Role of H2-calponin in regulating macrophage motility and phagocytosis

Abstract

The actin cytoskeleton plays a major role in cell motility that is essential for the function of phagocytes. Calponin is an actin-associated regulatory protein. Here we report the finding of significant levels of the h2 isoform of calponin in peripheral blood cells of myeloid lineage. To study the functional significance, h2-calponin gene (Cnn2) interrupted mice were constructed. Germ line transmission of the Cnn2-flox-neo allele was obtained in chimeras from two independent clones of targeted embryonic stem cells. The insertion of the neo(R) cassette into intron 2 of the Cnn2 gene resulted in a significant knockdown of h2-calponin expression. Removing the frt-flanked neo(R) cassette by FLP1 recombinase rescued the knockdown effect. Cre recombinase-induced deletion of the loxP-flanked exon 2 eliminated the expression of h2-calponin protein. H2-calponin-free mice showed reduced numbers of peripheral blood neutrophils and monocytes. H2-calponin-free macrophages demonstrated a higher rate of proliferation and faster migration than that of h2-calponin-positive cells, consistent with a faster diapedesis of peripheral monocytes and neutrophils. H2-calponin-free macrophages showed reduced spreading in adhesion culture together with decreased tropomyosin in the actin cytoskeleton. The lack of h2-calponin also significantly increased macrophage phagocytotic activity, suggesting a novel mechanism to regulate phagocyte functions.

FIGURE 1.

Expression of h2-calponin in myeloid cells. A, Western blots using anti-h2-calponin polyclonal antibody RAH2 and mAb CP22 on total protein extracted from human peripheral mononuclear cells, human myelogenous leukemia cell line K562, human monocyte line THP-1, and in vitro differentiated human myeloid leukemia cell line HL-60 detected significant amounts of h2-calponin. B, no calponin expression was detectable in the human T lymphocyte line Jurkat, mouse T lymphocyte line BW5147, mouse plasmacytoma cell line T1165, and mouse myeloma cell line NS-1 by RAH2 Western blot.

FIGURE 2.

Expression of h2-calponin during monocyte-macrophage differentiation. H2-calponin expression was examined during adhesion-dependent differentiation of human peripheral monocytes. Fresh isolated monocytes adhered to plastic dishes were cultured to differentiate in vitro into macrophages. A, the total cellular protein extracts from the cells before (0 day) and 1, 4, and 7 days in culture were examined by SDS-PAGE. Gel densitometry analysis demonstrates a decrease followed by significant increases in actin as normalized to histone levels that reflects cell numbers. *, p < 0.05, and **, p < 0.01 versus the day 0 level. B, Western blots using RAH2 antibody and densitometry quantification demonstrated that the expression of h2-calponin normalized to the level of cellular actin or histone showed a decrease in h2-calponin at the early stage of monocyte-macrophage differentiation followed by significant increases during the later stages of differentiation. The quantification was done from three sets of samples and two repeated analyses. *, p < 0.005, and **, p < 0.001 versus the day 0 level.

FIGURE 3.

Genotyping of Cnn2-targeted mouse ES cells. A, Southern blot screening of the targeted Cnn2 allele in mouse ES cells. Genomic DNA of transfected ES cell clones was digested by BamHI and hybridized with ³²P-labeled 5′- and 3′-flanking genomic DNA probes. The left panel shows that the 5′ probe detected a 9-kb band from the targeted allele (KO) in four representative positive clones. The presence of the 5-kb band from the wild-type (WT) allele indicates these cells are heterozygotes for the targeted Cnn2 allele. The right panel shows the same blot (after stripping the 5′ probe) re-probed with the 3′-flanking probe to detect the targeted allele as a 7-kb fragment together with the ∼9-kb WT allele band. B, maps of three modified Cnn2 alleles: Cnn2-flox-neo is the original Cnn2-targeted allele. The expression of exons 3–7 is negatively affected by the neo^R cassette inserted in intron 2, resulting in a knockdown of h2-calponin expression. The Cnn2-flox allele is derived from the Cnn2-flox-neo allele by FLP1-induced removal of the neo^R cassette, which restores the normal expression of h2-calponin. The Cnn2-KO allele is derived by further deletion of exon 2 through Cre-induced recombination, resulting in a reading frameshift in exons 3–7 and knock-out the expression of h2-calponin. The positions of 6 primers used as 4 pairs in PCR genotyping are outlined on the maps. C, PCR genotyping of the Cnn2-targeted alleles. The left panel shows agarose gels of PCR detections of the insertion of loxP sequence in intron 1 and the Cre-induced deletion of exon 2 (the PCR conditions for Primer pair 3 would detect the short product of Cnn2-KO allele but not the long segment in Cnn2-flox allele). The right panel shows agarose gels of PCR identification of the FLP1-induced removal of the neo^R cassette (the PCR conditions for Primer pair 2 would detect the short product of Cnn2-flox allele but not the long segment in Cnn2-flox-neo allele) together with detection of the loxP insertion. The presence or absence of neo^R cassette was confirmed by PCR using primer pair 4 (data not shown).

FIGURE 4.

Knock-out and reversible knockdown of h2-calponin in Cnn2 gene-targeted mice. A, knock-out of h2-calponin in representative mouse tissue and cell types. The SDS-PAGE and Western blots showed that h2-calponin was absent in stomach (smooth muscle), lung (alveolar cells), skin (keratinocytes), fibroblasts, peritoneal residential cells, and peripheral white blood cells (WBC) of Cnn2-knock-out (KO) mice in contrast to these tissue and cell types in wild type (WT) mice, which express h2-calponin at significant levels. B, densitometry quantification of multiple Western blots of total protein extracts showed very similar levels of h1-calponin in the stomachs of wild type and h2-calponin knock-out mice, indicating no compensatory up-regulation of h1-calponin when h2-calponin ceases expression. C, SDS-PAGE and Western blot showed that the level of h2-calponin significantly decreased in the stomach of a Cnn2-flox-neo heterozygous mouse and further down in Cnn2-flox-neo homozygotes, demonstrating a dominant knockdown effect. FLP1-induced removal of the neo^R cassette from the intron 2 of Cnn2 effectively restored the expression of h2-calponin to the wild type levels. +, wild type Cnn2 allele.

