Proinflammatory activation of macrophages by basic calcium phosphate crystals via protein kinase C and MAP kinase pathways: a vicious cycle of inflammation and arterial calcification?

Basic calcium phosphate (BCP) crystal deposition underlies the development of arterial calcification. Inflammatory macrophages colocalize with BCP deposits in developing atherosclerotic lesions and in vitro can promote calcification through the release of TNF alpha. Here we have investigated whether BCP crystals can elicit a proinflammatory response from monocyte-macrophages. BCP microcrystals were internalized into vacuoles of human monocyte-derived macrophages in vitro. This was associated with secretion of proinflammatory cytokines (TNFalpha, IL-1beta and IL-8) capable of activating cultured endothelial cells and promoting capture of flowing leukocytes under shear flow. Critical roles for PKC, ERK1/2, JNK, but not p38 intracellular signaling pathways were identified in the secretion of TNF alpha, with activation of ERK1/2 but not JNK being dependent on upstream activation of PKC. Using confocal microscopy and adenoviral transfection approaches, we determined a specific role for the PKC-alpha isozyme. The response of macrophages to BCP crystals suggests that pathological calcification is not merely a passive consequence of chronic inflammatory disease but may lead to a positive feed-back loop of calcification and inflammation driving disease progression.

Platelet-monocyte pro-coagulant interactions in on-pump coronary surgery.

OBJECTIVE: Platelets and monocytes possess haemostatic properties, but the clinical effect of platelet-monocyte interactions on haemostasis following coronary surgery is not known. The study characterises the platelet and monocyte responses in cardiac surgery and its impact on haemostasis. METHODS: In 1342 patients, changes in white blood cell counts (WBC), monocyte counts and platelet counts were measured. PMC formation was analysed by flow-cytometry using monoclonal antibodies against pan-leucocyte marker CD45, monocyte marker CD14 and platelet marker CD42. TF expression was determined using monoclonal antibodies against, CD45, CD14 and human-TF. Continuous variables were expressed as mean+/-SD. Changes in monocyte and platelet counts over time were considered as repeated measures data, and analysed using Generalised Estimating Equations (GEE). Multivariate regression analysis was used to evaluate the effect of several factors on blood loss. RESULTS: A monocytosis occurs with on-pump coronary surgery, but is less pronounced than with off-pump surgery. No difference was seen in patients having redo-surgery or more complex cardiac surgery. Factors associated with monocytosis on multivariate analysis were higher body mass index (p=0.02), diabetes (p=0.035) and smoking (p=0.01). Older patients manifested a lower response (p<0.001). Cross-clamp fibrillation was associated with a lower (p=0.048) monocytic response than was cardioplegia. PMC formation dropped following administration of heparin, peaked at 5 min of CPB, and declined by 2h of CPB (p=0.04). A return towards preoperative levels was found during postoperative days 1-5. No significant change in monocyte TF expression occurred. The mean postoperative blood loss was 581.2+/-292.8 ml, and inversely related to increasing preoperative platelet counts (p<0.001), and to higher monocyte % counts (p=0.012). Patients, who were female (p<0.001), had higher body mass indices (p<0.001), and higher core body temperatures during surgery (p=0.013), as well as patients having perioperative aprotinin (p<0.001) related to less blood loss. CONCLUSIONS: A higher postoperative platelet count as well as monocyte% significantly and independently decreases postoperative blood loss following cardiac surgery.

Monocyte Fc gamma receptor expression in patients undergoing coronary artery bypass grafting.

BACKGROUND: Cardiopulmonary bypass is associated with an inflammatory response with potential deleterious effects. The white cell subpopulation mostly investigated so far is the neutrophil. To date very little has been investigated regarding the role of the monocyte/macrophage. This study focuses on the expression of Fc gamma receptors I, II, and III by monocytes in patients undergoing cardiopulmonary bypass. METHODS: We studied the surface expression of Fc gamma receptors I, II, and III by flow cytometry on gated monocyte subpopulations in the whole blood of adult patients undergoing elective coronary artery bypass grafting. Blood samples were drawn preoperatively and at 15 minutes, 1, 2, 4, 24, 48, and 72 hours, and 6 days postoperatively. A second group of patients undergoing lung resection surgery were studied in a similar fashion. RESULTS: Neither Fc receptor I nor receptor II expression were significantly changed throughout the time points studied. Fc receptor III expression was reduced at 2 and 4 hours (p = 0.016 and 0.002) and increased at 24, 48, and 72 hours after commencement of CPB on a selected subpopulation (15%-35%) of monocytes (p = 0.004, < 0.001, and < 0.001, respectively). This expression returned to preoperative levels by the sixth postoperative day. There were no statistically significant changes in the lung resection group. CONCLUSIONS: Our study demonstrated that cardiopulmonary bypass is associated with a biphasic Fc gamma receptor III expression on a subpopulation of peripheral blood monocytes up to 3 days postoperatively.

