Differential immunostimulating effect of granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte colony-stimulating factor (G-CSF) and interferon gamma (IFNgamma) after severe trauma.

OBJECTIVE: Severe trauma leads to an increased vulnerability to bacterial sepsis. In the present study, we compared the immunostimulating potential of granulocyte-colony stimulating-factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF) and interferon-gamma (IFN-gamma). DESIGN: Prospective clinical experimental study. SETTING: University hospital intensive care unit and research facility. PATIENTS: 6 patients with an Injury Severity Score (ISS) of more than 25 points. INTERVENTIONS: Heparinized blood samples of severely injured patients and 12 healthy volunteers were incubated in vitro with 10 ng/ml GM-CSF, 10 ng/ml G-CSF or 10 ng/ml IFN-gamma for 6 h. MEASUREMENTS: Flow cytometry: HLA-DR expression on monocytes, B- and T-lymphocytes. ELISA: LPS-induced TNFalpha and IL-10 production. RESULTS: In all patients reduced cytokine production and HLA-DR expression on monocytes was established. After administration of GM-CSF and IFN-gamma it in vitro, the level of HLA-DR expression on monocytes and the it ex vivo TNFalpha-synthesis increased while only GM-CSF increased significantly IL-10-liberation after LPS-stimulation. However, only IFN-gamma had the capacity to enhance HLA-DR on B- and T-lymphocytes. G-CSF it in vitro had no significant effect on the measured parameter. CONCLUSIONS: These data suggest that GM-CSF and IFN-gamma may serve to support immune functions in severely injured patients.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) restores decreased monocyte HLA-DR expression after cardiopulmonary bypass.

OBJECTIVES: Cardiopulmonary bypass (CPB) is associated with a disturbed immune response, e.g., impaired HLA-DR expression on monocytes and the release of pro- and anti-inflammatory cytokines. Cytokine release plays a role in the pathogenesis of postoperative systemic inflammatory response syndrome (SIRS) and immune system deterioration, e.g., impaired monocyte and polymorphonuclear neutrophil (PMN) function, factors that ultimately lead to an increased susceptibility to infections. To gain a further understanding, we investigated HLA-DR expression on monocytes and on B- and T-lymphocytes. In addition, we investigated the IN VITRO effect of the immunostimulating hematopoietic growth factor granulocyte-macrophage colony-stimulating factor (GM-CSF) on HLA-DR expression of these cell types. Neither HLA-DR expression on B- and T-lymphocytes nor the effects of GM-CSF in cardiac surgical patients have been studied before. METHODS: In 16 patients undergoing elective cardiac surgery with CPB, counts of circulating leukocyte subsets as well as HLA-DR expression on monocytes, B- and T-lymphocytes were measured by flow cytometry before, immediately after CPB, and on the 2nd and 10th postoperative days. Treatment with GM-CSF was performed IN VITRO in whole blood cultures with 100 ng/ml recombinant human GM-CSF for 20 h. RESULTS: Monocyte HLA-DR expression was attenuated immediately after CPB (125 +/- 4 mean channel fluorescence [MCF] vs. 143 +/- 2 MCF preoperatively, mean +/- SEM, P < 0.001). HLA-DR expression further decreased on the 2nd day after CPB and did not normalize until the 10th day after the operation. In contrast, HLA-DR expression on T-cells was unchanged, whereas HLA-DR expression on B-cells did not decrease before the 2nd day after CPB (152 +/- 3 MCF vs. 170 +/- 2 MCF preoperatively, P < 0.001). IN VITRO GM-CSF treatment increased HLA-DR expression on monocytes prepared after CPB to a degree comparable to preoperative values. HLA-DR expression on B-lymphocytes could not be restored by GM-CSF. CONCLUSIONS: Immune system suppression after cardiac surgery is reflected in prolonged diminished HLA-DR expression on monocytes and B-lymphocytes. Suppression is not irreversible but can - at least IN VITRO - be overridden by the immunostimulating compound GM-CSF.

Nodular pulmonary vasculitis in a twelve-year-old boy.

A 12-year-old boy presented with left shoulder pain during physical exercise and complained of uncommon sweating and fatigue. Diagnostic evaluation revealed a solitary pulmonary nodule in the left upper lobe. All laboratory values were within normal limits, except for an elevated level of antineutrophil cytoplasmic antibodies directed against myeloperoxidase (p-ANCA). Surgery was performed, and pathological examination showed a localized granulomatous vasculitis. Antineutrophil cytoplasmic antibodies directed against affinity purified proteinase 3 (p-ANCA) concentrations returned to baseline within 6 months, and the patient has done well during a follow-up period of 2 years. While nodular vasculitis is known to occur in Wegener's granulomatosis, to the best of our knowledge, this case represents the first c-ANCA negative primary pulmonary vasculitis in childhood.

