Poor Mobilization in T-Cell-Deficient Nude Mice Is Explained by Defective Activation of Granulocytes and Monocytes

Abstract

It has been reported that both SCID mice and SCID patients poorly mobilize hematopoietic stem/progenitor cells (HSPCs) in response to granulocyte colony-stimulating factor (G-CSF). This defect has been proposed to result from a lack of naturally occurring IgM immunoglobulins to trigger activation of the complement cascade (ComC) and release of C5 cleavage fragments crucial in the mobilization process. However, SCID individuals also have T-cell deficiency, and T cells have been shown to modulate trafficking of HSPCs. To learn more about the role of T lymphocytes, we performed mobilization studies in T-lymphocyte-deficient nude mice and found that these mice respond poorly to G-CSF and zymosan but are normal mobilizers in response to AMD3100. Since nude mice have normal levels of IgM immunoglobulins in peripheral blood and may activate the ComC, we focused on the potential involvement of Gr1+ granulocytes and monocytes, which show defective maturation in these animals. Using a nude mouse mobilization model, we found further support for the proposition that proper function of Gr1+ cells is crucial for optimal mobilization of HSPCs.

Figure 1.

Nude mice are poor mobilizers in response to G-CSF and zymosan but normal mobilizers in response to AMD3100. Nude and BALB/c (control) mice were mobilized with six doses of granulocyte colony-stimulating factor (G-CSF) administered once a day, and 6 h after the last dose the peripheral blood was analyzed. Mice were injected with zymosan or AMD3100, and 1 h later the peripheral blood was analyzed. At the times indicated above, peripheral blood (PB) samples were collected and analyzed for the numbers of white blood cells (WBCs) (A), number (No.) of (Lin⁻/Sca-1⁺/c-kit⁺) (SKL) cells (B), hematopoietic stem cells (HSCs; Lin⁻/Sca-1⁺/CD45⁺) (C), and colony-forming unit-granulocyte/macrophage (CFU-GM) clonogenic progenitors (D). Experiments were performed three times with four mice per group. *p ≤0.05, differences between BALB/c and nude mice. Results are shown as percent of values of unmobilized mice.

Figure 2.

Hematological parameters in nude mice compared with BALB/c animals. Peripheral blood (PB) parameters were evaluated using a HemaVet 950FS analyzer, and nude mice had less white blood cells (WBCs), neutrophils (NEs), lymphocytes (LYs), and monocytes (MOs) (A). Compared with BALB/c (control) mice, nude mice had normal numbers of RBCs, hemoglobin content (HB), hematocrit (HCT), mean volume of erythrocytes (MCV), mean content of hemoglobin (MCH), mean concentration of hemoglobin in erythrocytes (MCHC), and red cell distribution width (RDW) (B). Under steady-state conditions, there were also no differences between nude and BALB/c mice in the numbers of Lin⁻/Sca-1⁺/c-kit⁺ (SKL) cells and hematopoietic stem cells (HSCs) circulating in PB (C). Bone marrow (BM) of nude and BALB/c mice was also isolated and evaluated for the numbers of colony-forming unit-granulocyte/macrophage (CFU-GM), erythroid progenitor cell (BFU-E), and CFU megakaryocyte (CFU-Meg) clonogenic progenitors in in vitro assays, in which nude mice showed higher levels of CFU-GM clonogenic progenitors (D). Data represent an average of at least eight mice tested per experimental group (*p ≤0.05).

Figure 3.

Chemotactic responsiveness of bone marrow-derived mononuclear cells (BM-MNCs) to SDF-1 and S1P gradients and of Gr-1⁺ cells and monocytes to SDF-1, S1P, and promobilizing C5a and _desArgC5a chemoattractant gradients. The chemotactic responsiveness of murine clonogenic colony-forming unit-granulocyte/macrophage (CFU-GM) progenitors to stromal cell-derived factor 1 (SDF-1) and sphingosine 1-phosphate (S1P) gradients (A). Gr-1⁺ cells (B) and monocytes (C) were employed for chemotaxis assays in response to S1P, SDF-1, complement component 5a (C5a), and _desArgC5a gradients using the Transwell system. The experiment was repeated twice, and the data were combined. Results are shown as the percentage of input which represents 5% of the insert (*p ≤0.05).

Figure 4.

Analysis of nude and BALB/c Gr-1⁺ cells in degranulation assays. Gr-1⁺ cells (granulocytes and monocytes) were isolated from the bone marrow (BM) of nude and BALB/c (control) mice and stimulated with medium alone or with G-CSF, AMD3100, C5a, or _desArgC5a. After stimulation, conditioned media were analyzed in degranulation assays by measuring elastase activity (A), myeloperoxidase (MPO) activity (B), and phospholipase C activity (C). The results are combined from three independent experiments and show changes as a percentage of the control. Statistical differences refer to the differences between BALB/c and nude mice after stimulation (*p ≤0.05).

Figure 5.

Analysis of Gr-1⁺ cells isolated from the bone marrow of nude and BALB/c mice for their ability to activate complement. Gr-1⁺ cells (granulocytes and monocytes) were isolated from bone marrow (BM) of nude and BALB/c mice and exposed to the plasma of BALB/c or nude mice. Complement activation was measured by enzyme-linked immunosorbent assay (ELISA) for C5a. Values are the fold increase of C5a compared with control plasma (*p ≤0.05).