Combined Hematopoietic Growth Factor Signaling Deficiencies

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By combining genetically based factor-deficiencies, the interacting roles of growth factors can be studied in vivo. Sometimes interactions have been achieved by combining ligand-deficiency for one factor and receptor deficiency for another, often for reasons of utility and availability. Occasionally, genetic constraints due to the proximity of loci influence the approach. Some combinations merely result in the simple addition of the phenotypic traits of the two individual factor deficiencies, suggesting independent roles for the two factors. Others result in the emergence of new phenotypic features, or the accentuation of component phenotype traits, suggesting that one factor can assume a compensatory role in the deficiency phenotype of another factor, although compensation requires that activation of a process over the usual normal amount be shown as well. The emergence of new phenotypic traits in combination with deficiency genotypes allows for the possibility that independent, separately regulated mechanisms may contribute to a particular process, and the integrity of the process requires one or the other mechanism to be intact, but only when both mechanisms are impaired does the process fail.

Table 5

Genetic Models of Deficiency of CSFs and Other Factors Affecting Hematopoiesis in Mice

Factor

Genetic basis (allele)

Major phenotypic featuresa

Reference to o

G-CSF Targeted gene disruption

GM-CSF Targeted gene disruption

M-CSF Natural point mutation (op)

SCF Natural mutation (Sl)b

LIF Targeted gene disruption

IL-1P Targeted gene disruption

IL-2 Targeted gene disruption

IL-3 Transgenesis (antisense RNA, partial IL-3 deficiency only) IL-3 Targeted gene disruption

-/- Chronic neutropenia i Progenitor cells Infection vulnerability -/- Unperturbed hematopoiesis Alveolar proteinosis Lung infections -/- Osteopetrosis i Monocyte/macrophages i Osteoclasts -/- Lethal in utero

Impaired hematopoiesis +/- Pale coat Mild macrocytic anemia Small gonads -/- Maternal infertility i Splenic CFC and CFU-S Normal peripheral blood -/- Fever-resistant i Acute-phase response Hematopoiesis not analyzed -/- Perturbed B-cell function

Ulcerative colitis +/- Lymphoproliferative disorder

Neurologic dysfunction -/- i Delayed-type hypersensitivity i Tissue mast cells in nematode infection

IL-4

IL-5

IL-6

IL-7

Targeted gene disruption

Targeted gene disruption

Targeted gene disruption

Targeted gene disruption

EPO Targeted gene disruption

TPO Targeted gene disruption

-/- I Th2 responses I Reactive eosinophilia I IgG1 switching I Mucosal immunity -/- Normal Th-dependent B-cell responses Normal immunoglobulins Normal eosinophils T in parasitic infection; normal parasite killing -/-4 Acute-phase and anti-infective response I Mucosal immunity

I Pre-CFU-S, CFU-S and lineage-committed CFCs T Bone turnover -/- IB lymphopoiesis I Thymic cellularity I Splenic lymphocytes -/- Lethal at E 13

Hepatic erythropoiesis fails -/-1 Platelets (>80%) I Marrow megakaryocytes and megakaryocyte-CFCs I Megakaryocyte ploidy +/-1 Platelets (67%)

180 181

182, 183 184

186 187

188, 189 188, 189

Abbreviations: CFC, colony-forming cell; CFU, colony-forming unit; EPO, erythropoietin; G-CSF, granulocyte colony-stimulating factor; GM-CSF, granulocyte-macrophage colony-stimulating factor; IL, interleukin; LIF, leukemia inhibitory factor; M-CSF, macrophage colony-stimulating factor; TPO, thrombopoietin; T, increased; decreased.

a Factor-deficient genotype indicated as heterozygous (+/-) or homozygous (-/-).

b Numerous different alleles exist. SI refers to that originally described (2,3). Other alleles are indicated by superscripts, e.g., Sld (Steel-Dickie): Sld/Sld and Sl/Sld mice are viable but severely anemic and sterile with black eyes and white coats (190).

