Mice with CCTα-null macrophages were derived as described previously (Zhang et al., 2000
). In brief, mice carrying a floxed Pcyt1a
gene were crossed with mice carrying a Cre
recombinase driven by the macrophage-specific LysM promoter. Two flox sites flanked a 12.5-kb fragment of the Pcyt1a
gene containing exons 5–9 (Karim et al., 2003
) and the intervening introns. Deletion of the region between the flox sites resulted in expression of a truncated CCTα protein that lacked the catalytic and regulatory domains located at the carboxy terminus. Mice that were homozygous for the floxed gene and heterozygous for the LysMCre gene were mated, and the pups were genotyped and used to obtain either CCTα knockout or littermate wild-type control macrophages. Peritoneal macrophages were elicited, and the CCTα-deficient macrophage population was confirmed to have reduced rates of de novo PtdCho synthesis as measured by [3
H]choline incorporation into the lipid fraction of adherent cells (). The deletion in this macrophage model was not completely penetrant (Zhang et al., 2000
). Analysis of the CCTα transcript level in CCTα-deficient macrophages by quantitative RT-PCR (qRT-PCR) revealed that the transcript level was significantly reduced to 30% of the wild-type level (). The CCTα-null cells were ≥95% macrophages as determined microscopically after staining with macrophage-specific anti–mouse CD11b. Cells were also stained for expression of CCTα protein, and 20–30% of the macrophages isolated from individual knockout mice (n
= 4) remained positive for expression, which confirms that the action of the Cre recombinase was not 100% penetrant. Thus, the residual PtdCho synthesis measured by metabolic radiolabeling in the CCTα-null populations was due not only to expression of the alternate isoform, CCTβ3 (), but also to the presence of cells that did not delete the CCTα gene. The qRT-PCR revealed that the CCTβ2 isoform was not expressed in macrophages, and that CCTβ3 expression was increased in the knockouts. CCTβ3 is equivalent to CCTα in its primary biochemical function, and expression of its activity can compensate for loss of CCTα to support bulk membrane synthesis in cultured cells (Karim et al., 2003
). It was previously found that CCTβ2 expression increased in the CCTα-deficient cells (Zhang et al., 2000
), but at the time, CCTβ3 had not been discovered, and both isoforms share the common epitope that was signaled in the previous immunoblots (Karim et al., 2003
). Total CCT activity was significantly lower in cell lysates prepared from CCTα-null macrophages compared with wild-type populations (), and these data agreed with the previously published study (Zhang et al., 2000
). The decrease in CCTα and the increase in CCTβ3 protein assessed by immunoblotting with isoform-specific antibodies reflected the changes in mRNA levels (). Macrophages were isolated from two knockout and two wild-type mice and pooled according to genotype, then immunoblots were prepared from six replicates of each genotype. The level of CCTα protein in the knockout population was 0.36 ± 0.08 compared with the wild-type population (level set at 1.00). The reduction in CCTα transcripts correlated with the protein level determined by immunoblots and with the number of macrophages devoid of CCTα expression as determined by microscopy of immunostained cell populations. These data confirm the loss of CCTα expression in the majority of cells and also provide the first example of CCTβ3 regulation as an adaptive response to PtdCho deficiency.
Figure 1. Reduced PtdCho synthesis in CCTα-deficient macrophages. (A) Wild-type (WT; n = 12) or CCTα-deficient (KO; n = 12) macrophages were labeled for 6 h with 1 μCi/ml [3H]choline, and incorporation into PtdCho was normalized (more ...)
