Immortalized Human Conjunctival Epithelial Cells Produce Functional Complement C3 and C4 Proteins.

PURPOSE: The aim of this study was to assess whether complement proteins C3 and C4 are produced by immortalized human conjunctival epithelial (HCjE) cells. METHODS: Supernatants and cell lysates from undifferentiated and differentiated HCjE cells were assayed for C3 and C4 by enzyme-linked immunosorbent assay. To measure complement protein function, supernatants and lysates were treated with heat-aggregated IgG, and soluble C5b-9 was measured. RESULTS: C3 was upregulated in supernatants from differentiated HCjE cells compared with undifferentiated HCjE cells (556.55 ± 91.75 vs. 56.95 ± 12.09 ng/mL, P <0.001). C4 was also increased in supernatants but to a much lesser extent (0.599 ± 0.476 vs. 0.172 ± 0.0133 ng/mL, P = 0.03). From HCjE cell lysates, total C3 production was 9.03 times higher in differentiated HCjE cells ( P <0.001), whereas total C4 remained relatively unchanged. After activation with heat-aggregated IgG, sC5b-9 could be detected from both undifferentiated and differentiated HCjE cell lysates, but not in the HCjE supernatants. CONCLUSIONS: HCjE cells produce C3 and C4 in sufficient quantities to support the formation of sC5b-9, confirming their biological activity and suggesting that HCjE cells likely produce all complement proteins C1 through C9.

Clustering Patients With Gout Based on Comorbidities and Biomarkers: A Cross-Sectional Study.

OBJECTIVE: This single-center clinical study identifies clusters of different phenotypes and pathophysiology subtypes of patients with gout and associated comorbidities. METHODS: Patients clinically diagnosed with gout were enrolled between January 2018 and December 2019. Hierarchical cluster analyses were performed using clinical data or biological markers, inflammatory markers, and oxidative stress pathway metabolites assayed from serum and plasma samples. Subgroup clusters were compared using ANOVA for continuous data and chi-square tests for categorical data. RESULTS: Hierarchical cluster analysis identified 3 clusters. Cluster 1 (C1; n = 24) comprised dyslipidemia, hypertension, and early-onset gout, without tophi. Cluster 2 (C2; n = 25) comprised hypertension, dyslipidemia, nephrolithiasis, and obesity. Cluster 3 (C3; n = 39) comprised multiple comorbidities and tophi. Post hoc comparisons of data obtained from samples of patients in C1, C2, and C3 revealed significant differences in the levels of oxidative stress and inflammation-related markers, including 3-nitrotyrosine, tumor necrosis factor, C-reactive protein, interleukin (IL) 1ß, IL-6, platelet-derived growth factor (PDGF)-AA, and PDGF-BB. Reclustering patients based on all markers as well as on the biological markers that significantly differed among the initial clusters identified similar clusters. CONCLUSION: Oxidative stress and inflammatory marker levels may affect the development and clinical manifestations (ie, clinical phenotypes) of gout. Measuring oxidative stress and levels of inflammatory cytokines is a potential adjunctive tool and biomarker for early identification and management of gout.

Therapeutic Lowering of C-Reactive Protein.

In the blood of healthy individuals C-reactive protein (CRP) is typically quite scarce, whereas its blood concentration can rise robustly and rapidly in response to tissue damage and inflammation associated with trauma and infectious and non-infectious diseases. Consequently, CRP plasma or serum levels are routinely monitored in inpatients to gauge the severity of their initial illness and injury and their subsequent response to therapy and return to health. Its clinical utility as a faithful barometer of inflammation notwithstanding, it is often wrongly concluded that the biological actions of CRP (whatever they may be) are manifested only when blood CRP is elevated. In fact over the last decades, studies done in humans and animals (e.g. human CRP transgenic and CRP knockout mice) have shown that CRP is an important mediator of biological activities even in the absence of significant blood elevation, i.e. even at baseline levels. In this review we briefly recap the history of CRP, including a description of its discovery, early clinical use, and biosynthesis at baseline and during the acute phase response. Next we overview evidence that we and others have generated using animal models of arthritis, neointimal hyperplasia, and acute kidney injury that baseline CRP exerts important biological effects. In closing we discuss the possibility that therapeutic lowering of baseline CRP might be a useful way to treat certain diseases, including cancer.

C-Reactive Protein Promotes the Expansion of Myeloid Derived Cells With Suppressor Functions.