Table 6

Genetic Models of Chronic Deficiency of Components of Receptors for Factors Affecting Hematopoiesis in Mice

Ligands

Receptor component

Genetic basis (allele)

Major phenotypic featuresa

Comments and comparison With ligand absence or impairment

Reference to to

IL-3

IL-3

c-kit

Spontaneous and mutagen-induced mutation (W)b

PIL3 (AIC2A) Targeted gene disruption

Pc (AIC2B) Targeted gene disruption

-/- Perinatal lethality

Severity macrocytic anemia Sterility

Absent coat pigmentation +/- Normal hematopoiesis Fertile

White spotting -/- Normal

-/- Lung alveolar proteinosis Normal CFC levels (assayed with SCF/IL-6/EPO) I eosinophils I reactive eosinophila aIL3

Spontaneous mutation -/- IL-3 hyporesponsivness

TPO c-mpl

Targeted gene disruption Targeted gene disruption (X-linked gene)

-/- Thrombocytopenia -/- Males lacking yc: Perturbed T lymphopoiesis Perturbed B lymphopoiesis

Similar

5,10,12

IL-3 signaling via aIL3 Pc receptor complex still possible; Basal hematopoiesis normal in IL-3-/- mice (82) Resembles GM-CSF-/- mice (60,61)

IL-5-/- mice have normal basal eosinophil numbers but I reactive eosinophila (181) Still have low numbers of high-affinity receptors on marrow cells; probably not a null allele Resembles TPO-- mice (195)

Reflects IL-2-/- mice (176) Reflects IL-7-receptor-/- mice (198)

68,69

191-194

196,197 203

IL-2

Targeted gene disruption

IL-8

mIL-8Rh

EPO-R

CSF-1 c-fms

Targeted gene disruption

Targeted gene disruption

Targeted gene disruption

Absent dendritic epidermal T cells Typhlitis and colitis

-/- i survival, activated CD4+ cells B-cell activation with IgG1 & IgE Perturbed T- and B-cell responses Hemolytic anemia Myeloproliferative disorder T Splenic granulopoiesis -/- Splenomegaly B-cell hyperplasia T Neutrophils and granulopoeisis i Neutrophil migration -/- Lethal at embryonic d 13 Failure of liver erythropoiesis CFU-E, BFU-E develop, but fail to survive -/- Osteopetrosis i monocytes and tissue macrophages reproductive defects T serum CSF-1 20-fold

Not ulcerative as in IL-2-/- mice (176) or T-cell receptor a-/-, P-/-and P-/-, 5-/- mice (199-202) No colitis as in IL-2-/- mice (176)

IL-8/- mice not yet described

Resembles op/op mice (14)

2G5-2G7

Abbreviations: SCF, stem cell factor; IL, interleukin; GM-CSF, granulocyte-macrophage colony-stimulating factor; TPO, thrombopoietin; CFU-E, colony-forming unit-erythroid; BFU-E, erythroid burst-forming unit; EPO, erythropoietin; CSF, colony-stimulating factor; T, increased; decreased. a Factor-deficient genotype indicated as heterozygous (+/-) or homozygous (-/-).

b At least 27 alleles exist. W refers to that originally described (10) and molecularly characterized by Nocka et al. (12). Other alleles are indicated by superscripts, e.g., Wv, a qualitatively different allele that occurred in C57BL/J6 results in homozygous mice that are viable but severely anemic, sterile, black-eyed, and white coated.

2.7.1. Combined Deficiencies Involving Erythropoietin

EPO-R-/- mice were interbred with GM-CSF-/- and IL-3-/- mice (31). A reduced frequency of marrow CFU-E was observed in EPO-R+/- haploinsufficient GM-CSF-/-or IL-3-/- mice, although CFU-E frequencies were reduced in mice with isolated GM-CSF or IL-3 deficiency. This finding was of functional significance in the mice with combined factor signaling deficiencies, since GM-CSF-/-EPO-R+/- and IL-3-/-EPO-R+/-mice were more anemic after exposure to phenylhydrazine than mice of the single-component genotypes.

