Annals of Vascular Surgery
Volume 23, Issue 2 , Pages 172-178, March 2009

Inflammatory Profiling of Peripheral Arterial Disease

  • Ramakrishna P.C. Chaparala

      Affiliations

    • Leeds Vascular Institute, Leeds General Infirmary, Leeds, UK
    • Corresponding Author InformationCorrespondence to: Ramakrishna P. C. Chaparala, Leeds Vascular Institute, Leeds General Infirmary, Great George Street, Leeds LS1 3EX, UK
    • ,
  • Nicolas M. Orsi

      Affiliations

    • Perinatal Research Group, YCR & Liz Dawn Pathology & Translational Sciences Centre, Leeds Institute of Molecular Medicine, St. James's University Hospital, Leeds, UK
    • ,
  • Nigel J. Lindsey

      Affiliations

    • Department of Biomedical Sciences, University of Bradford, Bradford, West Yorkshire, UK
    • ,
  • Raman S. Girn

      Affiliations

    • Leeds Vascular Institute, Leeds General Infirmary, Leeds, UK
    • ,
  • Shervanthi Homer-Vanniasinkam

      Affiliations

    • Leeds Vascular Institute, Leeds General Infirmary, Leeds, UK

published online 28 July 2008.

Article Outline

The progression of peripheral arterial disease (PAD) is poorly understood but may be caused by an underlying inflammatory dysfunction. This study therefore profiled interleukin (IL)-1β, IL-2, IL-4, IL-6, IL-8, IL-10, IL-13, anticardiolipin, and anti-β2-glycoprotein 1 antibody concentrations and characterized patients' inflammatory response in vitro. Patients were classified according to World Health Organization criteria and ankle-brachial pressure index into critical ischemics (n=20), stable claudicants (n=20), and controls (n=20). In vitro studies involved culturing whole blood with RPMI-1640 for 24hr with and without 1μg/mL lipopolysaccharide and profiling cytokine production. Autoantibody levels were measured using enzyme-linked immunosorbent assays, while cytokine profiles were determined by multiplex immunoassay. Serum IL-6, IL-10, IL-13, and anti-β2-glycoprotein 1 antibody levels were higher in PAD (p<0.05). In the case of IL-6 and anti-β2-glycoprotein 1 antibody, levels reflected increasing disease severity (p<0.05). In vitro studies revealed that IL-8 and IL-13 secretory capacities were significantly higher in PAD after 6hr. However, when these were standardized against patient leukocyte count, cytokine production profiles did not differ. PAD features an increased inflammatory burden irrespective of Th1:Th2 cytokine type; this is more pronounced with increasing disease severity. However, the inflammatory hyperresponsiveness of cultured whole blood from PAD patients probably relates to associated leukocytosis, rather than being attributable to an inherent inflammatory dysfunction.

 

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Introduction 

Peripheral arterial disease (PAD) affects 10% of men >65 years old, increasing to 20% of men and women ≥75 years old.1 Flow dynamics and stimulants involved in progression of PAD could differ from those of coronary and cerebral circulations.2 Factors like age, gender, smoking, diabetes, hypertension, low-density lipoprotein, and low ankle-brachial pressure index (ABPI) have been independently associated with progression.3, 4, 5, 6

However, these fail to account for the benign progression of the disease in the majority of patients and its aggressive nature in a few.7 Recently, progression of PAD has been associated with increased plasma fibrinogen, C-reactive protein, homocysteine, and lipoprotein (a).8, 9 With inflammation being recognized as a mediator of atherosclerosis, an array of T-helper (Th)1 and Th2 cytokines have been highlighted as predictive markers of disease progression.10 Despite these advances, there have been no studies to date investigating the role of multiple cytokines related to the severity of PAD.

There is evidence supporting a link between increased IL-6 levels and levels of endothelial cell reactive antibodies.11 These target antigens in endothelial cell membranes and have implications in accelerated atherosclerosis in autoimmune disorders.12, 13 Anticardiolipin antibodies and their major subpopulation, anti-β2-glycoprotein 1 antibodies, are two such autoantibodies which have been linked to PAD severity.11 Yet, little is known about the link between their profiles and those of Th1 and Th2 cytokines in PAD.

