Plasma cholesterol modulate functions of neutrophils in streptozotocin-induced type 1 diabetic rats

INTRODUCTION
Neutrophils, part of the innate immune response, are the first line of defense in hosts. Numerous studies have demonstrated altered neutrophil functions in diabetes and it is suggested that the impaired neutrophil functions [e.g., chemotaxis, phagocytosis, nitroblue tetrazolium (NBT) dye reduction ability etc.] cause the susceptibility to infections in diabetics (Coopan, 1985; Reeves and Wilson, 1992). The chemotactic activity of neutrophils from diabetic patients is significantly lower compared to those of healthy controls (Mowat and Baum, 1971). Studies of the phagocytic and microbicidal activities of neutrophils from diabetic patients reveal, with few contrasting results, an impairment of these functions. Decreased bactericidal activity (Tan et al, 1975), impairment of phagocytosis and decreased release of lysosomal enzymes (Bagdade et al, 1972), and reduced production of reactive oxygen species (Nielson and Hindson, 1989) by neutrophils of diabetic patients have been described. Furthermore, reduction in leukocyte phagocytosis and bactericidal activity showed a significant correlation with increases in blood glucose levels (Jakelic et al, 1995). 

Hyperglycemia, one of the characteristic manifestations of diabetes mellitus, has been found to be associated with neutrophil dysfunctions (Nabi et al, 2005). Formation of advanced glycation end products (AGEs) through an interaction between glucose and lipids and/or proteins is one of the causes of long-term complications in diabetes (Brownlee, 2001). Glycosylated proteins isolated from the serum of diabetic rats affect membrane permeability and migration, reduce the rolling and adhesion abilities of leukocytes in alloxan-induced diabetic rats (Sannomiya  et al., 1997; Masuda et al., 1990). Further, AGEs in human is linked to a rise in intracellular Ca2+ and to actin polymerization (Collison et al., 2002). Actin polymerization in neutrophil plays important role in chemotactic action of neutrophils that is crucial to exhibit its normal functions. A positive association between polyol pathway activation and leukocyte dysfunction in experimental diabetes mellitus has been reported and hypothesized that the accelerated formation of sorbitol in diabetic animals may increase the intracellular osmolarity or decrease the availability of the enzyme co-factor NADPH, leading to a disturbance of endothelial cell functions that might alter leukocyte-endothelial cell interactions (Cruz et al., 2000).

Epidemiological studies have established a strong correlation between elevated total cholesterol levels in serum and morbidity and mortality from myocardial infarction (Thomas et al, 1993). Elevated numbers of circulating neutrophils have been shown to be predictive of cardiovascular events independent of serum cholesterol levels (Guasti et al, 2011) In a more recent study, a direct mechanistic link between hypercholesterolemia and proliferation of myeloid progenitor cells and, hence, neutrophilia and monocytosis has been identified (Murphy et al, 2011 and Weber et al, 2011). High dose of Statin, a cholesterol lowering agent, has been demonstrated to lower the migration ability of neutrophils significantly in healthy volunteers (Kinsella et al, 2011). Further, free cholesterol has been found to be associated with the altered lipid raft structure of cell membrane and function regulating neutrophil ca2+ entry and respiratory burst (Kolenkode et al, 2007). Our previous study demonstrated neutrophil dysfunction in streptozotocin (STZ)-induced diabetic rats (Nabi et al., 2005). This study was aimed to investigate the biochemical indices such as plasma levels of cholesterol, triglycerides, creatinine, urea, alanine transaminase and aspartate transaminase in diabetic and control rats and thus, investigate their relationship with the previously studied neutrophil functions.

MATERIALS AND METHODS
Preparation for streptozotocin-induced type 1 diabetes mellitus in the rats and collection of blood and plasma 
A total of 30 Long Evan rats (Male: 15; Female: 15) each 2 weeks of age were kept in the plastic cages with even floors covered with wood shavings in the animal house of the department of Biochemistry and Molecular Biology, University of Dhaka, Bangladesh. The initial average body weight of the male rats was 145.3  5.6 gm and of the female rats was 140.85  4.3 gm. These animals were kept under constant temperature with a 14 hour light and 10 hour dark cycle. About 5-6 gm of balanced pelleted rat food was supplied thrice a day. These animals had also free access to drinking water. These conditions were maintained for the next 4 weeks.