2.7.2. Combined Deficiencies Involving Granulocyte Colony-Stimulating Factor

G-CSF-deficient mice were interbred with GM-CSF-deficient mice to create mice deficient in both factors (66). G-CSF-/-GM-CSF-/- mice were more neutropenic than G-CSF-/- mice in the early neonatal period, had higher neonatal mortality, and showed a propensity to the development of the amyloidosis evident in G-CSF-/- mice. Mice deficient in G-CSF and IL-6 signaling have been generated, both by creating mice deficient in both ligands (43) and by creating G-CSFR-/-IL-6-/- mice (79). G-CSFR-/-IL-6-/- mice had an exacerbated neutropenia compared with G-CSFR-/- mice (79). Although infection of G-CSF-/- mice with C. albicans resulted in a neutrophilia with increased amounts of serum IL-6, indicating that factors other than G-CSF can drive the emergency granulopoietic response, G-CSF-/-IL-6-/- mice also showed this phenomenon, indicating that IL-6 was not the sole driver of this infection-related granulopoietic response (43). Thrombopoietin (TPO)-deficient mice and G-CSFR-/- mice have been interbred, testing the role of either factor in modulating the other-factor defi-ciency-phenotype. G-CSF deficiency did not further exacerbate the thrombocytopenia of TPO-/- mice, but TPO deficiency augmented the granulopoietic defect of G-CSFR-/-mice, with a consequent increased early infective mortality (80).

2.7.3. Combined Deficiencies Involving Granulocyte-Macrophage Colony-Stimulating Factor

Since mice have a second IL-3 receptor (PIL-3), IL-3/GM-CSF/IL-5Rpc-deficient mice are not absolutely deficient in IL-3 signaling. IL-3-deficient mice have been generated by gene targeting (81,82), but the close chromosomal location of GM-CSF and IL-3 precluded bringing these mutations together efficiently by interbreeding. GM-CSF deficiency has been combined with IL-3 deficiency by a sequential gene targeting approach (83); these mice have a basal eosinophila but had impaired contact hypersensitivity reactions. They have also been used to evaluate the role of these cytokines in vivo in murine models of leukemia and myeloproliferative disease based on BCR-ABL and several leukemogenic TEL-tyrosine kinase fusion oncoproteins (84,85); in all models, combined deficiency of these two factors did not impact on the in vivo phenotype of the model leukemia. Mice completely lacking GM-CSF, IL-3, and IL-5 signaling were generated by creating IL-3-/-IL-3/GM-CSF/IL-5Rpc-/- mice; these mice have surprisingly normal basal hematopoiesis and showed normal hematopoietic responses to L. monocytogenes infection and after 5-FU administration (86). GM-CSF-/-IL-3-/-IL-3/GM-CSF/IL-5Rpc-/-mice have been created, which sum to the same growth factor signaling defect (83).

GM-CSF deficiency has been combined with CSF-1 (M-CSF) deficiency by interbreeding CSF-1-deficient op mutant mice with GM-CSF-/- mice (87,88). Concomitant

CSF-1 (M-CSF) deficiency accentuated the pulmonary disease of GM-CSF-deficient mice, but mice deficient in both factors still had residual macrophages, indicating that other factors are still able to affect macrophage development and differentiation in vivo (87). Conversely, GM-CSF deficiency was shown not to be the mediator of age-related corrections in macrophage development observed in op/op mice (88).

GM-CSF deficiency has also been combined with TPO signaling deficiency by generating GM-CSF-/-c-mpl-/- mice. On an inbred background, no further effect of GM-CSF deficiency on the thrombocytopenia of c-mpl-/- was observed. This study demonstrated one of the pitfalls of this approach: on a noninbred background, a partial amelioration of the c-mpl-- thrombocytopenia was seen, suggesting existence of other modifier genes of this phenotype.

2.7.4. Combined Deficiencies Involving Interleukin-11

To combine IL-11 and TPO deficiency, IL-11Ra-/- mice and mice deficient in the TPO receptor c-mpl were interbred (89). Despite the ability of pharmacologic doses of IL-11 to stimulate megakaryocytopoiesis and thrombopoiesis, combined IL-11Ra-/-c-mpl-/- mice did not have accentuation of the platelet and megakaryocyte production defects that characterize c-mpl deficiency.

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