Several macrophage functions were assessed to determine if they were compromised by reduced PtdCho synthesis. Phagocytosis was evaluated by quantifying the uptake of fluorescein-labeled Escherichia coli
, and the values were normalized to cell number by counterstaining and measuring DAPI fluorescence. The uptake of the bacteria was linear up to 2 h and was the same in both wild-type and CCTα-deficient cells (). Random migration and chemotaxis of the macrophages in response to the attractant formyl-methionylleucylphenylalanine (fMLP) was determined in modified Boyden chambers. The numbers of cells that crossed a membrane into an adjacent chamber containing medium alone or medium plus 10 nM fMLP were determined by staining with Calcein AM, and the data were normalized to the starting cell number. The assay was linear for at least 4 h and both the knockout and wild-type cell populations exhibited similar rates of random migration and chemotaxis (). Cytokine production by macrophages in response to E. coli
LPS was measured by sampling the culture medium 18 h after addition of the ligand. Secretion of TNFα, and interleukin-6 (IL-6) was reduced in the CCTα-deficient population, but the secretion of interleukin-1β (IL-1β) was comparable to that in wild-type cells (). Both TNFα and IL-6 are delivered to the medium by vesicle-mediated secretion from the Golgi apparatus. IL-1β is transported by ATP-dependent secretion of specialized lysosomes that bypass the Golgi compartment (Andrei et al., 2004
). The release of prostaglandin E2
) was also normal in the CCTα-null cells (). Altogether, these data indicated that LPS signaling was intact and that secretion of cytokines from the Golgi apparatus was impaired in the CCTα-deficient macrophages.
Figure 2. Functional comparison of wild-type and CCTα-deficient macrophages. (A) Phagocytosis of fluorescein-labeled E. coli was quantified after 2 h of incubation and then normalized to the cell number by staining with DAPI as described in Materials and (more ...)
A time course revealed reduced release of both TNFα and IL-6 from the knockout cells into the medium (). Reduced cytokine secretion was reflected in reduced total production of the cytokines (Fig. S1, available at http://www.jcb.org/cgi/content/full/jcb.200706152/DC1
). Immunoblotting of cell lysates at 12 and 24 h after stimulation showed that TNFα accumulated in the CCTα-deficient macrophages as both the precursor and the mature form (). In contrast, the membrane-bound precursor form of TNFα (pro-TNFα) was quantitatively processed to the soluble mature form and completely released from the wild-type cells by 24 h. TNFα processing is concurrent with its movement from the Golgi to the cell surface and mediated by a metalloprotease called TNFα-converting enzyme (TACE; Gearing et al., 1994
). A TACE inhibitor, N
-alanine, 2-aminoethylamide (TAPI; Crowe et al., 1995
), was added to wild-type macrophages after LPS addition to inhibit TNFα processing and confirm the identification of the pro-TNFα on the blots (). TAPI abolished TNFα release to the medium, but TAPI did not inhibit IL-6 secretion from wild-type cells (Fig. S2). The data demonstrated that LPS signaling and cytokine synthesis were intact in the CCTα-deficient macrophages. Because IL-6 secretion was not compromised, the inhibition of TACE activity by PtdCho deficiency was ruled out as a mechanism for the TNFα secretion defect. Constitutive secretion of apolipoprotein E (ApoE) was not dependent on LPS stimulation and was not impaired in the CCTα-null cells (). The synthesis of ApoE is repressed at the transcript level after LPS stimulation (Gafencu et al., 2007
), and, thus, the amount released to the medium was reduced in both wild-type and knockout cells at later times after LPS stimulation ( and S3 A). Imaging of cellular TNFα and ApoE in the same cells indicated that these secretory products were located in different compartments (Fig. S3 B), which supports the view that ApoE exited from the Golgi compartment via different vesicles. New PtdCho biosynthesis was not required for secretion of ApoE but was necessary for the release of TNFα and IL-6 after LPS stimulation.
Figure 3. Secretion and processing of TNFα. (A) TNFα in the culture medium from wild-type (○; n = 5) or CCTα-deficient (•; n = 10) macrophages was measured at indicated times after LPS stimulation. The anti-TNFα (more ...)
The process of cytokine synthesis and secretion in the macrophages was investigated using immunocytochemistry. Wild-type and knockout cells were treated with LPS for 6 and 18 h before fixation and then stained with anti-TNFα or anti–IL-6 antibodies (). The images confirmed that the CCTα-deficient cells were able to synthesize TNFα and IL-6. Intracellular TNFα and IL-6 were detected at 6 h, whereas wild-type cells were negative for these cytokines by 18 h after LPS stimulation. These results correlated with cellular cytokine synthesis and secretion to the medium. However, both cytokines were still retained within the CCTα-deficient cells up to 48 h after LPS stimulation.
