HLA-DR regulation and the influence of GM-CSF on transcription, surface expression and shedding.

Low surface HLA-DR expression is a feature in sepsis. However, the mechanisms that regulate HLA-DR expression have not been elucidated. The current study investigates regulation of HLA-DR gene transcription, post transcriptional events and shedding of surface HLA-DR, as well as the regulation of HLA-DR by GM-CSF and an immunomodulatory cytokine. Plasma and PBMC were collected from septic patients and healthy volunteers. An ELISA was developed to measure soluble HLA. PCR techniques were used to determine HLA-DR mRNA levels, and flow cytometry and fluorescent microscopy were used for measurement of surface expressed and intracellular HLA-DR. Septic patients fulfilling the criteria of the American College of Chest Physicians (ACCP) for sepsis were recruited for the study (n=70). HLA-DR was measured on three consecutive days, days seven and fourteen. Patients were excluded from the study if on immunosuppressive therapy. Results: Higher levels of shed HLA-DR were found in the plasma of septic patients compared to healthy controls. The level of HLA-DR mRNA was significantly lower in septic patients compared to healthy controls, however an increased intracellular HLA-DR expression was observed. When HL-60 cells were treated with GM-CSF, gene transcription, surface expression and shedding of HLA-DR were all up-regulated. These results indicate that the mechanisms involved in the regulation of HLA-DR in sepsis include shedding of HLA-DR from the cell surface and regulation of HLA-DR gene transcription. Post-translational processing of HLA-DR was also seen to be compromised. GM-CSF was shown to regulate HLA-DR at all these levels.

Is low monocyte HLA-DR expression helpful to predict outcome in severe sepsis?

OBJECTIVE: Low monocyte human leukocyte antigen-DR (HLA-DR) expression has been reported to be an indicator of poor survival in critically ill septic patients. We assessed its usefulness as a prognostic indicator in order to identify possible interventions to normalise HLA-DR expression in those patients with lowered monocyte HLA-DR. DESIGN: HLA-DR expression was measured on separated monocytes of septic patients, using flow cytometry, and HLA-DR upregulation was measured by the same techniques after ex vivo stimulation with granulocyte macrophage colony stimulating factor (GM-CSF). APACHE II score, age, sex and outcome were determined for all patients. SETTING: A single-centre study at the Royal Liverpool University Hospital in a medico-surgical 13-bed intensive care unit. PATIENTS AND PARTICIPANTS: All septic patients ( n=70) fulfilling the criteria of the ACCP for the diagnosis of sepsis were recruited for the study with informed consent from day 1 of diagnosis of sepsis and monocyte HLA-DR expression measured on 3 consecutive days. Patients were excluded from the study if they were on immunosuppressive therapy. Normal healthy volunteers ( n=45) were included. RESULTS: Low monocyte surface expression and median fluorescence density HLA-DR expression was not associated with a high mortality. High APACHE II scores were not correlated with low HLA-DR expression. However, in those patients where HLA-DR expression was lowered, this could be restored ex vivo by GM-CSF. CONCLUSIONS: In the group of septic patients under study, HLA-DR was not a useful prognostic marker of outcome. We did not find a higher mortality in the group of patients who had low expression. These findings are contradictory to some previously reported findings, and the possible reasons are discussed.

Low plasma granulocyte-macrophage colony stimulating factor is an indicator of poor prognosis in sepsis.

OBJECTIVE: Monocyte dysfunction has been shown to be associated with adverse consequences in septic patients. The cytokine growth factor granulocyte-macrophage colony stimulating factor (GM-CSF) may be required for optimal monocyte function in these patients. The current study investigates whether plasma GM-CSF levels were significantly different in septic patients and whether there was an association with prognosis. DESIGN: Plasma samples were collected from all septic patients from day 1 of the diagnosis of sepsis for 3 days. Healthy volunteer plasma served as control samples. A novel enzyme-linked immuno-adsorbent assay was developed with suitable sensitivity for detection of GM-CSF in patient and normal plasma. APACHE II score, age, sex and outcome were determined for all patients. SETTING: A single centre study at the Royal Liverpool University Hospital in a medico-surgical 13 bed intensive care unit. PATIENTS: All septic patients (n = 53) fulfilling the criteria of the APCC for the diagnosis of sepsis, were recruited for the study with informed consent from day 1 of the diagnosis of sepsis and plasma GM-CSF measured on three consecutive days. Patients were excluded from the study if on immunosuppressive therapy. Normal healthy volunteers (n = 33) were included in the study to serve as controls. RESULTS: Plasma GM-CSF levels were statistically significantly depressed in patients who died compared with those who survived, who had levels comparable with healthy controls. CONCLUSIONS: The results indicate that low plasma GM-CSF is associated with adverse consequences for septic patients. The measurement of GM-CSF in the plasma of septic patients merits further study for use as a prognostic marker and also to identify the type of immunotherapy the patient may benefit from.