Are wound healing disturbances and length of hospital stay reduced with minimally invasive vein harvest? A meta-analysis.

Minimally invasive great saphenous vein harvest for coronary artery bypass grafting aims to reduce post-operative leg-wound related morbidity. In a meta-analysis of randomised trials we have shown leg wound infection rates to be significantly lower in patients undergoing minimally invasive harvest. This study aims to use meta-analysis to compare the two techniques with regards to non-infective wound healing disturbances (NIWHD) (wound drainage, haematoma, dehiscence, necrosis, need for surgical debridement, and seroma formation). A meta-analysis of all studies published between 1995 and 2002 reporting a comparison between the two techniques was performed. Primary outcomes of interest were the six wound healing disturbances mentioned above and length of hospital stay. Heterogeneity was assessed using graphical exploration and sensitivity analysis with subgroup analysis. Twenty-seven studies published between 1997 and 2002 matched our selection criteria, with a combined total of 4953 subjects, of which 2442(49%) underwent minimally invasive harvest and 2511(51%) underwent conventional surgery. When considering only randomised studies, the total number of non-infective wound disturbances was lower in minimally invasive (4%) as compared to the conventional (13%) group (random effect OR 0.24, CI 0.16-0.38). Similar results were found when only fully matched studies were considered. The absolute risk reduction when comparing the two techniques was calculated to be 0.10, which translates to a number of patients needed to treat of 10. Length of stay was significantly reduced in the minimally invasive group in comparison to the conventional group (random effect weighted mean difference of -1.04, CI -1.92 to -0.16). Our results suggest that NIWHD all reduced with minimally invasive harvest techniques. Despite the limitations of this meta-analysis, we feel we have once again illustrated an important link between minimally invasive great saphenous vein harvest and improved tissue healing when compared to conventional open surgery. This has the potential to reduce wound-related morbidity, infection, post-operative pain, length of hospital stay, and re-admission rate.

Haptoglobin Genotype-Dependent Anti-Inflammatory Signaling in CD163(+) Macrophages.

Intraplaque hemorrhage causes adaptive remodelling of macrophages towards a protective phenotype specialized towards handling iron and lipid overload, denoted Mhem. The Mhem phenotype expresses elevated levels of hemoglobin (Hb) scavenger receptor, CD163, capable of endocytosing pro-oxidant free Hb complexed to acute phase protein haptoglobin (Hp). It is notable that individuals homozygous for the Hp 2 allele (a poorer antioxidant) are at increased risk of cardiovascular disease compared to the Hp 1 allele. In this study, we examined whether scavenging of polymorphic Hp:Hb complexes differentially generated downstream anti-inflammatory signals in cultured human macrophages culminating in interleukin (IL)-10 secretion. We describe an anti-inflammatory signalling pathway involving phosphatidylinositol-3-kinase activation upstream of Akt phosphorylation (pSer473Akt) and IL-10 secretion. The pathway is mediated specifically through CD163 and is blocked by anti-CD163 antibody or phagocytosis inhibitor. However, levels of pSer473Akt and IL-10 were significantly diminished when scavenging polymorphic Hp2-2:Hb complexes compared to Hp1-1:Hb complexes (P < 0.05). Impaired anti-inflammatory macrophage signaling through a CD163/pAkt/IL-10 axis may thus represent a possible Hp2-2 disease mechanism in atherosclerosis.

Anti-inflammatory haemoglobin scavenging monocytes are induced following coronary artery bypass surgery.