Immunosuppression with FK506 increases bone induction in demineralized isogeneic and xenogeneic bone matrix in the rat.

The aim of the present study was to investigate a systemic induction of bone formation in rats by immunosuppression with FK506 (1 mg/kg body weight intraperitoneally [ip]) in a model of osteoinduction of isogeneic and xenogeneic demineralized bone matrix (DBM) for a period of 28 days. In particular, alterations of in vitro cytokine synthesis and changes of lymphocyte subsets were studied. DBM was implanted intramuscularly in the abdominal wall of Lewis rats (seven per group). Blood was sampled on days -7, 0, 7, and 28 for determination of in vitro tumor necrosis factor a (TNF-alpha) synthesis and lymphocyte subsets by flow cytometry (CD3+, CD4+, CD8+, CD45+, ED9+, and Ia+ antibodies). Ossicles of de novo formed bone and the tibias were removed on day 28 after double tetracycline labeling for histomorphometric analysis. Immunosuppression with FK506 significantly decreased lipopolysaccharide (LPS)-stimulated in vitro cytokine synthesis after 7 days and 28 days (p < 0.05). Compared with control animals FK506 treatment significantly increased the volume of induced bone in isogeneic (2.1 +/- 0.3 mm3 vs. 10.8 +/- 0.9 mm3) and xenogeneic (O mm3 vs. 4.7 +/- 0.8 mm3) DBM. Bone histomorphometry of the tibias revealed that immunosuppression increased both bone formation and bone resorption, accompanied by a significant reduction in the relative trabecular area (Tb.Ar). FK506 caused a decrease in the counts of CD8+ T cells probably because of destruction or dislocation of these cells. This suggests that the amount of CD8+ cells and the degree of T cell activation in terms of mean fluorescence intensity (MFI) may be associated with bone metabolism. In support of this, statistical analysis revealed a significant positive correlation between parameters of bone formation as well as bone resorption and the CD4+/CD8+ ratio. There was a significant negative correlation between parameters of remodeling of the metaphysis of the tibia and induced bone volume (BV), respectively, and MFI values of CD3+/Ia+ cells. These findings suggest an important role of T lymphocytes in bone formation and bone resorption in vivo. FK506 caused a marked increase of bone formation in DBM. However, the conclusion that immunosuppression increases fracture healing warrants further investigation.

Influence of granulocyte-macrophage colony-stimulating factor (GM-CSF) on whole blood endotoxin responsiveness following trauma, cardiopulmonary bypass, and severe sepsis.

Major surgery, multiple injury, and severe sepsis lead to an impaired immune response. The suppressed status of the immune system is reflected by a reduced TNFalpha production of whole blood after stimulation with endotoxin in vitro and by a decreased HLA-DR expression on monocytes. In the present study, the effect of the immunostimulating hematopoetic growth factor GM-CSF on whole blood cultures of multiple injury, cardiac surgery, and severe sepsis patients was investigated. Endotoxin-induced TNFalpha production and HLA-DR expression was reduced in blood cultures of these patients compared to healthy donors. Preincubation with GM-CSF in vitro increased cytokine production in volunteers' and all patients' blood specimens in a dose-dependent manner. The elevation of cytokine response in cardiopulmonary bypass patients' blood, caused by in vitro preincubation with GM-CSF, equaled that of normal patients, whereas GM-CSF caused a lower rise of TNFalpha-producing capacity in blood of multiple-injury and sepsis patients. Further, GM-CSF treatment in vitro increased the down-regulated HLA-DR expression on monocytes prepared after cardiac surgery to a degree comparable to preoperative levels. Finally, GM-CSF incubation in vitro elevated TNFalpha synthesis in normal monocytes and in cells treated with a combination of the anti-inflammatory mediators IL-10, TGFbeta, and PGE2. These experiments show that hyporesponsiveness of whole blood induced by trauma, sepsis, or cardiac surgery is not irreversible but can be, at least in vitro, overridden by the immunostimulating compound GM-CSF.

Impact of localized radiotherapy on blood immune cells counts and function in humans.