Figure 4. TNFα and IL-6 accumulation in the CCTα-deficient macrophages. (A) Wild-type (WT) or CCTα-deficient (KO) macrophages were treated with LPS, fixed, and stained with anti-TNFα antibody (green) at 0, 6, or 18 h. (B) Wild-type (more ...)
The results described thus far were obtained using thioglycolate-elicited macrophages that were isolated by peritoneal lavage and cultured in vitro. The physiological relevance of the phenotype was tested in vivo to ensure that the findings were not artifacts of manipulation of the cells or the in vitro system. The numbers of circulating monocytes, peritoneal macrophages, and bone marrow macrophages in the knockout animals were the same or slightly greater than those from the wild-type mice (Fig. S4, available at http://www.jcb.org/cgi/content/full/jcb.200706152/DC1
). We subjected both wild-type and knockout mice to infection with an intranasal dose (107
colony-forming units) of luminescent Streptococcus pneumoniae
strain T4X and followed the course of the infection in five to six individual littermate mice of each genotype (). Wild-type mice developed mild pneumonia and between 10 and 50% succumbed in three independent experiments. In contrast, mice with CCTα-deficient macrophages developed more serious diffuse pneumonia, with higher bacterial counts, and the disease quickly progressed to sepsis (), with a mean 70% mortality rate (). In lungs of knockout animals, there were extensive areas of interstitial pneumonia with consolidation, necrosis, and marked fibrinopurulent pleuritis (, top). The inflammatory infiltrate in the pneumonic areas was composed of viable and degenerate neutrophils, macrophages, and perivascular cuffs of lymphocytes and plasma cells. In lungs of wild-type mice, there were patchy mild interstitial inflammatory infiltrates composed primarily of neutrophils with fewer macrophages and perivascular and peribronchiolar infiltrates of lymphocytes and plasma cells. A Gram's stain to detect bacteria in the lung tissue revealed abundant cocci in the knockout animals (, bottom), particularly embedded in the layers of fibrin along the pleural surface (arrows), whereas Gram-positive cocci were rarely observed in the wild-type lungs. The numbers of macrophages in the knockout model were equivalent to the numbers in wild-type animals (Fig. S4, A–C), and the knockout macrophages at the site of infection accumulated and retained TNFα, in contrast to the wild-type macrophages (). The macrophages that infiltrated the lungs 48 h after infection were identified in cryosections with an anti–MAC-1 antibody and counterstained with an anti-TNFα antibody. The wild-type macrophages were devoid of the cytokine by this time, but the majority of knockout cells still retained a strong signal, which indicates that the cytokine had not yet been released into the extracellular space (). These data supported the hypothesis that CCTα deficiency caused the macrophages to accumulate TNFα and impaired cytokine secretion in vivo.
Figure 5. Aberrant cytokine release from CCTα-deficient macrophages after bacterial infection. (A) Distribution of S. pneumoniae LUX at 3 d after intranasal challenge. Bacterial numbers are proportional to the color bar on the right. Note the small focus (more ...)
The reorganization of the Golgi apparatus after LPS stimulation was compared between wild-type and CCTα-deficient macrophages using an antibody that recognizes the ER–Golgi intermediate compartment 53-kD protein (ERGIC-53), a protein specifically localized in the ER and cis-Golgi compartment (Litvak et al., 2005
). The images indicated only limited rearrangement of the marker protein after LPS stimulation and a similar distribution in both wild-type and knockout cells (). Next, the trans-Golgi compartment was imaged with an antibody that recognizes the K58 marker protein (Bloom and Brashear, 1989
). K58 was located proximal to the cell nucleus and surrounded 30–50% of the nucleus before LPS stimulation. In wild-type cells, the K58 protein coalesced into a focused structure at a distinct perinuclear site by 6 h and then returned to the more diffuse distribution characteristic of unstimulated cells at 18 h after LPS activation (). In the CCTα-deficient cells, the K58 marker behaved similarly to wild-type cells, except that the K58 protein was still intensely focused in a single region adjacent to the nucleus by 18 h after LPS stimulation. Interestingly, the CCTα protein in the wild-type, stimulated primary macrophages was associated with the Golgi compartment (). The CCTα signal converged by 6 h after LPS treatment and then redistributed to the more diffuse distribution surrounding up to 50% of the nucleus, similar to the K58 marker. The CCTα protein was not visualized in ~80% of the knockout macrophage population, and cells that expressed CCTα did not retain TNFα 18 h after LPS (Fig. S4 E). The CCTβ proteins did not redistribute after LPS treatment (Fig. S4 D). These data indicated that the CCTα protein in primary macrophages localized to the trans-Golgi region and that this compartment was the site of the Golgi dysfunction in the cytokine secretion pathway of CCTα-deficient cells.