Consequently, this study had three principal aims: (1) to determine the serum levels of IL-1β, IL-2, IL-4, IL-6, IL-8, IL-10, and IL-13; (2) to profile anticardiolipin and anti-β2-glycoprotein 1 antibody levels; and (3) to characterize the inflammatory response (in terms of IL-6, IL-8, IL-10, and IL-13 secretory capacity) in PAD and control patients using a whole blood stimulation model in vitro.

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Materials and Methods 

Study Patients 

Patients were recruited following ethics approval and written consent. The PAD subgroups were (1) stable claudicants (SC, n=20) with a history of intermittent claudication as defined by the World Health Organization/Rose questionnaire and an ABPI<0.9 or, in the case of diabetics, radiologically proven occlusion or stenosis of the iliac, femoral, or popliteal arteries (angiography or duplex ultrasound) and (2) critical limb ischemia (CI, n=20). CI was defined as chronic ischemic rest pain, ulcers, or gangrene attributable to objectively proven arterial occlusive disease (i.e., an ankle systolic pressure of <50mm Hg for rest pain, 60mm Hg for nonhealing ulcers, and/or radiologically proven occlusion or stenosis of the iliac, femoral, or popliteal arteries). Controls (n=20) were recruited synchronously on an age and gender-matched basis after ensuring that they had no symptoms of PAD and exhibited an ABPI of 0.9-1.2 or, for diabetics, a toe pressure of >70mm Hg. Those who suffered an acute myocardial infarction or cerebrovascular accident in the previous 3 months, involvement in other studies during this period, or confirmed diagnosis of vasculitis, systemic autoimmune condition, or malignancy were excluded. Patient demographics are outlined in Table I.

Table I. Study group demographics and characteristics
GroupControlSC (Fontaine II)CI (Fontaine III–IV)
Patients (n)202020
Mean age (years)6769.672.6
ABPI1.10.690.33
Hypertension (n)0910
Smoking (n)01114
Diabetes (n)011
Hypercholesterolemia (n)0911
Heart disease (n)099
Aspirin usage (n)01412
Statin usage (n)0910
Warfarin usage (n)033
Other antiplatelet agent usage (n)011
Antihypertensive agent usage (n)087
β-blocker usage (n)058
Mean white cell count (x106 white cells/mL)6.47.408.50

Whole Blood and Plasma Collection 

Following a 12hr overnight fast, patients avoided physical exercise in the morning and rested for 30min prior to whole blood collection from the antecubital fossa with a 16G needle into heparinized tubes. The majority of the sample was retained for lipopolysaccharide (LPS) stimulation, while 1mL was centrifuged at 5,000rpm for 10min in order to isolate plasma. This was stored at –80°C until assayed to determine circulatory cytokine profiles.

LPS Stimulation of Whole Blood In Vitro 

LPS stimulation is a standard assay for monitoring inflammatory response in an array of cell types. Heparinized whole blood (4mL) was diluted 1:1 with RPMI-1640 medium (with l-glutamine, without phenol red; Invitrogen, Paisley, UK) supplemented with 2.5 U/mL penicillin, 2.5μL/mL streptomycin, and 6.25 ng/mL amphotericin B (Invitrogen), with (LPS challenge) or without (baseline) 1μg/mL LPS (from Salmonella enterica serotype abortus equi) (Sigma-Aldrich, Poole, Dorset, UK) in vented culture dishes (SLS, Nottingham, UK). The dishes were incubated on a platform rocker (SLS) to ensure mixing at 37°C for 24hr under a humidified 5% CO2 atmosphere. Serial 350 μL samples were collected at 0, 3, 6, and 24hr of culture and centrifuged at 5,000rpm for 3min. The supernatant was frozen at –80°C until analyzed for cytokine profiles.

Cytokine Profiling 

Cytokine concentrations were determined by fluid-phase multiplex immunoassay using custom kits (Upstate, Milton Keynes, UK), run on a Luminex (Austin, TX) 100 cytometer, equipped with StarStation software (version 2.0; Applied Cytometry Systems, Dinnington, UK). Plasma samples were analyzed for interleukin (IL)-1β, IL-2, IL-4, IL-6, IL-8, IL-10, and IL-13, while cultured whole blood was analyzed for IL-6, IL-8, IL-10, and IL-13 levels.

