Phagocytosis of the malarial pigment, hemozoin, impairs expression of major histocompatibility complex class II antigen, CD54, and CD11c in human monocytes

Abstract

In Plasmodium falciparum malaria, large proportions of resident macrophages and circulating monocytes and leukocytes contain massive amounts of the malarial pigment, hemozoin. Previous studies have shown that important functions (e.g., the generation of the oxidative burst, the ability to repeat phagocytosis, and protein kinase C activity) were severely impaired in hemozoin-loaded monocytes. Expression of membrane antigens directly involved in the immune response and in the phagocytic process, and/or under protein kinase C control, in hemozoin-loaded human monocytes was studied. Expression of major histocompatibility complex (MHC) class II after gamma interferon stimulation was blocked in hemozoin-loaded monocytes at the protein expression and gene transcription levels but was preserved in control monocytes loaded with opsonized latex beads or anti-D(Rho)-immunoglobulin G (IgG)-opsonized human erythrocytes. Expression of CD54 (intracellular adhesion molecule 1) and CD11c (p150,95 integrin) was also decreased in hemozoin-loaded monocytes. Expression of MHC class I, CD16 (low-affinity Fc receptor for aggregated IgG), CD32 (low-affinity Fc receptor for aggregated IgG), CD64 (high-affinity receptor for IgG), CD11b (receptor for complement component iC3b [CR3]), CD35 (receptor for complement components C3b and C4b [CR1]), and CD36 (non-class-A scavenger receptor) was not specifically affected by hemozoin loading. These results suggest that hemozoin loading may contribute to the impairment of the immune response and the derangement of antigen presentation reported in previous studies of P. falciparum malaria.

FIG. 1

Expression of MHC class II antigens in human monocytes after phagocytosis of serum-opsonized HE or latex beads with or without IFN-γ stimulation. Suspended human monocytes were allowed to phagocytose HE (left panel) or latex beads (right panel) in Teflon-bottom dishes kept in a humidified incubator at 37°C. Twelve hours after the start of phagocytosis, 200 U of IFN-γ/ml was added to a part of each dish. Forty-eight hours after the start of phagocytosis, monocytes were harvested by aspiration. After being washed, monocytes were immunostained with anti-MHC class II MAb and bound MAb was revealed by FITC-conjugated F(ab′)₂ goat anti-mouse Ig. Flow cytometry analysis of surface antigens was performed on a FACScan flow cytometer (Becton Dickinson). The first peak at the left in both panels represents MHC class II antigen expression in unstimulated monocytes incubated for 48 h without phagocytosis. The y axis represents cell number and the x axis represents fluorescence intensity. This was one of four similar experiments.

FIG. 2

Quantification of MHC class I and class II mRNA by RT-PCR. Increasing amounts (7.5 to 60 ng, quantified by OD, as indicated under the lanes) of total RNA extracted from human monocytes were employed in reverse transcription with Moloney murine leukemia virus reverse transcriptase for 1 h, followed by PCR in the same tube (100-μl final volume, 25 cycles). RT-PCR was performed in duplicate with each of the indicated total RNA amounts. Specific primers for conserved regions in MHC class I and class II genes were utilized to obtain PCR products of 118 and 81 bp, respectively. PCR products were separated on a 3% agarose gel and stained with ethidium bromide. DNA size standards are indicated on the right.

FIG. 3

(Upper panel) steady-state levels of MHC class I and class II mRNA in human monocytes (C) and monocytes after phagocytosis of serum-opsonized HE (H) with (+) or without (−) IFN-γ stimulation. After the start of HE phagocytosis, monocytes were incubated for 24 h before addition of IFN-γ (200 U/ml). After a further 24 h of incubation, total RNA was extracted, RT-PCR was performed in duplicate with 15 ng of total RNA, and amplified MHC class I and class II gene products of 118 and 81 bp, respectively, were analyzed as indicated in the legend to Fig. 2. DNA size standards are indicated on the right. This was one of five similar experiments. (Lower panel) steady-state levels of MHC class I and class II mRNA in human monocytes (C) and monocytes after phagocytosis of opsonized latex beads (L) or opsonized erythrocytes (R) with (+) or without (−) IFN-γ stimulation. After the start of phagocytosis of the latex beads or erythrocytes, monocytes were incubated for 24 h before addition of IFN-γ (200 U/ml). After a further 24 h of incubation, total RNA was extracted, RT-PCR was performed with 15 ng of total RNA, and amplified MHC class I and class II gene products of 118 and 81 bp, respectively, were analyzed as indicated in the legend to Fig. 2. DNA size standards are indicated on the right. This was one of five similar experiments.

FIG. 4

Expression of surface antigens in unstimulated human monocytes after phagocytosis of serum-opsonized HE or latex beads. Suspended monocytes were allowed to phagocytose HE or latex beads in Teflon-bottom dishes kept in a humidified incubator at 37°C. Forty-eight hours after the start of phagocytosis, monocytes were harvested by aspiration. After being washed, monocytes were immunostained with purified MAb and bound MAb was revealed by FITC-conjugated goat anti-mouse Ig. Flow cytometry analysis of the surface antigens MHC class I, CD32 (low-affinity receptor for aggregated Ig), CD11c (p150,95 integrin), and CD54 (ICAM-1) was performed on a FACScan (Becton Dickinson) flow cytometer. (A) unfed monocytes at time zero (——) and after 48 h in culture (); (B) HE-fed (——) and unfed () monocytes after 48 h in culture; (C) latex-fed (——) and unfed () monocytes after 48 h in culture. Black profiles on the left represent the fluorescence background of monocytes incubated with irrelevant mouse Ig. The y axis represents cell number and the x axis represents fluorescence intensity. This was one of four similar experiments.