Figure 6. Distributions of Golgi markers in LPS-treated macrophages. Wild-type (WT) and knockout (KO) peritoneal macrophages were treated with LPS at 10 ng/ml for 0, 6, and 18 h. After immediate fixation in 4% paraformaldehyde, cells were permeabilized and stained (more ...)
The biochemical defect underlying the phenotype in the knockout cells could be caused by an imbalance in several different lipids. PtdCho is metabolized to PtdOH in the Golgi organelle by a PLD, which is activated by secretory stimuli. PtdOH is in turn rapidly converted to DAG by a PtdOH P'tse. PtdCho is also the metabolic precursor to sphingomyelin (SM), a lipid that is synthesized in the Golgi compartment and then transported to the cell surface. Measurement of the amount before and 18 h after LPS stimulation revealed that the PtdCho was significantly reduced in the CCTα-deficient cells after LPS, in contrast to the wild-type cells, where the PtdCho level was maintained after stimulation (). In contrast, the amount of total protein per cell was the same in wild-type and knockout cells both before and after LPS (Fig. S4 F). Phosphatidylethanolamine (PtdEtn), the second most abundant phospholipid in intracellular membranes, did not change significantly in the knockout cells after LPS stimulation (Fig. S5 A, available at http://www.jcb.org/cgi/content/full/jcb.200706152/DC1
). The DAG levels in CCTα-deficient cells increased significantly but remained the same in wild-type after LPS stimulation (). SM was reduced to the same apparent level with no statistical difference between the wild-type and knockout cells (). These data indicated a biochemical imbalance in the PtdCho and DAG but normal SM metabolism in the CCTα-deficient cells. To confirm that SM synthesis was unaffected in the knockout cells, macrophages were radiolabeled with [3
H]choline for 6 h with or without LPS. The data showed that SM synthesis did not change after LPS treatment of wild-type cells and that SM synthesis in the knockouts was the same as the wild type (Fig. S5 B). Meanwhile, data from the same experiments demonstrated that the rate of PtdCho synthesis was reduced in the CCTα-null macrophages both in the unstimulated cells and after LPS addition. Thus, the amount of PtdCho in the CCTα-null cells was sufficient for SM production despite the reduced rate of PtdCho synthesis.
Figure 7. Amounts of PtdCho, DAG, and SM after LPS stimulation. Wild-type (WT; white) and CCTα-deficient (KO; black) macrophages were incubated with 10 ng/ml LPS or without LPS for 18 h, and total lipids were extracted. The identification of PtdCho (A) (more ...)
Both CDP–Cho and DAG are substrates for the CPT that makes PtdCho. Limitation of the CDP–Cho supply by genetic inactivation of the CCTα accounted for the reduced PtdCho and for the metabolic accumulation of DAG after LPS. Affymetrix microarray data, available at the National Center for Biotechnology Information website (Gene Expression Omnibus; http://www.ncbi.nlm.nih.gov/
; Shell et al., 2005
), revealed that expression of a select group of lipid metabolic genes increased 18 h after LPS stimulation of wild-type macrophages, including those encoding the PLD1, the CCTα, and the choline/ethanolamine phosphotransferase (C/EPT; Table S1, available at http://www.jcb.org/cgi/content/full/jcb.200706152/DC1
; Shell et al., 2005
). We confirmed the data using real-time qRT-PCR on total RNA extracted from wild-type macrophages before and 18 h after LPS stimulation, and found a statistically significant increase in the expression of these genes (Fig. S5 E). This information indicated the isoforms of each enzyme that worked together with the CCTα to control phospholipid metabolism during LPS-stimulated cytokine secretion. Taken together, the data support the existence of a cycle of PtdCho degradation and resynthesis that accompanies cytokine secretory vesicle formation and budding from the Golgi complex ().