Determination of Anticardiolipin and Anti-β2-Glycoprotein 1 Antibodies 

Antibodies to anticardiolipin and anti-β2-glycoprotein 1 were measured using a commercially available enzyme-linked immunosorbent assay (ELISA; Bindazyme, Birmingham, UK).

Data Presentation and Statistical Analysis 

Cytokine concentrations were expressed in picograms per milliliter, while autoantibody levels were given in units per milliliter. Cytokine production in vitro was expressed as secretory capacity (=LPS stimulated profile – baseline production levels [pg/mL]). Values were also standardized according to white cell count (picograms/million white cells) because there was a very significant degree of leukocytosis associated with CI subgroup patients compared to controls (p<0.001). All data were expressed as mean ± standard error of the mean. Significance between groups was assessed by Kruskall-Wallis tests and Mann-Whitney U-tests post hoc, having determined that data were not normally distributed by Anderson-Darling tests (Minitab, State College, PA; version 14.1).

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Results 

Circulatory Cytokine Profiles 

Markedly higher (66-fold) levels of IL-6 were found in the CI compared to the SC group (p<0.001), which in turn were moderately higher than in controls (p<0.05), where IL-6 concentrations were largely undetected (Table II). Moreover, while the circulatory concentrations of IL-10 and IL-13 were higher (approximately 1.1- and 2-fold, respectively) in both PAD groups when compared to controls (p<0.05), there was no evident difference in levels between the CI and SC populations. By contrast, there were no significant differences in IL-1β, IL-2, and IL-8 circulatory profiles between any of the study populations.

Table II. Serum cytokine profiles
Cytokine (pg/mL)ControlSCCI
IL-1β0.93±0.061.09±0.150.86±0.07
IL-21.94±0.832.34±0.921.65±0.54
IL-40.107±0.100.347±0.220.173±0.10
IL-60.00±0.00a0.43±0.43b28.54±9.81c
IL-80.42±0.265.00±2.983.84±1.76
IL-101.94±0.05a2.16±0.07b2.18±0.06b
IL-130.75±0.07a1.35±0.16b1.25±0.23b

Different superscripts (a and b) indicate statistically significant differences between groups, ∗p<0.05.

Circulatory Autoantibody Levels 

There was an increase in anti-β2-glycoprotein 1 antibodies in association with increasing severity of PAD (Fig. 1). In particular, levels in CI patients were significantly higher than those of controls (p<0.05). SC anti-β2-glycoprotein 1 antibody levels were intermediate between CI and control group levels and significantly different from both of these (p<0.05). Anticardiolipin antibody levels, however, were unaffected by PAD for both SC and CI populations (Fig. 2).

  • View full-size image.
  • Fig. 1 

    Anti-β2-glycoprotein 1 antibody levels in the different study populations (different superscripts indicate statistically significant differences between groups, p<0.05).

Cytokine Profiles following LPS Stimulation In Vitro 

Following LPS stimulation in vitro, IL-8 secretory capacity was significantly higher in CI patients as well as in the PAD population as a whole (i.e., SC+CI) after 6hr of incubation (p<0.05) (Table III). Following LPS stimulation in vitro, there was a trend approaching significance for IL-13 production to be higher in all PAD patients (i.e., SC+CI) after 6hr of incubation (p=0.06). However, only the CI population displayed a significantly greater IL-13 secretory capacity at this time point (p<0.05) (Table IV). By contrast, there was no significant increase in IL-6 or IL-10 secretory capacities allied to either of the PAD subgroups (Table V, Table VI). However, when all secretory capacities were standardized for white cell count, there was no significant difference in any cytokine production profile across all study groups.

Table III. IL-8 secretory capacities in different study groups
Time (hr)ControlSCCIPAD (SC+CI)
Standardized secretory capacity (pg/million white cells)
00.0±0.00.0±0.00.0±0.00.0±0.0
315.9±2.617.3±3.116.4±2.116.8±1.8
620.5±2.432.4±6.726.1±2.729.1±3.5
2458.3±11.771.7±19.050.0±6.460.2±9.6
Uncorrected secretory capacity (pg/mL)
00.0±0.00.04±0.00.0±0.00.0±0.0
399.8±15.0119.2±20.1143.1±20.0131.8±14.1
6130.0±15.8218.5±38.9216.1±21.8∗217.2±21.3∗
24371.9±75.3472.6±107.1422.9±55.2446.3±57.5

∗Significantly different from control, p<0.05.