Immune cells subsets were prospectively analyzed after localized radiotherapy (LRT). LRT reduced the levels of all lymphocyte subsets, with B-cells and naive T-cells being most sensitive. Lymphocyte function was suppressed, but still within the normal range. Rapid recovery of cytotoxic T-cells/natural killer cells after LRT and the functional suppression within normal levels explains the low incidence of infections after LRT.

The extent of traumatic damage determines a graded depression of the endotoxin responsiveness of peripheral blood mononuclear cells from patients with blunt injuries.

OBJECTIVE: To study whether the endotoxin responsiveness of peripheral blood mononuclear cells correlates with the severity of injury in trauma patients. DESIGN: Prospective, observational study. SETTING: University trauma center. PATIENTS: Fifty-nine patients with blunt trauma (Injury Severity Score [ISS] 4 to 57 points). INTERVENTIONS: Standard emergency department care, surgical care, and postoperative intensive care unit treatment. MEASUREMENTS AND MAIN RESULTS: Whole blood and serum were obtained 94+/-89 (SD) mins post trauma (day 0) and during a 14-day period postinjury. Endotoxin-induced tumor necrosis factor-alpha (TNF-alpha) synthesis of peripheral blood mononuclear cells ex vivo was tested using a whole blood assay. Serum samples were assayed for TNF-alpha concentrations. A reduced capacity of whole blood to produce TNF-alpha ex vivo with endotoxin treatment was found to be closely correlated with the ISS. The capacity to produce TNF-alpha on endotoxin stimulation of whole blood from patients with an ISS > or =16 points was depressed immediately after trauma and did not reach normal values during the observation period. In patients with an ISS >22 points, maximum depression of the capacity of whole blood to produce TNF-alpha occurs within 100 mins post injury. In contrast, in patients with an ISS <22 points, maximal depression of whole blood TNF-alpha production occurs with a delay of 24 to 48 hrs after trauma. Based on pre- and postoperative values, primary surgical intervention caused a decrease of the endotoxin-stimulated TNF-alpha production of whole blood in the latter patient subgroup, as well as in the entire patient population (ISS 4 to 57) when secondary surgical treatment was necessary 5 to 13 days after trauma. CONCLUSIONS: The extent of traumatic tissue damage leads to a graded depression of immunocyte function and appears to be amplified by surgical treatment. The endotoxin responsiveness of peripheral blood mononuclear cells displays a functional marker of the anatomically defined severity of injury and gives insights into the regulation of immunocyte function after severe blunt trauma.

HLA-DR expression and soluble HLA-DR levels in septic patients after trauma.

OBJECTIVE: To determine if cellular and soluble HLA-DR molecules may be relevant in severely injured patients for the development of gram-positive or gram-negative sepsis. SUMMARY BACKGROUND DATA: HLA-DR molecules play a central role in the specific immune response to infection. The reduced HLA-DR expression on monocytes is considered to correlate with infectious complications and the development of sepsis. Data on the role of HLA-DR expression on T cells and soluble HLA-DR molecules are rare. METHODS: HLA-DR expression on monocytes and T cells was measured by flow cytometry. Plasma levels of soluble HLA-DR were studied by enzyme-linked immunosorbent assay. RESULTS: HLA-DR expression on circulating T cells, calculated as mean fluorescence intensity in channels, was reduced at day 1 after admission in 20 patients with subsequent severe sepsis compared with 46 patients without sepsis. The septic patients immediately after trauma had significantly lower soluble HLA-DR plasma levels than the nonseptic patients. At day 2 after admission, HLA-DR expression on monocytes was significantly lower in the severe sepsis group than in the patients without sepsis, and lasted until day 14 after injury. CONCLUSIONS: In severely injured patients, decreased levels of cellular and soluble HLA-DR appear as early indicators of an immune deviation associated with the development of severe sepsis. Moreover, immune alterations of different cell types may promote distinct kinds of septicemia.

Reduced B cell HLA-DR expression and natural killer cell counts in patients prone to sepsis after injury.

