Granulocyte Colony Stimulating Factor

Neutralizing polyclonal (33) and monoclonal (34) antibodies (MAbs) to HuG-CSF have been available; they formed the basis for determination of immunoreactive HuG-CSF levels and for showing specificity in HuG-CSF bioassays (35). A polyclonal neutralizing antiserum to murine (Mu)G-CSF has been used for G-CSF neutralization in vitro (36). Despite the availability of these reagents, no attempts to neutralize endogenous MuG-CSF in vivo were reported. One experiment in rats involved passive immunization with a rabbit anti-G-CSF Ab 2 h before pulmonary challenge with Pseudomonas aeruginosa (37). Anti-G-CSF Ab pretreatment reduced pulmonary neutrophil recruitment and intrapulmonary bactericidal activity at 4 h after infection without affecting the number of circulating neutrophils, suggesting that a local pulmonary G-CSF response to the infection had been impaired.

The hematologic consequences of neutralization of endogenous G-CSF were first observed in dogs, resulting from Ab induced to HuG-CSF crossreacting against canine G-CSF (38) (Table 3). Dogs administered HuG-CSF developed an initial neutrophilia, but with ongoing HuG-CSF administration, neutropenia supervened. On cessation of HuG-CSF administration, neutrophil counts slowly returned to normal, but after a non-treatment interval, neutropenia rapidly recurred upon retreatment with HuG-CSF. Anti-HuG-CSF Abs in serum were seen, and passive immunization of dogs by plasma infusion was achieved.

Induction of autoimmunity to murine MuG-CSF required the use of immunostimu-latory MuG-CSF conjugates (39). Immunized mice developed neutropenia coincident with an IgG autoantibody response, without effect on other peripheral blood parameters or on the number of marrow progenitor cells. The neutropenia was sustained for >9 mo. Hematologically, these mice phenocopied mice with absolute G-CSF deficiency owing to disruption of either the G-CSF ligand (40) or receptor (41) genes.

Mice with absolute G-CSF deficiency induced by targeted disruption of either the G-CSF or G-CSF receptor (G-CSFR) gene have similar hematologic phenotypes (40,41). G-CSF-/- mice display chronic neutropenia, reduced marrow granulopoiesis, and impaired G-CSF-provoked neutrophil mobilization (40). Kinetic analysis of granulopoiesis revealed a reduced transit time through the mitotic compartment of G-CSF-/- mice, a normal transit time through the postmitotic compart-

Table 3

Animal Models of Reduced G-CSF Levels or Signaling

Animal Method of reduced G-CSF signaling Major phenotypic consequences Reference

Table 3

Animal Models of Reduced G-CSF Levels or Signaling

Animal Method of reduced G-CSF signaling Major phenotypic consequences Reference

Dog

Immunization during HuG-CSF administration resulting in anti-HuG-CSF antibodies

Transient neutropenia

Rapid neutropenia on rechallenge

38

crossreacting with canine G-CSF

i Local response to pulmonary bacterial infection

Rat

Passive immunization with anti-MuCSF antibodies

37

Mouse

Active immunization with MuG-CSF-conjugates resulting in anti-MuG-CSF autoantibodies

Prolonged neutropenia

39

Mouse

Targeted disruption of G-CSF gene

Chronic neutropenia i marrow granulopoiesis Pathogen susceptibility T neutrophil apoptosis Haploinsufficiency

40

Mouse

Targeted disruption of G-CSF receptor gene

Chronic neutropenia i marrow granulopoiesis i progenitor cell and neutrophil mobilization i neutrophil chemotaxis Haploinsufficiency

41

Abbreviations: G-CSF, granulocyte colony-stimulating factor; Hu, human; T, increased; 4-, decreased.

Abbreviations: G-CSF, granulocyte colony-stimulating factor; Hu, human; T, increased; 4-, decreased.

ment, and an increase in the proportion of Gr-1+ cells that have initiated apoptosis as detected by mercocyanine 540 staining (42). G-CSF deficiency results in increased susceptibility to pathogens including Listeria monocytogenes and Candida albicans (43). Surprisingly, despite the unexpected impairment of monocyte/macrophage responses in G-CSF-/- mice during Listeria infections (40,44,45), Mycobacterium avium infections were not exacerbated in G-CSF-/- mice, and high levels of interferon (IFN)-y production accompanied infection with this pathogen (46). Candida infection of G-CSF-/- mice was accompanied by a vigorous neutrophilia, exceeding the magnitude of that in wild-type mice, and early control of the pathogen load. However, after 1 wk of infection, deep tissue infection with high Candida pathogen loads persisted in G-CSF-/- mice at a time the infection was resolving in wild-type mice (43).

The hematologic profile of G-CSFR-/- mice largely resembled that of the ligand-deficient mice, with chronic neutropenia, reduced marrow granulopoiesis, and a propensity of Gr-1+ marrow cells to undergo apoptotic death in vitro (41). The G-CSFR-/- mice have enabled distinctions to be drawn between G-CSF-dependent and G-CSF-independent neutrophil functions. Neutrophil primary granule myeloperoxidase activity was normal, and neutrophil migration induced by chemical peritonitis was preserved. However, progenitor cell and neutrophil mobilization into the peripheral blood by cyclophosphamide and IL-8 was impaired (47). Neutrophils from G-CSFR-/- mice had defective chemotactic responses to IL-8 and other chemoattractants in vitro, despite intact metabolic responses to several agents (48). The intrinsic defect in G-CSFR-/-cells has enabled experiments to be designed to distinguish between cell-autonomous and -nonautonomous functions. For example, radiation chimeras were established with either wild-type or G-CSFR-/- hematopoietic cell populations in wild-type or G-CSFR-/- stromal backgrounds to study the phenomenon of G-CSF-stimulated progenitor cell mobilization. Expression of the G-CSFR on the hematopoietic cells (and then only a subpopulation of them) and not the stromal cells was necessary for G-CSF-stim-ulated mobilization to occur (49), although interpretation of this experiment assumes that little reconstitution of the marrow stroma by the transplanted marrow cells occurred.

To define signals mediated specifically by the G-CSFR, gene-targeted mice have been generated in which the G-CSFR was replaced by a chimeric receptor comprising the extracellular and transmembrane portions of the G-CSFR (capable of binding G-CSF) connected to the intracellular portion of the EPOR (50). Hematologically, these mice resemble G-CSFR-/- mice with peripheral blood neutropenia and a modest marrow granulopoietic defect. Although this chimeric receptor supported granulocytic lineage commitment and differentiation, some specific defects were demonstrable: there was impaired G-CSF-stimulated progenitor cell mobilization and reduced IL-8-induced chemotaxis (50,51).

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