Table IV. IL-13 secretory capacities in the different study groups
Time (hr)ControlSCCIPAD (SC+CI)
Standardized secretory capacity (pg/million white cells)
00.0±0.00.0±0.00.5±0.30.3±0.1
30.0±0.00.2±0.10.4±0.20.3±0.1
60.4±0.40.4±0.20.7±0.30.6±0.2
240.2±0.00.3±0.10.1±0.00.2±0.0
Uncorrected secretory capacity (pg/mL)
00.1±0.10.1±0.03.8±2.12.0±1.1
30.1±0.11.4±1.32.2±1.31.9±0.9
62.4±2.32.3±1.25.8±2.4∗4.1±1.4
241.1±0.32.5±1.31.1±0.31.8±0.6

∗Significantly different from control, p<0.05.

Table V. IL-6 secretory capacities in the different study groups
Time (hr)ControlSCCIPAD (SC+CI)
Standardized secretory capacity (pg/million white cells)
00.0±0.00.0±0.00.0±0.00.0±0.0
3386.4±77.3389.4±50.8334.9±31.0360.6±29.0
6496.8±84.0648.7±73.7499.5±46.6569.9±43.8
24594.5±93.9742.7±98.2564.9±46.2648.9±53.7
Uncorrected secretory capacity (pg/mL)
00.0±0.00.0±0.00.2±0.10.1±0.1
32,457.5±486.32,593.5±280.72,694.4±188.82,646.7±163.6
63,178.5±536.54,304.2±338.53,916.7±198.94,099.7±191.2
243,880.9±613.34,902.3±485.54,480.3±201.24,679.6±251.3
Table VI. IL-10 secretory capacities in the different study groups
Time (hr)ControlSCCLIPAD (SC+CLI)
Standardized secretory capacity (pg/million white cells)
00.0±0.00.0±0.00.0±0.00.0±0.0
30.5±0.40.07±0.00.2±0.10.1±0.0
64.0±1.03.3±0.73.5±0.83.4±0.5
2425.5±6.134.2±5.231.7±4.632.9±3.4
Uncorrected secretory capacity (pg/mL)
00.0±0.00.0±0.00.0±0.00.0±0.0
33.1±2.30.5±0.12.0±0.81.3±0.4
625.0±6.023.5±4.732.4±7.828.2±4.7
24164.7±41.1230.1±30.6270.9±40.0251.6±25.5

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Discussion 

The progressive increase in circulatory IL-6 concentration with increasing PAD severity is in agreement with previous studies.11, 14 IL-6 induces acute-phase proteins such as C-reactive protein (CRP), (thereby accounting for its higher circulatory levels and that of other interleukins), activates platelets and increases procoagulant activity.15, 16 While baseline IL-6 levels were higher in association with CI, this did not translate into greater secretory capacity following LPS stimulation. This may be due to the fact that increased circulatory levels reflect high local levels at the atherosclerotic site.17 Alternatively, the raised plasma levels of IL-6 may come from a different cell source, such as damaged endothelial cells consequent to stimulation by antiendothelial antibodies.18 This increase in IL-6 levels is lost in the in vitro system, partly through further dilution of blood samples with RPMI and partly through standardization of secretory capacity against leukocyte profile. The subsequent failure of LPS to stimulate overproduction of IL-6 argues against the notion of an aberrant systemic inflammatory response underlying individual susceptibility to PAD. However, given that our model focuses only on whole blood, it is possible that any putative underlying inflammatory dysfunction may be due to increased monocyte cytokine production and/or endothelial cell activation.19

In SC, repeated inflammatory episodes occurring in association with walking may contribute to a chronic low-grade inflammation resulting in endothelial dysfunction. This is supported by studies showing neutrophil activation, increases in adhesion molecules, and release of IL-8 following exercise.20, 21, 22 The increase in inflammatory markers such as IL-6 may also reflect a concomitant low-grade activation of the immune system.23 In this scenario, a more pronounced inflammatory profile may cause a greater atherosclerotic burden. Circulatory IL-8 levels were not associated with severity of PAD, in contrast to previous reports,22 although these discrepancies may be accounted for by the fact that the cited studies focused on exercising patients, which may result in neutrophil activation and increased cytokine production following ischemia–reperfusion. In addition, half of the patients in the present study (Table I) were on statin therapy, which is increasingly being found to modulate the levels of inflammatory markers such as cytokines.24