OBJECTIVE: To examine the influence of natural killer (NK) cells and HLA-DR molecules on B cells in the development of severe sepsis after injury. DESIGN: Prospective study. SETTING: Medical school, Germany. SUBJECTS: 46 severely injured (Injury Severity Score >16) patients. INTERVENTIONS: Blood samples were taken immediately after admission and subsequently for 14 days. MAIN OUTCOME MEASURES: HLA-DR expression on B cells and counts of B and NK cells measured by flow cytometry, and morphological estimation of large granular lymphocytes by microscopy. RESULTS: HLA-DR expression on circulating B cells was significantly reduced from days 6-14 after admission in 13 patients with subsequent severe sepsis compared with 33 patients who did not develop sepsis. In septic patients NK cell counts were significantly decreased from day 4 onwards (p < 0.05). CD16+/CD56+ cells correlated with the morphology of large granular lymphocytes. CONCLUSION: In severely injured patients reduced counts of NK cells and HLA-DR molecules on B lymphocytes seem to be part of an immune deviation that is associated with the development of severe sepsis.

Alveolo-capillary protein permeability and lung function in patients with late pulmonary complications after allogeneic bone marrow transplantation.

A prospective study was performed to identify markers predictive for the development of pulmonary complications in the early (<50 days) and late (>50 days) phase after bone marrow transplantation (BMT). The characterization of BMT patients with early or late pulmonary complications revealed clear-cut differences. Early and long term increase of alveolo-capillary protein permeability was associated with smoking and was found in 20 patients developing pulmonary complications within 50 days after BMT (group 1). The 22 patients who developed such complications thereafter (group 2) had more acute graft vs host disease than 66 patients who remained free of these complications for a minimum of 1 year. Concentrations of bronchoalveolar lavage (BAL) fluid albumin (alb) and serum beta2-microglobulin (S-beta2m) were determined 10 days before BMT, on days 1, 30, and 40 after BMT, whereas lung function tests were performed before BMT, after discharge from the hospital, and 6 months as well 1 year after BMT. Using cut-off values for BAL fluid alb (>2.3 mg/dl) and S-beta2m (>0.8 mg/liter) we could significantly discriminate 12 patients out of 19 group 1 patients (early pulmonary complications) as well as 9 out of 21 group 2 patients (late pulmonary complications) from 12 out of 64 group 3 patients (without such complications) 1 day after BMT. Our results demonstrate that early increased alveolo-capillary protein permeability defines a patient population at risk to develop pulmonary complications later than 50 days after BMT with up to 1 year significantly decreased lung volumes (FEV1, 73% predicted, VC, 85% predicted).

Immunoglobulin and beta 2-microglobulin concentrations in bronchoalveolar lavage of children and adults.

Immunoglobulins play an important role in the pulmonary host defense, but little information is available about immunoglobulin and beta 2-microglobulin concentrations in the lung of normal children. Using bronchoalveolar lavage (BAL) we have studied immunoglobulin and beta 2-microglobulin levels in 30 children 3-15 years old undergoing elective surgery for nonpulmonary illnesses and in 15 healthy adult volunteers. BAL was performed with 3 x 1 ml/kg of body weight normal saline through an endotracheal tube after induction of anesthesia in children and under local anesthesia in adults. Similar concentrations of IgA and IgG were found in BAL fluid of children and adults even though serum levels were lower in children. As comparable results were obtained for albumin, a serum-derived protein, these data suggest that the permeability of the alveolar membrane is higher in children. IgE and IgM were detected in BAL fluid in only a fraction of children. beta 2-microglobulin levels were higher in both blood and BAL fluid of children. These data provide the first reference data for immunoglobulin and beta 2-microglobulin in children and can serve as a basis for future studies of children with pulmonary diseases.

Association of increased bronchoalveolar lavage fluid albumin and serum beta 2-microglobulin with pulmonary complications after allogeneic bone marrow transplantation.

The aim of this prospective study was to identify markers in bronchoalveolar lavage fluid (BAL fluid) and serum predictive for the development of pulmonary complications in the early phase (< 50 days) post-BMT. Concentrations of BAL fluid albumin (alb) and serum beta 2-microglobulin (S-beta 2m,) were determined 10 days before BMT (BAL-B, baseline) and on day 1 post-BMT (BAL-1) in 20 patients who subsequently developed pulmonary complications (group 1) and in 66 patients who remained free of complications for a minimum of 12 months (group 2). Median BAL fluid alb concentrations were significantly (P < 0.05) higher in group 1 patients as compared to group 2 patients at BAL-B (40 vs 28 mg/l) and at BAL-1 (30 vs 15 mg/l). S-beta 2m at BAL-1 was also significantly elevated in group 1 patients (median 1.3 mg/l) compared to group 2 patients (median 1.15 mg/l). Using cut-off values for BAL fluid alb (> 23 mg/l) and S-beta 2m (> 0.8 mg/l) we identified 12 patients out of 19 who developed subsequent pulmonary complications from 12 out of 62 patients without such complications, 1 day post-BMT.

