with 50-65% of monocytes staining positive. In parallel with its increased intracellular presence, TNF alpha was also found in the culture supernatant. The intracellular accumulation of IL-1 beta in monocytes became detectable after 2 h of culture in the presence of LPS. After 4 h, a plateau was reached, with 90% of the monocytes being positive. In parallel, but with a little delay, IL-1 beta could be detected in the culture supernatant. TNF alpha and IL-1 beta can be produced simultaneously in the same monocytes as was shown by a three-color-immunofluorescence technique. It is concluded that TNF alpha and IL-1 beta are good parameters for the early measurement of monocyte activation and that both the intracellular accumulation in monocytes and the amount of secreted cytokines can be used for such a purpose. The intracellular accumulation in monocytes can be measured by the three-color-immunofluorescence technique described.

Increased plasma concentrations of interleukin-1 receptor antagonist in neonatal sepsis.

Newborns are prone to severe infections and sepsis. Cytokines such as tumor necrosis factor-alpha and IL-1 beta play a major role in the initiation of the host response to infections. IL-1 receptor antagonist (IL-1ra) is a naturally occurring antagonist of IL-1 beta. We hypothesized that low IL-1ra plasma concentrations might contribute to the high morbidity and mortality of neonatal sepsis. We studied IL-1ra plasma concentrations during neonatal sepsis. Eleven newborns with severe infection or sepsis, 28 newborns suspected as having sepsis, and eight healthy newborns were enrolled in the study. IL-1ra plasma concentrations proved to be increased in the newborns with severe infections or sepsis (5635 +/- 411 ng/L) versus the concentrations in the suspected group (2597 +/- 433 ng/L) and the control group (273 +/- 88 ng/L) (p < 0.001). After the start of antibiotic therapy, the IL-1ra plasma concentrations remained high during the first 16 h. The IL-1 beta plasma concentrations were increased in the group with a proven infection (78 +/- 27 ng/L) versus the suspected group (37 +/- 7 ng/L) (p < 0.05). Interestingly, the mean Il-1RA plasma concentration is a factor 50-100 higher than the IL-1 beta plasma concentrations. We conclude that IL-1ra in newborns is produced in an amount equal to that in adults. An inadequate IL-1ra response does not seem to contribute to the increased morbidity and mortality of neonatal sepsis.

Diagnostic value of plasma levels of tumor necrosis factor alpha (TNF alpha) and interleukin-6 (IL-6) in newborns with sepsis.

The aim of this study was to examine if TNF alpha and IL-6 plasma levels could be of value in diagnosing neonatal sepsis. Tumor necrosis factor alpha (TNF alpha) and interleukin-6 (IL-6) plasma levels were determined in 15 newborn infants with confirmed sepsis (group I), 18 with suspected sepsis (group II) and 22 control infants (group III). In 33 newborns, initially suspected of having sepsis (groups I and II), a positive test result for plasma concentration of TNF alpha (> 70 pg/ml) had a sensitivity of 73% and a specificity of 94%. A positive test result for IL-6 (> 500 pg/ml) had a sensitivity of 80% and a specificity of 78%. When plasma levels of TNF alpha and IL-6 were combined for the diagnosis of neonatal sepsis, a positive test result for both tests had a sensitivity of 60% and a specificity of 100%. When both tests are positive the diagnosis of neonatal sepsis is almost certain (likelihood ratio = infinity). The combination of TNF alpha and IL-6 determinations appears to be a good predictor of neonatal sepsis.

Tumor necrosis factor-alpha, interleukin-1 beta, and interleukin-6 plasma levels in neonatal sepsis.

Tumor necrosis factor-alpha, IL-1 beta, and IL-6 are thought to be involved in the pathogenesis of sepsis with gram-negative bacteria. We studied these cytokines during neonatal sepsis with mainly gram-positive bacteria. Ten newborns with clinical sepsis and 22 healthy controls were enrolled in the study. TNF alpha plasma levels proved to be increased in the newborns with sepsis up to 560 +/- 234 pg/mL (ng/L) versus 36 +/- 4 pg/mL (ng/L) in the control group (p < 0.005), whereas IL-6 plasma levels in newborns with sepsis were 79.700 +/- 37.500 pg/mL (ng/L) versus 55 +/- 28 pg/mL (ng/L) in the control group (p < 0.01). The IL-1 beta plasma levels were only slightly elevated in the group newborns with sepsis [up to 18 +/- 5 pg/mL (ng/L) versus 7 +/- 1 pg/mL (ng/L) in the control group (p < 0.01)]. After the start of therapy with antibiotics, both TNF alpha and IL-6 plasma levels decreased concomitantly with the improvement of the clinical situation within 2 d. These data confirm the abundant presence of TNF alpha and IL-6 during neonatal sepsis, whereas IL-1 beta appeared to be present in small amounts only. Nevertheless, the IL-1 beta but not the TNF alpha plasma level appeared to correlate inversely with the decrease in diastolic tension as standardized according to birth weight (R = 0.66, p = 0.04). TNF alpha, IL-1 beta, and IL-6 were not correlated with any febrile response in the group with sepsis.(ABSTRACT TRUNCATED AT 250 WORDS)

Recovery from cytomegalovirus infection is associated with activation of peripheral blood lymphocytes.

The number of CD8bright and CD56+ lymphocytes in the peripheral blood and their activation status were monitored by flow cytometry in 23 renal transplant recipients with cytomegalovirus (CMV) infection and were correlated with the virus load (as determined by CMV antigenemia) and clinical symptoms. Recovery from CMV infection coincided with expansion of the CD8bright and CD56+ subsets and with increased expression of the activation marker HLA-DR. Primary infection was associated with activation of both subsets, whereas during secondary infection, mainly CD8bright cells responded. Progressive CMV disease (requiring antiviral treatment) and relapse occurred in association with low numbers of activated CD8bright and CD56+ cells. Lymphocyte activation and antibody responses against CMV often occurred simultaneously, but different kinetics of these responses in some patients indicated that cellular responses are necessary to control viral replication, whereas humoral responses alone may be insufficient. Monitoring of lymphocyte activation may provide clinically useful information during CMV infection.