FIGURE 5.

Increased diapedesis of h2-calponin-free macrophages. The left panels show representative flow cytometry histograms of 72-h elicited peritoneal cells for the identification of macrophages (A) and granulocytes (B). The Mac-1+ population is indicated on the x axis (FITC) and the Gr-1 (phycoerythrin-Cy7) or F4/80 (allophycocyanin) is indicated on the y axis. Macrophages were identified by strong Mac1-positive, F4/80-positive, and Gr1-negative stains, whereas granulocytes were identified by strong Mac1-positive, F4/80-negative, and Gr1-positive stains. The upper right quadrant represents the double positive cells that represent macrophages (A) and granulocytes (B). The right panels show the summarized results in which the percent of 72-h elicited macrophages was significantly increased in h2-calponin knock-out (KO) mice compared with the wild type (WT) control (**, p < 0.01 in two-tail Student's t test) (A), whereas the percent of 72-h elicited granulocytes showed a corresponding decrease in h2-calponin knock-out mice than that in WT mice (B) (*, p < 0.05 in one-tail Student's t test). n = 7 mice in each experimental group except for n = 5 or the 72 h elicited h2-calponin knock-out group.

FIGURE 6.

Faster migration of h2-calponin-free macrophages during in vitro wound healing. A, scratch wounds were made in monolayer cultures of peritoneal residential macrophage 24 h after plating. Healing of the wound by cell migration was monitored for 6 h. The micrographs showed an earlier closure of the wound in the h2-calponin-free macrophage culture than the wild type control. B, SDS-PAGE and Western blot using RAH2 antibody on total protein extracts from cells collected at the end of the wound healing experiments confirmed the presence and absence of h2-calponin in the wild type and Cnn2 knock-out cells, respectively. C, quantification of the wound healing data demonstrates the faster migration rate of the h2-calponin knockout macrophages than the wild type control. *, p < 0.05.

FIGURE 7.

Faster proliferation of h2-calponin-free cells. A, Western blots showed that bone marrow cells isolated from adult wild type (WT) and h2-calponin knock-out (KO) mice had positive and negative, respectively, expression of h2-calponin. B, the rate of bone marrow cell proliferation in L929 cell-conditioned media was examined by Crystal Violet staining and the results showed a significantly faster proliferation of h2-calponin-free bone marrow cells than that of wild type cells. C, Western blots showed that the RAW264.7 mouse macrophage line had lost endogenous h2-calponin expression in contrast to primary mouse peritoneal macrophages. Stable transfective expression of mouse h2-calponin cDNA under the cytomegalovirus promoter produced a significant level of h2-calponin. D, RAW264.7 cells stably transfected with sense or antisense h2-calponin cDNA expression vector and non-transfected control were examined for the rate of cell proliferation as in B. The results demonstrated a significant decrease in the h2-calponin sense cDNA-transfected cells in comparison to that of the antisense-transfected and non-transfected RAW cells. The slightly lower proliferation rate of the antisense cDNA-transfected cells than that of the non-transfected cells reflected the effect of G418 in the culture of the transfected cells. **, p < 0.001.

FIGURE 8.

Removal of h2-calponin from macrophages reduces cell spreading area in adhesion cultures. A, immunofluorescence microscopy using anti-h2-calponin RAH2 and anti-tropomyosin CG3 antibodies followed by TRITC-conjugated secondary antibodies and TRITC-phalloidin staining of F-actin examined the cellular localization of h2-calponin in macrophages and the effect of its removal on the actin cytoskeleton. The phase-contrast and fluorescence images demonstrate that h2-calponin-free macrophages had a smaller spreading area, although the structure of actin cytoskeleton was not significantly altered. B, the reduced spreading area of h2-calponin-free macrophages in adherent culture was quantified using NIH Image software version 1.61. **, p < 0.01.

FIGURE 9.

Removal of h2-calponin resulted in a decrease of tropomyosin in macrophages. A, Western blots showed a lower level of tropomyosin versus actin in the h2-calponin-free macrophages than that in wild type cells. B, this decrease in the tropomyosin level was quantitatively demonstrated by densitometry analysis (*, p < 0.05).

FIGURE 10.

H2-calponin-free macrophages had enhanced phagocytosis. Residential (A) and elicited (B) peritoneal cells isolated from h2-calponin knock-out (KO) and wild type (WT) mice were incubated with red fluorescent carboxyl microspheres to test phagocytosis activity. In the representative flow cytometric histograms of the F4/80-positive WT and KO macrophages, the x axis represents the fluorescent intensity indicating phagocytosis of the beads and the y axis indicates the gating for macrophages (upper panels) and the relative cell counts (lower panels). The peaks from left to right represent cell populations with no bead-ingested, 1 bead ingested, and 2 to more than 5 bead ingested. C, the phagocytosis activities measured on peritoneal residential (n = 7 for both WT and KO) and 72-h elicited (n = 7 for WT, n = 5 for KO) macrophages are summarized as phagocytosis index (percent of beads-ingested cells × mean florescence intensity of cells containing beads). The data demonstrated an enhanced phagocytosis activity of both residential and elicited macrophages when h2-calponin was absent (*, p < 0.05).