Protocol for the preparation of streptozotocin-induced type 1 diabetic rats has been described in our previous study (Nabi et al., 2005) using intraperitoneal injection of streptozotocin (STZ) dissolved in citrate buffer (65 mg/Kg body weight) at the age of 4 weeks (185.5  10.2 gm and 180.7  11.2 gm body weights for 10 male and 10 female rats, respectively). Age matched control rats (5 male and 5 female) were injected with citrate buffer only. After anesthetizing in a chamber containing diethyl ether, blood samples were collected by sacrificing each diabetic and control rats into a heparin-containing falcon tube. Immediately after collection, 2.0 ml of blood was transferred into fresh tube and centrifuged at 3000 rpm for 10 minutes. The plasma was collected and stored at –20OC until further analysis.

Isolation of neutrophils, polarization assay and NBT dye reduction tests
Neutrophils were isolated from the freshly collected blood samples of control and diabetic rats by dextran (Sigma) sedimentation followed by centrifugation on Ficoll-Hypaue (Pharmacia, Uppsala, Sweden) as described elsewhere (Islam and Nabi, 2003; Nabi et al, 2005). The separated neutrophils were resuspended in a minimal volume (1.0 – 1.5 ml) of Hank's Balanced Salt Solution mixed with 3-(N-morpholino)-propanesulfonic acid (HBSS-MOPS). The percentage of polarized cells was assessed by viewing the cell preparation under Light microscope using a 40X objective (Nabi and Islam, 2001; Islam and Nabi, 2003; Nabi et al, 2005). At least 300 cells were counted from each preparation. NBT dye reduction test was performed according to the protocol described earlier (Nabi and Islam, 2001; Nabi et al, 2005). Briefly, neutrophils (2 x 106 cells/mL) treated with yeast activated serum, incubated for 30 minutes at 37OC and then, aliquots of NBT (Sigma) solution was added into the cells and incubated for 1 hour at 37OC. The unused NBT was removed through washing and the reduced dye was extracted in dioxan (Sigma) and quantitated at 520 nm.
Biochemical analyses of the plasma samples
Total plasma cholesterol, triglycerides, plasma creatinine, urea and activities of plasma AST and ALT were measured by standard colorimetric method in an Autoanalyzer (UK) using kits purchased from RANDOX, UK. Briefly, plasma cholesterol was measured by oxidation of cholesterol using cholesterol oxidase, triglycerides by enzymatic hydrolysis using lipases, creatinine by Jaffe method using alkaline picrate, urea using urease method, ALT and AST by kinetic methods using lactate dehydrogenase and NADH.By means of respective units, results of each parameter were expressed. 
Phagocytosis assay
Baker’s yeasts (Saccaromyces cerevisiae; Gist brocades, Holland) were preincubated with fresh control serum for opsonization to perform phagocytosis assay. Neutrophils (1 x 106cells/mL, from control and diabetic rats) were taken onto clean glass slides and incubated for 5 minutes at 37OC. A few drops of prepared yeasts at 1 x 108/mL were then added to the neutrophils and incubated for a further 5 minutes at 37OC. The scoring was done according to our previous protocol (Islam and Nabi, 2003; Nabi et al, 2005). The percentage of phagocytic cells and the number of yeast cells attached per 100 randomly chosen neutrophils were counted by examining at least 300 neutrophils from each preparation (controls and diabetics) under the oil immersion lens.
Statistical analyses
The results were expressed as mean  SD. To compare the differences between neutrophils from the control and diabetic rats, independent Student's t-test was performed. Correlation was determined by using non-parametric Spearman's rho test. A p value of less than 0.05 was considered significant. Multi dimensional scaling or Euclidean distance model was performed using SPSS package. 

