Effects of acute and chronic low density lipoprotein exposure on neutrophil function

Abstract

Mounting evidence suggests that obesity and the metabolic syndrome have significant but often divergent effects on the innate immune system. These effects have been best established in monocytes and macrophages, particularly as a consequence of the hypercholesterolemic state. We have recently described defects in neutrophil function in the setting of both obesity and hypercholesterolemia, and hypothesized that exposure to elevated levels of lipoproteins, particularly LDL its oxidized forms, contributed to these defects. As a model of chronic cholesterol exposure, we examined functional responses of bone marrow neutrophils isolated from non-obese mice with diet-induced hypercholesterolemia compared to normal cholesterol controls. Chemotaxis, calcium flux, CD11b display, and F-actin polymerization were assayed in response to several chemoattractants, while neutrophil cytokine transcriptional response was determined to LPS. Following this, the acute effects of isolated LDL and its oxidized forms on normal neutrophils were assayed using the same functional assays. We found that neutrophils from non-obese hypercholesterolemic mice had blunted chemotaxis, altered calcium flux, and normal to augmented CD11b display with prolonged actin polymerization in response to stimuli. In response to acute exposure to lipoproteins, neutrophils showed chemotaxis to LDL which increased with the degree of LDL oxidation. Paradoxically, LDL oxidation yielded the opposite effect on LDL-induced CD11b display and actin polymerization, and both native and oxidized LDL were found to induce neutrophil transcription of the monocyte chemoattractant MCP-1. Together these findings suggest that chronic hypercholesterolemia impairs neutrophil functional responses, and these defects may be in part due to protracted signaling responses to LDL and its oxidized forms.

Figure 1. Serum levels of LDL cholesterol are similarly elevated in obese mice compared to lean mice on high cholesterol diet

(A) Serum levels of total, LDL and HDL cholesterol in lean and obese db/db mice compared to wild type mice fed a normal or high cholesterol diet. (B) Oxidation of serum LDL in lean and obese db/db mice was assayed by heparin/manganese precipitation followed by TBARS and normalized to LDL content. n = 3-5 mice/group. * p<0.05, ** p<0.01 compared to control (lean or normal cholesterol). Cholesterol levels in high cholesterol diet mice are not significantly different from the corresponding levels found in obese mice.

Figure 2. Chronic hypercholesterolemia impairs neutrophil chemotaxis

Chemotaxis of density centrifugation-isolated mature bone marrow neutrophils from lean hypercholesterolemic mice was compared to normal cholesterol controls using a modified Boyden chamber with KC (50ng/ml) and formyl-Met-Leu-Phe (fMLP, 1 μM). Four separate experiments were performed. ns = not significant, * p<0.05, ** p<0.01. All fMLP and KC conditions were significantly higher than respective control (p<0.01).

Figure 3. Chronic hypercholesterolemia attenuates neutrophil calcium flux in response to chemoattractants

Cellular calcium flux response to fMLP (1 μM) was determined in mature bone marrow neutrophils isolated from lean hypercholesterolemic mice was compared to normal cholesterol controls using Indo-1AM cytosolic dye-loading and flow cytometry. Three separate experiments were performed on high cholesterol diet mouse isolated neutrophils and normal cholesterol control; representative runs are shown for each source.

Figure 4. Stimulated neutrophils from hypercholesterolemic mice show normal to elevated CD11b expression and F-actin polymerization, and actin polymerization is slow to reverse over time

(A) CD-11b expression by neutrophils from normal vs. high cholesterol diet mice was assessed by flow cytometry following stimulation with phorbol myristate acetate (PMA, 1 μM) for 30min. Values are expressed as percent of CD-11b expressed in normal cholesterol diet mice. Three separate experiments were performed. (B) F-actin polymerization in neutrophils from normal vs. high cholesterol diet mice was assessed by phalloidin staining and flow cytometry following stimulation with fMLP (1 μM) for 30s or 5min. Values are expressed as percent of F-actin polymerization at 30 seconds in normal cholesterol diet mice. Four separate experiments were performed. ** p<0.01 lower compared to 30s, ns = not significantly different compared to 30s. NCD = normal cholesterol diet; HCD = high cholesterol diet.

Figure 5. LDL induces neutrophil chemotaxis, which increases with level of LDL oxidation

Chemotaxis of density centrifugation-isolated mature bone marrow neutrophils from normal cholesterol diet mice was examined using a modified Boyden chamber with native LDL (nLDL), minimally oxidized LDL (mmLDL), moderately oxidized LDL (ox-LDL) (all 100 μg/mL), and the chemoattractants KC (50ng/ml) and fMLP (1 μM). Four separate experiments were performed. ** p<0.01 by 1-way ANOVA with post-test for linear trend, *** p<0.001 compared to control.

Figure 6. Native LDL but not oxidized LDL induces neutrophil calcium flux

Cellular calcium flux response to nLDL (100μg/mL), ox-LDL (100μg/mL), or fMLP (1 μM) was determined in mature bone marrow neutrophils isolated from normal cholesterol diet mice using Indo-1AM cytosolic dye-loading and flow cytometry. Five separate experiments were performed; representative runs are shown for each stimulus.

Figure 7. Native LDL induces neutrophil CD11b surface display and intracellular F-actin polymerization, and is more effective than ox-LDL in this regard

(A) CD-11b expression by neutrophils from normal cholesterol diet mice was assessed following stimulation with PMA (1 μM), nLDL (100μg/mL), or ox-LDL (100μg/mL) for 30min. (B) F-actin polymerization in neutrophils from normal cholesterol diet mice was assessed following stimulation with fMLP (1 μM), nLDL (100μg/mL), or ox-LDL (100μg/mL) for 30s. Values are expressed as ratio mean fluorescence intensity normalized to buffer control in each experiment. Four separate experiments were performed for each assay. * p<0.05 lower compared to control or indicated comparison, ** p<0.01, ns = not significant.

Figure 8. Oxidized LDL induces neutrophil apoptosis

Neutrophils from mice fed normal cholesterol diet were treated with nLDL, ox-LDL (both 100μg/mL), or buffer control for 4h in culture, fixed and stained for caspase 3, and scored by fluorescent microscopy. The numbers of apoptotic neutrophils (per 100 counted) on low powered fields were determined on 5 fields and averaged for each experimental condition, and the experiment was repeated 3 times. * p<0.05. ns = not significant.