Search

Septic complications have been major problems in the management of patients with obstructive jaundice and neonatal jaundice. This study investigates effects bilirubin on human T lymphocyte responses against allogeneic mixed lymphocyte reaction. In vitro exposure of human peripheral blood mononuclear cells (PBMNC) with unconjugated bilirubin at pathological levels (6 to 12 mg/dl) did not alter the subsets of CD3, CD4, CD8, CD14, CD19 and CD56 positive populations, or expression of costimulatory surface molecules CD2, CD3, CD4 and CD8. Further incubation of bilirubin-treated PBMNC with irradiated B lymphoid Raji cells after removal of the extracellular bilirubin resulted in a dose-dependent decrease of cytotoxic T lymphocyte (CTL) activity, DNA synthesis, and expression of Tac antigen (CD25) and transferrin receptor (CD71). However, no significant change of interleukin-2 (IL-2) production was observed after this incubation between bilirubin-treated and -untreated PBMNC. These results suggest that bilirubin inhibits the induction of CTL activity, and this defect may result from the impaired responsiveness against IL-2. These observations may help explain the increased infection observed in hyperbilirubinemic patients.

Decreased immune responses have been documented in hyperbilirubinemic patients. This study investigates the effects of intracellular bilirubin accumulation on lymphoproliferative response to phytohemagglutin A (PHA). Human peripheral blood mononuclear cells (PBMNC) were preincubated with unconjugated bilirubin dissolved in bovine albumin solution at pathological levels seen in clinical hyperbilirubinemia (0-12 mg/dl), washed, and further cultured with PHA. DNA synthesis was measured by [3H]thymidine uptake. Interleukin-2 (IL-2) activity was determined by the CTLL proliferation assay. The amount of intracellular bilirubin and expression of cell surface antigens were analyzed by flow cytometry. In vitro exposure of normal PBMNC to bilirubin resulted in the accumulation of intracellular bilirubin and a decrease in DNA synthesis after PHA stimulation in a time- and dose-dependent manner. Addition of autologous untreated monocytes could not correct the decreased DNA synthesis of bilirubin-treated lymphocytes. IL-2 production by bilirubin-treated PBMNC after PHA stimulation was significantly decreased compared to bilirubin-untreated PBMNC. However, addition of exogenous IL-2 to pretreated PBMNC could not correct the decreased DNA synthesis. Expression of Tac antigen and transferrin receptor on bilirubin-treated lymphocytes after PHA stimulation was not significantly different from bilirubin-untreated cells. These results suggest that decreased PHA-induced T-lymphocyte proliferation following bilirubin-pretreatment may result from impairment of proliferation at a step beyond transferrin receptor expression. These observations may help explain the increased susceptibility to infection often observed in hyperbilirubinemic patients.

Septic complications have been major problems in the management of patients with obstructive jaundice and neonatal jaundice. This study investigates effects of unconjugated bilirubin on lymphocyte-mediated cytotoxicity against human tumor target cells. In vitro exposure of human peripheral blood lymphocytes (PBL) with bilirubin IX alpha in bovine albumin solution resulted in a dose-dependent decrease of both natural killer activity and antibody dependent cellular cytotoxicity (ADCC) activity. Inhibition of both activities correlated with the amounts of intracellular bilirubin. Expression of cell surface CD16, CD56 antigen, and IL-2 receptor beta chain was unchanged in bilirubin-treated PBL as compared to bilirubin-untreated PBL. When bilirubin-treated PBL were cultured with interleukin-2 (IL-2), a dose-dependent decrease of lymphokine-activated killing activity, ADCC activity, and DNA synthesis was observed. Expression of CD56 antigen and IL-2 receptor alpha chain was unchanged in bilirubin-treated PBL following IL-2 stimulation as compared to bilirubin free control. These results suggest that bilirubin inhibits major histocompatibility complex-unrestricted cytotoxicity in both unstimulated and IL-2 stimulated lymphocytes. These observations may help explain the increased susceptibility to infection observed in hyperbilirubinemic patients.

Human peripheral blood mononuclear cells were incubated at 37 degrees C with bilirubin in bovine albumin solution. Histological analysis of bilirubin-treated cells demonstrated a prominent brown pigment deposited in the cytoplasm. Homogenates of these cells in chloroform-methanol solution showed an identical absorption spectrum with pure bilirubin dissolved in the same solution. When bilirubin-treated cells were excited at 488 nm, a significant autofluorescence was detected by flow cytometry at 585 nm in a dose-dependent manner, which had a significant correlation with the amount of bilirubin chemically extracted from the cells (r = 0.963, n = 34, p less than 0.001). Intraassay and interassay variability of the autofluorescence by flow cytometric analysis was small (both less than 5%). When bilirubin-treated cells were stained with fluorescein-labeled anti-CD14 antibody, the CD14 positive cell population can be fractionated without interference of autofluorescence derived from intracellular bilirubin. These results suggest that flow cytometric analysis of bilirubin-treated cells can quantitate intracellular bilirubin, and that bilirubin does not interfere with analysis of surface antigens.

Decreased immune responses have been observed in hyperbilirubinemic patients. This study investigates bilirubin transport into human peripheral blood mononuclear cells (PBMNCs). In vitro incubation of PBMNCs at 37 degrees C with 0-12 mg/dl bilirubin in solution with a fixed bovine serum albumin (BSA) concentration (3.0 g/dl) resulted in a dose-dependent increase of intracellular bilirubin in both monocytes and lymphocytes. Bilirubin uptake in monocytes was significantly higher (up to 2.7 times) than in lymphocytes under the same culture conditions. When PBMNCs were incubated with varying concentrations of bilirubin (0-16 mg/dl) in fixed BSA (3.0 g/dl) solution or at a fixed bilirubin/albumin molar ratio (0.4), the initial velocity of uptake in both cell fractions was proportional to the free (unbound to albumin) bilirubin concentration rather than the total bilirubin concentration. Bilirubin uptake by both cell fractions was significantly inhibited by treatment with metabolic inhibitors. Bilirubin uptake by monocytes continued to increase in parallel with incubation temperature from 0 degrees C to 40 degrees C, whereas uptake by lymphocytes reached a maximal level at 20 degrees C and remained constant thereafter. These results suggest that monocytes and lymphocytes incorporate bilirubin in proportion to the free bilirubin concentration and this function may rely on different energy-dependent mechanisms.