Samuel R. Wellhausen

HLA-DR antigen expression on peripheral blood monocytes correlates with surgical infection

Monocyte human leukocyte antigen-DR (HLA-DR) expression has correlated closely with clinical outcome in severely injured patients at high risk for infection. Monocytes from 77 asymptomatic volunteers expressed HLA-DR antigen with minimal variability in respect to age, gender, race, time of day or year, or serum alcohol level. Patients who developed infection after elective laparotomy had a significantly lower mean percentage of monocytes expressing HLA-DR antigen and a lower mean fluorescent intensity than uninfected patients (p less than 0.05). Severely infected nonsurgical patients had significantly lower values than normal volunteers (p less than 0.01), and the mean fluorescent intensity of those who died from infection was significantly lower than that of those who survived (p less than 0.05). Patients on immunosuppressive regimens after renal transplantation had levels of HLA-DR expression similar to those of the volunteers. Monocyte HLA-DR expression was found to be a reliable marker of clinical infection and showed remarkable reproducibility within the normal uninfected study population.

Polymorphonuclear leukocyte function during hemodialysis: relationship to complement activation

Phagocytosis, H2O2 production, and C3bi receptor (CR3) expression by polymorphonuclear leukocytes (PMN) obtained from patients before, during, and after a hemodialysis treatment were evaluated by flow microfluorometry. The results were compared to changes in plasma levels of C3ades Arg and C5ades Arg. Prior to hemodialysis C3ades Arg and C5ades Arg levels, CR3 expression and phagocytosis were not different from normal controls. However, both basal and phagocytosis-induced H2O2 production were increased. C3ades Arg and C5ades Arg were increased after 15 min of dialysis; this was accompanied by transient but significant reductions in PMN count and phagocytosis and increased CR3 expression. No changes in basal or stimulated H2O2 production were observed. We conclude that PMN of hemodialysis patients are primed for an enhanced respiratory burst before dialysis is initiated. Dialysis-induced complement activation after the initiation of dialysis does not further stimulate H2O2 production or enhance the response to phagocytosis. However, complement activation may cause leukopenia and CR3 expression.

Experimental and clinical significance of endotoxin-dependent HLA-DR expression on monocytes

For much of the last decade, an increasing number of surgeons have been interested in objective assessment of cellular contributors to host defense function. In order to study many of these processes, it is apparently desirable that the cells be isolated to the extent feasible for the purpose of analyzing a more or less pure population of cellular elements. The purpose of this paper is to describe the physiologic activation of mononuclear cells that occurs as a result of the isolation process. Therefore, it follows logically that such cells are therein intrinsically less responsive to further physiologic manipulation in vitro. Analyses of such data without an awareness of this intrinsic aberration will undoubtedly lead to misinterpretation of the capacity of such cells for further modulation by immunostimulants or by the intrinsic processes related to injury, anesthesia, and operation. Furthermore, it may indicate that certain agents, e.g., cytokines, are unable to stimulate cellular function when, in fact, the defense function of the cell has been initially stimulated by the isolation procedure. Fractionation of human peripheral blood over Hypaque-Ficoll and subsequent purification of monocytes by adherence to plastic lead to an increase in the relative density of HLA-DR on monocytes. This increase occurred when carried out in endotoxin lipopolysaccharide (LPS)-contaminated or LPS-depleted reagents. LPS, added experimentally to whole blood, enhanced HLA-DR expression on monocytes without further manipulation. Monocyte HLA-DR expression measured in whole blood was reduced in patients with major sepsis (n = 19) compared to normal subjects (n = 10).(ABSTRACT TRUNCATED AT 250 WORDS)

Evaluation of intraoperative autotransfusion filtration for hepatectomy and pancreatectomy

