Sustained IL-4 exposure leads to a novel pathway for hemophagocytosis, inflammation, and tissue macrophage accumulation

Abstract

Erythrophagocytosis and inflammation from activated macrophages occur in distinct clinical scenarios. The presence of CD8(+) T cells and interferon-γ (IFN-γ) production is required to induce disease in mouse models of hemophagocytic lymphohistiocytosis. We investigated the roles of a different class of proinflammatory cytokines, interleukin-4 (IL-4) and IL-13, in the induction of inflammatory tissue macrophage accumulation and/or hemophagocytosis. We found that large amounts of IL-4, but not IL-13, delivered via an implanted mini-pump or IL-4/anti-IL-4 complexes, lead to substantial YM1(+) tissue macrophage accumulation, erythrophagocytosis within the liver, spleen, and bone marrow, decreased hemoglobin and platelet levels, and acute weight loss. This effect is not dependent on the presence of antibody or T cells, as treatment of Rag2(-/-) mice leads to similar disease, and IFN-γ neutralization during IL-4 treatment had no effect. IL-4 treatment results in suppression of IL-12, elevation of serum IFN-γ, IL-10, and the murine IL-8 homolog KC, but not IL-6, IL-1β, or tumor necrosis factor-α. Finally, mice transgenic for IL-4 production developed tissue macrophage accumulation, disruption of splenic architecture, bone marrow hypocellularity, and extramedullary hematopoiesis. These data describe a novel pathophysiologic pathway for erythrophagocytosis in the context of tissue macrophage accumulation and inflammation involving elevations in IL-4 and alternative macrophage activation.

Figure 1

Induction of histiocytosis and erythrophagocytosis by IL-4. (A) Hematoxylin and eosin stain (×100) of tissue from day 3 of IL-4 pump (1 μg/hour), showing activated luminal macrophages and erythrophagocytosis (arrows) within the liver. (B) Hematoxylin and eosin stain (×100) of erythrophagocytosis within the bone marrow. Immunohistochemistry (×40) for F4/80 showing increased cellular density within red pulp of IL-4 mini-pump–treated spleen (D) compared with control (C). Immunohistochemistry (×100) for F4/80 showing more diffuse and larger F4/80⁺ Kupffer cells within the liver of IL-4 mini-pump–treated mice (F) compared with controls (E). Ym1 immunohistochemistry in (G) liver and (H) spleen (×100) of IL-4 mini-pump–treated mouse.

Figure 2

IL-4–treated mice develop weight loss, signs of HLH, and evidence of accumulation of alternatively activated macrophages. (A) Wild-type B6 mice were treated with 1 μg/hour IL-4 mini-pumps or PBS control (5 in each group) for 3 days. The indicated serum and blood cell indices were measured at that time. This experiment was repeated with similar results. (B) Mononuclear cells were isolated from the livers of these mice, enumerated and stained for F4/80 to determine liver macrophage burden (shown are total macrophages/liver), and real-time PCR was performed to evaluate markers of classic and alternative activation (expression levels of untreated mice set at 1; shown are fold increases of expression of the indicated gene over control by IL-4–treated mice).

Figure 3

Multiplex serum cytokine profile of Rag2^−/− mice receiving PBS, IL-4 (1 μg/hour), or IL-13 (1 μg/hour) via micro-osmotic pump for 3 days. Serum cytokine concentrations in B6 Rag2^−/− mice treated 3 days earlier with an IL-4– or IL-13–containing mini-pump. Three to 5 mice were used per group, and similar results were obtained in a second similar experiment. *P < .05.

Figure 4

Neutralization of interferon-γ does not ameliorate IL-4–induced disease. Hemoglobin, polymorphonuclear leukocytes, and platelet values for mice treated with micro-osmotic pumps containing PBS or IL-4 (1 μg/hour, 3 mice in each group) for 3 days with or without neutralizing anti–IFN-γ monoclonal antibody. When the neutralizing antibody was present, IFN-γ levels were not detectable in the serum for the duration of the experiment. ns indicates not significant.

Figure 5

Mice transgenic for IL-4 expression have liver erythrophagocytosis, splenic histiocytosis, and extramedullary hematopoiesis. (A) Hematoxylin and eosin stains of B6 WT spleen (left) and spleen (×4) from IL-4 TG.UG mice (right) showing areas of normal white pulp (arrows). Histiocytosis and extramedullary hematopoiesis are only seen in the TG.UG spleen as shown in areas marked with yellow stars. (Insets) Original magnification ×100 (view of red pulp). Stars in wild-type mice indicate normal red pulp. (B) Hematoxylin and eosin stain (×40) of liver showing erythrophagocytosis (arrows). (Inset) Original magnification ×100 from a representative 11-month-old B6 IL-4 transgenic mouse. (C) Bone marrow cellularity from 1 long bone of IL-4 transgenic and age-matched wild-type B6 mice (4 from each group evaluated).