RESULTS
Biochemical analyses
The development of diabetes was confirmed by the presence of hyperglycemia (blood glucose level > 230 mg/dL) as described previously (Nabi et al, 2005). Plasma glucose levels were determined by the glucose oxidase method using blood samples obtained from the animal tail. The rats were used for the experiments 1 week after receiving STZ injection. The average levels of glucose in the plasma of diabetic and control rats were 302.6 ± 87.5 and 100.7 ± 11.5 mg/dL, respectively. Other biochemical indices examined from the plasma of both the groups of rats have been presented in Table 1 and shown in Figure 1. In diabetic and control rats, the average levels of plasma triglycerides were 174.9 ± 18.6 vs 82.2 ± 10.2 mg/dL; total cholesterol were 250.8 ± 22.3 vs 165.2 ± 24.1 mg/dL; plasma creatinine were 0.94 ± 0.19 vs 0.81 ± 0.05 mg/dL; plasma urea were 77.1 ± 9.7 vs 26.8 v 5.8 mg/dL, respectively. The mean values of aspartate transaminase and alanine transaminase in diabetic and control rats were 141.4 ± 28.0 vs 61.6 ± 18.6 IU/L and 61.4 ± 13.6 vs 48.5 ± 6.0 IU/L, respectively.
 
Table 1. Levels of plasma glucose, creatinine, urea, alanine transaminase (ALT), aspartate transaminase (AST) in control and STZ-induced diabetic Long Evan rats.

Long Evan rats	Glucose (mg/dL)	Creatinine (mg/dL)	Urea (mg/dL)	ALT (IU/L)	AST (IU/L)
Control	100.7 ± 11.5	0.81 ± 0.05	26.8 v 5.8	48.5 ± 6.0	61.6 ± 18.6
Diabetic	302.6 ± 87.5	0.94 ± 0.19	77.1 ± 9.7	61.4 ± 13.6	141.4 ± 28.0
p values	< 0.001	< 0.001	< 0.001	< 0.001	< 0.001

Statistical analysis using student’s t test showed an increase levels of all the biochemical parameters that measured in diabetic rats varied significantly (p < 0.001) compared to those of control rats (Table 1 and Figure 1).

Association of biochemical indices with polarization of neutrophils and NBT dye reduction 
Our previous reports revealed that at the base line level, neutrophils from diabetic rats were significantly more polarized (p < 0.001) compared to those from control rats (30 ± 4 vs 13 ± 3) (Nabi et al, 2005). We also showed that neutrophils from diabetic rats could reduce significantly more NBT dye (0.12 ± 0.03 vs 0.04 ± 0.01, respectively; p < 0.001) than those from control rats at the baseline level (Nabi et al, 2005). These functions exhibited by diabetic and control neutrophils did show any relationship with the biochemical indices examined (Table 2 and 3). However, Euclidean distance model represented in Figure 2 indicated that the ability to reduce nitroblue terazolium dye by the neutrophils (e) of the study rats was closely related to the percentages of the polarized neutrophils (f). 

Association of biochemical indices with the phagocytic activity of neutrophils
According to our previous study, it was perceived that neutrophils from the diabetic rats were statistically less phagocytic than those from the control rats (61 ± 7% vs 87 ± 4%, respectively; p < 0.001) by phagocytosing significantly (p < 0.001) less number of opsonized yeast particles (282 ± 16) compared to those of the control cells (381 ± 17) (Nabi et al, 2005). Though our previous findings demonstrated that percentages of phagocytic neutrophils as well as efficiency of the neutrophils i.e., number of yeast particles phagocytosed by the cells were inversely related to the levels of plasma glucose in diabetic rats (Nabi et al, 2005), we did not find any relationship between the immune function mediated by the neutrophils and plasma cholesterol/triglycerides using bivariate Spearman’s rho analysis in case of neither diabetic nor control rats (Table 2 and 3). Interestingly, multidimensional scaling plot demonstrated a close relation between the levels of plasma total cholesterol and the percentages of phagocytic neutrophils as shown in Figure 2.