Following LPS challenge, IL-8 secretory capacity increased after 6hr incubation, in particular in patients with CI and in the PAD population overall. If this early increase in IL-8 production also occurs following inflammatory stimuli in vivo, it may contribute to neutrophil chemotaxis to sites of inflammation, such as atherosclerotic plaques, where it may also play a role in triggering firm adhesion of rolling monocytes to the vascular endothelium and their infiltration of the vessel wall as part of the early stages in atherosclerotic progression.25

The present study showed higher circulatory IL-10 levels in both PAD populations compared to controls, in contrast to DePalma and coworkers.26 Rather than supporting a Th1:Th2 imbalance as an underlying mechanism for PAD, these findings support a regulatory role for IL-10, which compensates for the higher levels of Th1-type cytokines such as IL-6. This is also the first report of the measurement of IL-13 in PAD. It was present at low concentrations, and production was largely unresponsive to LPS challenge in vitro. Although there was a significant increase in circulatory levels of IL-13 allied to PAD, it was quantitatively minor. This could simply reflect a low-grade Th2-type modulatory mechanism geared to counteract the higher IL-6 levels associated with PAD, as described for IL-10. Alternatively, it may reflect the paradoxically proinflammatory properties of IL-13, as demonstrated by its potentiation of IL-8 receptor expression, low-density lipoprotein (LDL) oxidation by monocytes, and enhanced leukocyte transmigration.27, 28, 29

The low IL-1β and IL-2 circulatory levels in all study groups suggest that these cytokines may play a limited role in the pathophysiology of PAD. While these low levels are consistent with previous findings,26, 30 it is also possible that peripheral concentrations are poorly representative of their local involvement with atherosclerotic plaques. Circulatory antiatherogenic IL-4 levels were also very low in all groups, perhaps because its expression in atherosclerosis is limited.31

There are several potential inflammatory triggers in PAD. Patients with PAD who have higher atherosclerotic burdens are likely to have higher levels of antibodies against oxidized LDL.32 These antibodies against oxidized LDL may cross-react with endothelial cells along with antibodies against cardiolipin, β2-glycoprotein 1, and heat shock proteins.33 While high levels of anticardiolipin antibodies have been shown to be an independent risk factor for myocardial infarction and cardiac death in middle-aged men,34 no raised levels of these were noted in this study of patients with PAD. However, these negative findings are supported by other reports.35, 36 In the present study, anti-β2-glycoprotein 1 antibody levels were elevated in patients with CI and SC compared to controls. These data are consistent with studies showing elevated levels of anti-β2-glycoprotein antibodies, with a number of animal and human studies revealing the correlation between increased anti-β2-glycoprotein 1 antibody titers and accelerated atherosclerosis.11, 13, 37

Another potential source of inflammation in PAD may relate to exercise. In claudicants, repeated inflammatory episodes allied to walking may result in low-grade inflammation and endothelial dysfunction. This is supported by studies indicating neutrophil activation as well as increased adhesion molecule and IL-8 production following exercise.21, 22, 38 Finally, inflammation may also reflect low-grade activation of the immune system.23 In this scenario, the more pronounced inflammatory profile of claudicants may be both the cause as well as the consequence of a greater atherosclerotic burden.

In conclusion, this study does not support either Th1 or Th2 cytokines as being predominant in PAD (i.e., representing a Th1:Th2 imbalance), although inflammatory burden is more pronounced in severe manifestations of the disease. A minor degree of inflammatory hyperresponsiveness associated with cultured whole blood from SC and CI patients appeared to relate to leukocytosis rather than being attributable to an inherent inflammatory dysfunction per se. The raised levels of endothelial cell reactive antibodies in PAD coupled with their actions to increase IL-6 could contribute to the increased inflammatory burden and may be responsible for the accelerated atherosclerosis seen in CI patients.

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PII: S0890-5096(08)00214-8

doi:10.1016/j.avsg.2008.06.005

Annals of Vascular Surgery
Volume 23, Issue 2 , Pages 172-178, March 2009

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