Human Granulocytic Anaplasmosis and Anaplasma phagocytophilum

Understanding how Anaplasma phagocytophilum alters neutrophils will improve diagnosis, treatment, and prevention of this severe illness

RAGE Expression in Human T Cells: A Link between Environmental Factors and Adaptive Immune Responses

The Receptor for Advanced Glycation Endproducts (RAGE) is a scavenger ligand that binds glycated endproducts as well as molecules released during cell death such as S100b and HMGB1. RAGE is expressed on antigen presenting cells where it may participate in activation of innate immune responses but its role in adaptive human immune responses has not been described. We have found that RAGE is expressed intracellularly in human T cells following TCR activation but constitutively on T cells from patients with diabetes. The levels of RAGE on T cells from patients with diabetes are not related to the level of glucose control. It co-localizes to the endosomes. Its expression increases in activated T cells from healthy control subjects but bystander cells also express RAGE after stimulation of the antigen specific T cells. RAGE ligands enhance RAGE expression. In patients with T1D, the level of RAGE expression decreases with T cell activation. RAGE+ T cells express higher levels of IL-17A, CD107a, and IL-5 than RAGE− cells from the same individual with T1D. Our studies have identified the expression of RAGE on adaptive immune cells and a role for this receptor and its ligands in modulating human immune responses

Diminished Adhesion of Anaplasma phagocytophilum-Infected Neutrophils to Endothelial Cells Is Associated with Reduced Expression of Leukocyte Surface Selectin

Anaplasma phagocytophilum propagates within neutrophils and causes a disease marked by inflammatory tissue injury or complicated by opportunistic infections. We hypothesized that infection with A. phagocytophilum modifies the binding of neutrophils to endothelial cells and the expression of neutrophil adhesion molecules and studied these changes in vitro. Infected dimethyl sulfoxide-differentiated HL-60 cells and neutrophils showed reduced binding to cultured brain and systemic endothelial cells and lost expression of P-selectin glycoprotein ligand 1 (PSGL-1, CD162) and L-selectin (CD62L) (to 33 and 5% of control values, respectively), at a time when the levels of β(2) integrin and immunoglobulin superfamily adhesion molecules and activation markers Mac-1 and intercellular adhesion molecule 1 increased (5 to 10 times that of the control). The loss of CD162 and CD62L expression was inhibited by EDTA, which suggests that neutrophil activation and sheddase cleavage occurred. The loss of selectin expression and the retained viability of the neutrophils persisted for at least 18 h with A. phagocytophilum infection, whereas Escherichia coli and Staphylococcus aureus rapidly killed neutrophils. The adhesion defect might increase the numbers of infected cells and their persistence in the blood prior to tick bites. However, decreased CD162 expression and poor endothelial cell binding may partly explain impaired host defenses, while simultaneous neutrophil activation may aggravate inflammation. These observations may help us to understand the modified biological responses, host inflammation, and immune response that occur with A. phagocytophilum infections

Defective Phagocytosis in Anaplasma phagocytophilum- Infected Neutrophils

Anaplasma phagocytophilum infection induces functional neutrophil changes. Using both Candida albicans and fluorescent-aggregate phagocytosis assays, we examined whether neutrophil and dimethyl sulfoxide-differentiated HL-60 cell infection impairs internalization. A. phagocytophilum infection significantly decreased phagocytosis compared to that of controls (P < 0.05). This further impairment of neutrophil function may promote opportunistic infections and exacerbate disease

RAGE is seen in a granular pattern in T cells and colocalizes with endosomes.

<p>A: Jurkat cells were transfected with GFP-RAGE (A) or control GFP vector (B) and photographed. A granular pattern of staining is seen within the RAGE transfected cells. C–E: HEK293 cells were transfected with GFP-RAGE (D and E) and fixed and stained with RhoB (C and E). Panel E shows the merged staining. The arrows indicate cells+ for RhoB and RAGE.</p

Expression of RAGE on human APC's and T cells.

<p>A: Surface RAGE expression was studied on CD11c+ PBMC before (top) and after (bottom) culture with LPS for 7 days. (solid line = staining with anti-RAGE antibody, dashed line = staining with isotype control) B: Cell surface (L and intracellular RAGE expression was studied on CD4+ T cells before (top) and after 7 days in culture with anti-CD3 mAb (bottom). A single experiment representative of cultures with more than 4 donors is shown. C: PBMC were activated with anti-CD3 mAb for 48 hrs and lysed or separated into CD4+ and CD8+ T cells with magnetic beads and lysed. A blot of the lysates was probed with anti-RAGE antibody. The arrow identifies RAGE in the cells.</p

Changes in RAGE expression on activated T cells from patients with T1D and healthy control subjects.

<p>RAGE expression was studied on CD4+ or CD8+ T cells before and 48 hrs after culture with anti-CD3 mAb. RAGE expression was higher on CD4+ (p<0.001) and CD8+ (p<0.001) T cells from patients with T1D vs healthy controls. While the level of RAGE expression increased in CD4+ and CD8+ T cells from healthy control subjects (p<0.05), it decreased in the patients with T1D (p<0.05).</p

Phenotype of RAGE+ T cells.

<p>CD8+ T cells, that were not activated from a patient with T1D (R column) or a CD8+ T cells from a HLA-A2+ healthy control subject, activated with anti-CD3+28 mAbs (L column) or from the same HC subject activated with EBV peptide (middle column) were compared. The RAGE+ T cells from the patient with T1D do not express CD25, are CCR7+ and have a more uniform distribution of CD45RA. Results from a single donor representative of 3 is shown.</p