DISCUSSION
The levels of biochemical indices such as important metabolites of lipid profile panel e.g., plasma cholesterol, triglycerides; of kidney function e.g., creatinine and urea; of liver function activities of serum enzymes such as aspartate transaminase and alanine transaminase were studied in streptozotocine-induced type 1 diabetic rats and thus, association of these indices with the possible alteration of the functions of neutrophils were investigated in this study. Plasma ALT and AST levels were measured to evaluate the hepatic functions. This study reveals statistically significant (p < 0.001) increased levels of AST and ALT in the plasma of diabetic rats compared to that of control rats (Table 1). The increase in aminotransferases levels may be due to the cellular damage in the liver caused by STZ-induced diabetes. Thus, our result supports the data of other researchers who found elevated levels of ALT and AST in diabetic rats (Zafar et al, 2009; Baxter and Schofield, 1980). Voss et al. (1988) also reported similar finding by proposing time dependent rise in AST, ALT, and alkaline phosphates (ALP) levels in STZ-induced diabetic rats. Moreover, we also found that levels of creatinine and urea were significantly elevated in the plasma of diabetic rats (Table 1) which matched with the data reported by Voss et al. (1980). Thus, this data indicated that normal metabolic processes are altered in STZ-induced diabetic rats by affecting both liver and kidneys.
 

Table 2. Correlation of the plasma glucose, triglycerides, total cholesterol, creatinine and urea with functional indices of neutrophils such as percent polarized, percentages of phagocytic neutrophils, total number of opsonized yeasts phagocytosed and ability to reduce NBT dye by neutrophils from diabetic rats.

	Glucose (mg/dL)	Creatinine (mg/dL)	Urea (mg/dL)	%Phagocytic cells	Total number of yeast phagocytosed	NBT reduction	%polarized cells	Plasma triglyceride (mg/dL)	Plasma cholesterol (mg/dL)
Glucose (mg/dL)	1.000
Creatinine (mg/dL)	0.133	1.000
Urea (mg/dL)	0.170	-0.081	1.000
%Phagocytic cells	-0.497*a	0.013	-0.406	1.000
Total number of opsonized yeast phagocytosed	-0.500*a	0.264	-0.230	0.245	1.000
NBT reduction	0.044	-0.132	0.111	0.132	-0.179	1.000
%polarized cells	-0.193	0.007	-0.034	0.343	0.113	-0.230	1.000
Plasma triglyceride (mg/dL)	-0.175	-0.012	0.449*	0.163	-0.026	0.003	0.172	1.000
Plasma cholesterol (mg/dL)	0.185	-0.429	-0.213	0.067	-0.193	0.225	0.030	-0.017	1.000
*Correlation is significant at the 0.05 level (2-tailed). aNabi et al, 2005.

Table 3. Correlation of the plasma glucose, triglycerides, total cholesterol, creatinine and urea with functional indices of neutrophils such as percent polarized, percentages of phagocytic neutrophils, total number of opsonized yeasts phagocytosed and ability to reduce NBT dye by neutrophils from control rats.

	Glucose (mg/dL)	Plasma triglyceride (mg/dL)	Plasma cholesterol (mg/dL)	Creatinine (mg/dL)	Urea (mg/dL)	%Phagocytic cells	Total number of yeast phagocytosed	NBT reduction	%polarized cells
Glucose (mg/dL)	1.000
Plasma triglyceride (mg/dL)	0.078	1.000
Plasma cholesterol (mg/dL)	0.267	0.237	1.000
Creatinine (mg/dL)	-0.237	-0.585	0.061	1.000
Urea (mg/dL)	-0.284	0.030	0.607	0.469	1.000
%Phagocytic cells	-0.103	-0.201	-0.568	0.116	-0.390	1.000
Total number of opsonized  yeast phagocytosed	0.187	-0.369	-0.158	0.048	-0.296	0.024	1.000
NBT reduction	0.466	-0.090	0.413	0.012	0.163	0.310	-0.248	1.000
%polarized cells	0.613	0.328	0.103	-0.733*	-0.492	-0.397	0.200	0.042	1.000
*Correlation is significant at the 0.05 level (2-tailed).