Lupus-like autoimmune disease induced by interferon therapy for myeloproliferative disorders.

Symptoms of autoimmune disease were evaluated in 125 patients with chronic myelogenous leukemia (CML) and in 12 patients with essential thrombocythemia undergoing treatment with recombinant interferon (IFN)-alpha-2b plus/minus low-dose recombinant IFN-gamma. Twenty-seven of 137 patients (20%) developed rheumatoid symptoms. Furthermore, the incidence of antinuclear antibody (ANA) formation was studied. Elevated ANA titers were found in 5/19 (26%) of CML patients at the time of diagnosis and in 3/18 (17%) of patients treated with hydroxyurea or busulfan. During IFN treatment, 18 of 25 tested patients (72%) had elevated ANA titers. In 15 of these ANA-positive patients, clinical signs of autoimmune disease appeared. All these patients were under long-term IFN treatment and were in remission of disease. In three patients criteria for systemic lupus erythematosus were fulfilled. Severity of side effects had led to the discontinuation of IFN treatment in these patients. The data indicate that IFN-alpha and IFN-gamma can induce ANA associated with autoimmune disease in patients with myeloproliferative disorders.

[Post-traumatic alveolar changes in lung contusion]. / Posttraumatische alveoläre Veränderungen nach Lungenkontusion.

Bronchoalveolar lavage (BAL) specimens taken from nine patients with lung contusion following multiple trauma were compared with specimens from different control groups. Early interstitial and intra-alveolar reactions are PMN degranulation, mediator release and high protein leakage. The alveolar reactions are similar in extent to the reaction found in post-traumatic ARDS.

Antagonism of the estradiol-mediated repression of microsomal 3 beta-hydroxysteroid dehydrogenase activity in rat liver by antiestrogenic substances.

5 alpha-Dihydrotestosterone, 17-hydroxyprogesterone caproate, 2-methoxyestrone and a number of nonsteroidal antiestrogens (clomiphene citrate, nafoxidine hydrochloride, tamoxifen, MER-25) were tested for their ability to block estradiol-mediated repression of the androgen-dependent 3 beta-hydroxysteroid dehydrogenase activity of male rat liver. With the exception of 5 alpha-dihydrotestosterone, which induced activity in females, none of these substances affected 3 beta-hydroxysteroid dehydrogenase activity when administered alone to otherwise untreated male and female rats. Tamoxifen (100 or 500 micrograms/day) was the only substance which prevented a decrease in enzyme activity when given simultaneously with estradiol (5 micrograms/day). The estradiol-mediated decrease in activity was not antagonized by a 100-fold higher dose of androgen (5 alpha-dihydrotestosterone, 0.5 mg/day), demonstrating the potent antiandrogenic effect of estradiol on this hepatic androgen-dependent enzyme activity.

Time-studies of the changes in the sex-dependent activities of enzymes of hepatic steroid metabolism in the rat during oestrogen administration.

This investigation was undertaken to elucidate the amount of oestradiol and duration of its administration necessary to cause complete feminization of the activities of cytoplasmic 3 alpha- and 17 beta-hydroxysteroid dehydrogenase, microsomal 3 alpha- and 3 beta-hydroxysteroid dehydrogenase and microsomal 5 alpha-reductase in male rat liver. With the exception of cytoplasmic 3 alpha-hydroxysteroid dehydrogenase, 5 microgram oestradiol/d for 8 days and less was sufficient to cause complete feminization. The order of oestrogen sensitivity was cytoplasmic 3 alpha-hydroxysteroid dehydrogenase greater than microsomal 3 beta-hydroxysteroid dehydrogenase greater than microsomal 3 alpha-hydroxysteroid dehydrogenase greater than microsomal 5 alpha-reductase greater than cytoplasmic 17 beta-hydroxysteroid dehydrogenase. Although the changes occurring after oestradiol administration are qualitatively the same as after testectomy, they occur more rapidly. This rules out the possibility that oestradiol exerts its effect via androgen deprivation. Diethylstilboestrol administration causes the same changes in cytoplasmic 17 beta- and microsomal 3 beta-hydroxysteroid dehydrogenase activity as oestradiol, although the dosage must be increased 100 fold. The effect of diethylstilboestrol on 5 alpha-reductase activity changes with the dose applied. Doses up to 100 microgram/d partially feminize the activity, but at higher doses the enzyme activity is repressed.