BackgroundHepatectomy and pancreatectomy are often associated with significant intraoperative blood loss leading to postoperative anemia, which has been demonstrated to lead to increased perioperative morbidity, a prolonged hospital stay, and decreased overall survival. Cancer has remained an absolute contraindication to autotransfusion because of the unproven concern about reinfusion of malignant cells. Thus, the aim of this study was to test for the presence of malignant cells in autotransfused filtered blood in patients undergoing major pancreatic and liver resection.MethodsA prospective study of 20 consecutive patients evaluated the presence of malignant cells from autotransfusion filtered blood after resection by flow cytometric and immunohistochemical methods.ResultsTen patients underwent major hepatectomy for metastatic colorectal cancer, with a median blood loss of 500 mL (range, 200–700 mL). Three patients received a total of six units of packed red blood cells. Ten patients underwent pancreaticoduodenectomy for adenocarcinoma with a median blood loss of 400 mL (range, 200–1300 mL). Five patients received a total of nine units of packed red blood cells. Flow cytometry did not demonstrate the presence of any cytokeratin-positive carcinoma cells in filtered blood.ConclusionsIntraoperative autotransfusion for major hepatectomy in metastatic colorectal cancer and pancreatectomy for adenocarcinoma is safe and should begin to be evaluated in a phase II study for efficacy.

Role of intracellular calcium in priming of human peripheral blood monocytes by bacterial lipopolysaccharide

To determine the role of intracellular calcium ([Ca2+]i) in the priming of monocytes (MΦ) by bacterial lipopolysaccharide (LPS), the membrane expression of two functional proteins and phagocytosis and respiratory burst were examined by microfluorimetry. LPS induced a significant increase in HLA-DR and C3bi receptor (CR3) expression within 2 h of its addition to whole blood. The enhanced expression of both antigens by LPS was dose-dependent, with concentrations as low as 0.1 ng/ ml producing a response. The involvement of [Ca2+]i was demonstrated by loading isolated MΦ with the intracellular calcium chelator quin-2 or the inhibitor of intracellular calcium redistribution TMB-8 prior to addition of LPS. Both compounds inhibited the LPS-induced increase in HLA-DR and CR3 expression. No role for extracellular calcium, for calcium slow channel flux, or for the calcium-calmodulin complex in LPS priming was demonstrated when LPS was added in the presence of EGTA, trifluperazine (TFP), or verapamil. The addition of the calcium ionophores A23187 or ionomycin failed to increase expression of either antigen. Prior exposure to LPS primed MΦ for enhanced phagocytosis and respiratory burst activity. These functions were inhibited by TMB-8, but not by TFP or verapamil. Addition of LPS to isolated MΦ increased [Ca2+]i by 23% at 30 sec and 42% at 5 min, as measured by the calcium-sensitive, intracellular probe indo-1. These results suggest that intracellular Ca2+ mobilization is necessary, but not sufficient, for LPS-induced priming of human peripheral blood monocytes.

The capacity of serum to support neutrophil phagocytosis is a vital host defense mechanism in severely injured patients.

The opsonic capacity of patient serum was studied in 43 trauma patients of whom 13 recovered uneventfully, 21 developed major infection, and nine died, mostly of infection. Blood samples were taken within 24 hours of injury. Fifteen patients were studied serially of whom 14 developed severe infection and/or died. Opsonic capacity was determined by flow cytometry and measured as the ability of normal neutrophils to phagocytose killed bacteria previously incubated with patient serum. The most dilute sera reflected changes for better and worse most clearly. On initial assessment, those who died of sepsis showed a 61% mean fluorescent intensity (MFI), which was significantly lower than the 99% MFI for those who survived infection (p < 0.01) and the 78% MFI of those who developed no infection (p < 0.05). Serial samples demonstrated a super serum response in four of seven patients surviving major sepsis but in none of the seven who died of infection.

Biochemical basis of HLA-DR and CR3 modulation on human peripheral blood monocytes by lipopolysaccharide

The biochemical events leading to enhanced membrane expression of HLA-DR and CR3 by human peripheral blood monocytes (MO) following exposure to bacterial lipopolysaccharide (LPS) were examined. In a previous study we demonstrated that an increase in intracellular calcium was necessary, but not sufficient, for MO to increase membrane expression of both antigens within 1 hr of addition of LPS. The present study was initiated to examine the other biochemical requirements which lead to the MO response to LPS. Enhanced expression of both antigens following addition of LPS was dependent on microfilament function, but independent of microtubule function and of protein synthesis. Inhibition of formation of cyclooxygenase or lipoxygenase metabolites of arachidonic acid had no effect on HLA-DR or CR3 modulation by LPS. A role for phosphatidylinositol metabolism was suggested by the inhibition of the MO response to LPS by dibutyryl cAMP and theophylline and by the enhanced expression of both antigens following addition of phorbol diesters. However, H-7, a putative inhibitor of protein kinase C, did not alter the MO response to LPS or phorbol diesters. These results suggest that LPS enhances expression of HLA-DR and CR3 by inducing redistribution of these antigens from an intracellular pool. The data also support a role for the generation of hydrolysis products of phosphatidylinositol, leading to calcium redistribution and activation of protein kinase C or other kinases, in the MO response to LPS.