Figure 8. Cycle of PtdCho degradation and resynthesis associated with secretion from the Golgi apparatus. PtdCho is degraded by a PLD1 to PtdOH, which in turn is converted to DAG by a PtdOH P'tse during secretion from the trans-Golgi compartment. The C/EPT utilizes (more ...)
A pharmacological approach was used to test several aspects of this hypothesis. Et-18-OCH3
(edelfosine), an inhibitor of CCT (Boggs et al., 1995a
), dramatically reduced TNFα secretion in cells of both genotypes (). Incubation with lysophosphatidylcholine (lysoPC), which is rapidly converted to PtdCho (Baburina and Jackowski, 1999
), bypassed the CCTα genetic defect (Esko et al., 1982
; Boggs et al., 1995b
; Baburina and Jackowski, 1998
) and partially restored TNFα secretion from CCTα-deficient cells (). The LysoPC pathway would replenish PtdCho but would not remove DAG. In fact, measurement of the DAG level in lysoPC-treated knockout cells revealed that the DAG level increased (Fig. S5 F). This result was probably caused by inhibition of CCTβ3 by lysoPC, which has been shown to interfere with the membrane association and activation of the enzyme (Boggs et al., 1995a
; Attard et al., 2000
). Treatment of cells with either 1-butanol to inhibit phospholipase D activity, or propranolol to inhibit PtdOH P'tse activity, also inhibited TNFα secretion (). These results support the involvement of the phospholipase D pathway in secretion. However, fumonisin B1, an inhibitor of ceramide and SM synthesis, had no effect. These results confirmed that the cellular defect was caused by reduced PtdCho synthesis and did not extend to an imbalance in SM synthesis (). SM synthesis and TNFα and IL-6 release to the medium were measured in the same experiment in the presence and absence of fumonesin B1 in wild-type cells. Although SM synthesis was severely reduced, the amount of either cytokine in the medium was the same with or without inhibitor treatment (Fig. S5, C and D). Nor did 2-butanol have an effect, which was a treatment control for the specificity of the phospholipase D inhibition by 1-butanol (). Taken together, these data support the idea that de novo PtdCho synthesis was required to maintain cytokine secretion from the Golgi apparatus in LPS-stimulated macrophages.
Figure 9. Response of TNFα secretion to different treatments. (A) Wild-type (WT; white bars) or CCTα-deficient (KO; black bars) macrophages were treated with 10 ng/ml LPS for 18 h. At the same time, 100 μM LysoPC, 15 μM edelfosine, (more ...)
DAG levels were increased to test whether impaired secretion would be the result. At 3 h after LPS stimulation, wild-type cells were incubated with increasing concentrations of a phospholipase C isolated from Bacillus cereus. The phospholipase C in the medium converted surface membrane PtdCho to DAG. We reasoned that at least a portion of the DAG would relocate intracellularly to the Golgi apparatus, similar to the supplemental lysoPC in the medium that resulted in rescue of the phenotype (). At 6 and 18 h after addition of the enzyme, the amount of TNFα in the medium and the amount of DAG in the cells was quantified (). Despite substantial increases in DAG that correlated with the amount of phospholipase C added (), TNFα secretion was not impaired (). These data argued that increased DAG was not the critical biochemical factor leading to the knockout phenotype. Rather, depletion of the PtdCho supply impeded vesicular transport, resulting in accumulation of cytokines in the Golgi apparatus.
Figure 10. Cell-associated DAG and TNFα release to the medium after LPS and exogenous phospholipase C. Wild-type macrophages were treated with LPS for 3 h, then B. cereus phospholipase C was added at the indicated concentrations for the remainder of the (more ...)