In vitro animal and clinical research indicated extensive relationship between serum lipids and the immune system (Sullivan, 1994; Kelley and Bendich, 1996). From the basic metabolic standpoint of view, we know that lipoprotein particles deliver cholesterol, various phospholipids and fat soluble antioxidants to cells which are essential for maintaining the integrity of cell membrane and optimal immune function thorugh the production of eicosanoid and anti-oxidants (Heiniger et al, 1978; Pace and Eshfahani, 1987; Bendich, 1990). Antigen presenting activity of monocytes and chemotactic activity of some cell lines can be increased by cholesterol (Hughes et al, 1992; Kreuzer et al, 1991). Another study reported that diet-induced hypercholesterolemia in animals reduce macrophage response and phagocytic function (Kos et al, 1979).

Cholesterol is present in human plasma at concentrations of milligrams per milliliter.  The vast majority is complexed in lipoproteins. However, although clinical free cholesterol is albumin-bound and albumin can delay cholesterol’s effects on signaling in vitro, cells are continuously exposed to cholesterol in vivo in equilibrium where cholesterol desorbs from albumin to cell membranes. Moreover, albumin concentrations vary markedly from plasma to the interstitium in health, and may vary further as a function of disease states. It is reported that high serum or tissue-free cholesterol availability could activate immune cells that contribute to the progression of atherosclerotic lesions (Kolenkode et al, 2007). With respect to lipid effects on neutrophil phagocytosis, the phagocytic capacity in most human and animal studies appears to be decreased in vitro by lower chain triglycerides or LCT (Wiernik et al, 1983; Usmani et al, 1988; Rasmussen et al, 1988). Also, depressed phagocytosis, oxygen radical production and Fc-receptor expression have been observed after exposure of neutrophils to LCT (Cleary and Pickering, 1983).

The earliest morphological response of leucocytes to a chemoattractant is a change from a spherical to a polarized shape with formation of an extended, ruffled, anterior veil or lamellipodium. This polarization is accompanied by functional polarization, since the lamellipodium remains organelle-free, is rich in filamentous actin, and several cell surface proteins have been reported to become concentrated at the front of the cell. In various leucocytes these proteins include Fc receptors (Walter et al., 1980; Wilkinson et al., 1980), Thy-1 (Shields and Haston, 1985), CD15 and CD45 (Haston and Maggs, 1990), and urokinase-type plasminogen activator receptor (Estreicher et al. 1990). In the present study, it has been demonstrated through multi-dimensional scaling that phagocytic activity of the neutrophils are closely related to that of the levels of plasma cholesterol (Figure 2). Thus, it is possible that the equilibrium between the albumin-bound cholesterol and membrane cholesterol has been disturbed which might have altered baseline morphology of neutrophils followed by change in the normal distribution of receptors responsible for phagocytosis as reported by Cleary and Pickering (1983). This has also been reflected by the significantly lower percentages of phagocytosis mediated by diabetic neutrophils. Previous findings revealed enhanced phagocytic activity by the activated neutrophils (Kozel et al, 1987). Thus, it was expected that activated diabetic neutrophils at the baseline level should demonstrate higher phagocytic activity. However, decreased phagocytic activity of the diabetic neutrophils as well as their decreased efficiency and its close association with plasma cholesterol might be attributed to the possible i) shedding of the receptors and/or ii) sequestration of the receptors and/or iii) alteration of membrane-bound receptor mediated cell signaling required to maintain optimal phagocytic activity. Thus, endogenous plasma cholesterol elevated due to type 1 diabetes in STZ-induced diabetic rats may alter the functions of neutrophils in vitro. The short coming of the current study is that we could not evaluate the effects of exogenous cholesterol on the neutrophil functions. However, future research work should be performed to observe the dose-dependent and time-dependent effects of cholesterol on neutrophil functions, status of different functional receptors on the plasma membrane as well as on the cell signaling mechanism.

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