Mechanism by which methylprednisolone inhibits acute immune complex-induced changes in vascular permeability

Intravital microscopy was used to quantitate protein leakage which resulted from the deposition of immune complexes in the vasculature of the rat cremaster muscle. Immune complex deposition was initiated by the addition of 80 μg/ ml of ovalbumin to the bath surrounding the muscle, followed by the intravenous administration of antiovalbumin. Administration of 25 mg/kg of antiovalbumin produced significant leakage of protein from the third-order venules, while 7.5 and 2.5 mg/kg had no effect. Administration of methylprednisolone (MP), 30 mg/kg, 1 h prior to the deposition of immune complexes significantly inhibited protein leakage. In separate experiments, MP inhibited intradermal edema formation and protein exudation induced in rats by histamine, platelet activating factor, or C5a. However. MP had no effect on protein exudation or edema produced by xanthine oxidase or glucose oxidase. Intravenous administration of MP inhibited the ability of polymorphonuclear leukocytes (PMNs) to phagocytize bacteria, but failed to alter hydrogen peroxide production. These results suggest that MP prevents acute changes in vascular permeability following immune complex deposition by inhibiting the effects of soluble mediators of edema on vascular endothelium and by inhibiting PMN phagocytosis.

IL-4 production by CD8+ lymphomatoid papulosis, type C, attracts background eosinophils

There are two subsets of CD8+ T cells: Tc1 and Tc2. INF‐γ production by Tc1 cells causes granulomatous inflammation. IL‐4 production by Tc2 cells attracts eosinophils. A 76‐year‐Caucasian female presented with CD8+ lymphomatoid papulosis (LyP), type C. We hypothesized that the LyP cells belonged to the Tc2 subset because of abundant background eosinophils. Hematoxylin and eosin and immunohistochemical stains were carried out on tissue sections from a skin punch biopsy. Antibodies for immunohistochemical stains included CD3, CD4, CD5, CD7, CD8, CD30, CD56, ALK‐1, clusterin and IL‐4. There was involvement of the dermis by a dense lymphoid infiltrate composed of large atypical cells and numerous eosinophils. The LyP cells expressed CD5, CD8, CD30 and IL‐4. Keratinocytes showed a membranous pattern of immunoreactivity for IL‐4. IL‐4 production by CD8+ LyP, type C indicates that it belongs to the Tc2 subset. The cytokine milieu produced by the LyP cells attracted eosinophils. The IL‐13R complex on keratinocytes bound IL‐4 and produced a membranous staining pattern. Although CD8+ LyP is rare, we believe that this CD30+ lymphoproliferative disorder should be included in the World Health Organization‐European Organization for Research and Treatment of Cancer classification of cutaneous T‐cell lymphomas.

Interleukin-10 inhibits interleukin-2-induced tumor necrosis factor production but does not reduce toxicity in C3H/HeN mice.

Immunotherapy with interleukin‐2 (IL‐2) is limited by severe side effects thought to be mediated by the activation of immune effector cells and the induction of secondary cytokines including tumor necrosis factor‐alpha (TNF‐α) and interferongamma (IFN‐γ). In C3H/HeN mice the primary IL‐2 toxicity is the production of pleural effusion with subsequent respiratory compromise. IL‐10 is a cytokine that has been shown to inhibit the generation of secondary cytokines in vitro and in vivo. In this study, in C3H/HeN mice, we tested the ability of IL‐10 to inhibit IL‐2‐induced mononuclear cell and alveolar macrophage activation and IL‐2‐induced increases in serum TNF‐α and IFN‐γ, all of which may contribute to the generation of toxicity. IL‐10 was ineffective at reducing IL‐2‐induced pleural effusion. However, IL‐10 did inhibit IL‐2‐induced increases in serum TNF‐α, which was accompanied by a decrease in Golgi apparatus and rough endoplasmic reticulum in alveolar macrophages. In addition, ILIO combined with IL‐2 increased mononuclear cell activation, which may limit the ability of IL‐10 to inhibit IL‐2‐induced IFN‐γ production and pulmonary injury.