OBJECTIVE: Circulating monocytes may counter systemic pro-inflammatory and haemolytic insults through the expression and shedding of the haemoglobin scavenger receptor (CD163). This prospective study aims to assess the effects of coronary artery bypass grafting with and without cardiopulmonary bypass (CPB) on haemoglobin scavenging and anti-inflammatory monocyte behaviour. METHODS: Forty consecutive patients underwent coronary surgery using CPB (n=20) and off-pump (n=20) techniques. Peri-operative blood samples were taken until the fifth day following surgery and statistical comparison performed to baseline using group- and subject-based analysis. Monocyte receptor expression and plasma concentrations of anti-inflammatory molecules were measured by flow cytometry and enzyme-linked immunosorbent assay, respectively. RESULTS: Monocyte CD163 expression was significantly elevated post-operatively in both surgical groups with (p=0.001) and without CPB (p=0.000) at 24-48h. By contrast, shed CD163 (p=0.02) and haemoglobin-haptoglobin complexes (p=0.000) in plasma were only significantly elevated in the on-pump group at 2-4h. Subject-based analysis demonstrated that CPB is an independent predictor of monocyte CD163 (p=0.002) and plasma sCD163 (p=0.01) concentration adjusting for the measurement timing. Significant downregulation of the monocyte major histocompatibility complex II receptor was observed in both surgical groups, whereas lipopolysaccharide receptor (CD14) expression only declined following on-pump surgery. The anti-inflammatory cytokine interleukin (IL)-10 was elevated post-operatively in both surgical groups peaking earlier in the on-pump group (2h) versus off-pump group (4h). The immunoregulatory IL-6 cytokine was significantly upregulated at 4h in both groups. CONCLUSION: Coronary artery bypass surgery induces monocyte-associated anti-inflammatory and immunoregulatory responses. There was no significant difference in haemolysis although the effect on monocyte CD163 and plasma sCD163 concentration was significantly different between the two groups. Compensation for varying pro-inflammatory and haemolytic insults may explain the differences observed. Therapeutic strategies for inducing such desirable circulating monocytes may potentially improve surgical outcome and patient recovery.

Effect of particle size on hydroxyapatite crystal-induced tumor necrosis factor alpha secretion by macrophages.

Macrophages may promote a vicious cycle of inflammation and calcification in the vessel wall by ingesting neointimal calcific deposits (predominantly hydroxyapatite) and secreting tumor necrosis factor (TNF)alpha, itself a vascular calcifying agent. Here we have investigated whether particle size affects the proinflammatory potential of hydroxyapatite crystals in vitro and whether the nuclear factor (NF)-kappaB pathway plays a role in the macrophage TNFalpha response. The particle size and nano-topography of nine different crystal preparations was analyzed by X-ray diffraction, Raman spectroscopy, scanning electron microscopy and gas sorbtion analysis. Macrophage TNFalpha secretion was inversely related to hydroxyapatite particle size (P=0.011, Spearman rank correlation test) and surface pore size (P=0.014). A necessary role for the NF-kappaB pathway was demonstrated by time-dependent I kappaB alpha degradation and sensitivity to inhibitors of I kappaB alpha degradation. To test whether smaller particles were intrinsically more bioactive, their mitogenic activity on fibroblast proliferation was examined. This showed close correlation between TNFalpha secretion and crystal-induced fibroblast proliferation (P=0.007). In conclusion, the ability of hydroxyapatite crystals to stimulate macrophage TNFalpha secretion depends on NF-kappaB activation and is inversely related to particle and pore size, with crystals of 1-2 microm diameter and pore size of 10-50 A the most bioactive. Microscopic calcific deposits in early stages of atherosclerosis may therefore pose a greater inflammatory risk to the plaque than macroscopically or radiologically visible deposits in more advanced lesions.

Safe disposal of inflammatory monosodium urate monohydrate crystals by differentiated macrophages.

OBJECTIVE: Although monosodium urate monohydrate (MSU) crystals have been recognized since the 18th century as the etiologic agent of gout, it is still unknown why certain hyperuricemic individuals remain asymptomatic, and how an acute attack of gout spontaneously resolves. We hypothesized that mononuclear phagocytes hold the key to these questions, and that the state of monocyte/macrophage differentiation is critical. METHODS: Human peripheral blood monocytes were differentiated for 1-7 days in vitro and examined with respect to 1) uptake of MSU crystals, 2) expression of macrophage, dendritic cell, and activation markers, 3) secretion of tumor necrosis factor alpha (TNFalpha), interleukin 1beta (IL-1beta), IL-6, and IL-10, 4) activation of endothelial E-selectin expression, and 5) enhancement of secondary neutrophil recruitment by endothelial cells. RESULTS: MSU crystals induced TNFalpha, IL-1beta, and IL-6 (but not IL-10) secretion in undifferentiated monocytes, which in turn promoted endothelial cell E-selectin expression and secondary neutrophil capture under shear flow. In contrast, differentiation over 3-5 days led to development of a noninflammatory phenotype characterized by a lack of proinflammatory cytokine secretion, lack of endothelial cell activation, and lack of secondary neutrophil recruitment. Acquisition of the noninflammatory phenotype correlated with expression of macrophage antigen but not with expression of dendritic cell marker or activation marker. Monocytes and macrophages were similarly phagocytic, and a control particle, zymosan, elicited secretion of the full panel of cytokines in both cell types. However, coincubation with MSU led to a significant suppression of zymosan-induced TNFalpha secretion (P = 0.009) from macrophages but not monocytes. CONCLUSION: These findings imply that differentiated macrophages provide a safe-disposal mechanism for the removal of inflammatory urate crystals. This may be of clinical relevance to the maintenance of asymptomatic hyperuricemia and the resolution of acute gout.