Previously we established that human C-reactive protein (CRP) exacerbates mouse acute kidney injury and that the effect was associated with heightened renal accumulation of myeloid derived cells with suppressor functions (MDSC). Herein we provide direct evidence that CRP modulates the development and suppressive actions of MDSCs in vitro. We demonstrate that CRP dose-dependently increases the generation of MDSC from wild type mouse bone marrow progenitors and enhances MDSC production of intracellular reactive oxygen species (iROS). When added to co-cultures, CRP significantly enhanced the ability of MDSCs to suppress CD3/CD28-stimulated T cell proliferation. Experiments using MDSCs from FcγRIIB deficient mice (FcγRIIB-/-) showed that CRP's ability to expand MDSCs and trigger their increased production of iROS was FcγRIIB-independent, whereas its ability to enhance the MDSC T cell suppressive action was FcγRIIB-dependent. Importantly, CRP also enabled freshly isolated primary human neutrophils to suppress proliferation of autologous T cells. These findings suggest that CRP might be an endogenous regulator of MDSC numbers and actions in vivo.

C-reactive protein promotes acute kidney injury via Smad3-dependent inhibition of CDK2/cyclin E.

Acute kidney injury (AKI) is exacerbated in C-reactive protein transgenic mice but alleviated in Smad3 knockout mice. Here we used C-reactive protein transgenic/Smad3 wild-type and C-reactive protein transgenic/Smad3 knockout mice to investigate the signaling mechanisms by which C-reactive protein promotes AKI. Serum creatinine was elevated, and the extent of tubular epithelial cell necrosis following ischemia/reperfusion-induced AKI was greater in C-reactive protein transgenics but was blunted when Smad3 was deleted. Exacerbation of AKI in C-reactive protein transgenics was associated with increased TGF-ß/Smad3 signaling and expression of the cyclin kinase inhibitor p27, but decreased phosphorylated CDK2 and expression of cyclin E. Concomitantly, tubular epithelial cell proliferation was arrested at the G1 phase in C-reactive protein transgenics with fewer cells entering the S-phase cell cycle as evidenced by fewer bromodeoxyuridine-positive cells. In contrast, the protection from AKI in C-reactive protein transgenic/Smad3 knockout mice was associated with decreased expression of p27 and promotion of CDK2/cyclin E-dependent G1/S transition of tubular epithelial cells. In vitro studies using tubular epithelial cells showed that C-reactive protein activates Smad3 via both TGF-ß-dependent and ERK/MAPK cross talk mechanisms, Smad3 bound directly to p27, and blockade of Smad3 or the Fc receptor CD32 prevented C-reactive protein-induced p27-dependent G1 cell cycle arrest. In vivo, treatment of C-reactive protein transgenics with a Smad3 inhibitor largely improved AKI outcomes. Thus, C-reactive protein may promote AKI by impairing tubular epithelial cell regeneration via the CD32-Smad3-p27-driven inhibition of the CDK2/cyclin E complex. Targeting Smad3 may offer a new treatment approach for AKI.

C-reactive protein exacerbates renal ischemia-reperfusion injury: are myeloid-derived suppressor cells to blame?

Myeloid-derived suppressor cells (MDSCs) are a CD11b(+)Gr1(+) population in mice that can be separated into granulocytic (g-MDSC) and monocytic (m-MDSC) subtypes based on their expression of Ly6G and Ly6C. Both MDSC subtypes are potent suppressors of T cell immunity, and their contribution has been investigated in a plethora of diseases including renal cancer, renal transplant, and chronic kidney disease. Whether MDSCs contribute to the pathogenesis of acute kidney injury (AKI) remains unknown. Herein, using human C-reactive protein (CRP) transgenic (CRPtg) and CRP-deficient mice (CRP(-/-)) subjected to bilateral renal ischemia-reperfusion injury (IRI), we confirm our earlier finding that CRP exacerbates renal IRI and show for the first time that this effect is accompanied in CRPtg mice by a shift in the balance of kidney-infiltrating MDSCs toward a suppressive Ly6G(+)Ly6C(low) g-MDSC subtype. In CRPtg mice, direct depletion of g-MDSCs (using an anti-Gr1 monoclonal antibody) reduced the albuminuria caused by renal IRI, confirming they play a deleterious role. Remarkably, treatment of CRPtg mice with an antisense oligonucleotide that specifically blocks the human CRP acute-phase response also led to a reduction in renal g-MDSC numbers and improved albuminuria after renal IRI. Our study in CRPtg mice provides new evidence that MDSCs participate in the pathogenesis of renal IRI and shows that their pharmacological depletion is beneficial. If ongoing investigations confirm that CRP is an endogenous regulator of MDSCs in CRPtg mice, and if this action is recapitulated in humans, then targeting CRP or/and MDSCs might offer a new approach for the treatment of AKI.

Associations between abnormal rod-mediated dark adaptation and health and functioning in older adults with normal macular health.

PURPOSE: Delayed rod-mediated dark adaptation (DA) is characteristic of early age-related macular degeneration (AMD) and also can be observed in some older adults in normal macular health. We examine cross-sectional associations between rod-mediated DA and risk factors for AMD in older adults in normal macular health. METHODS: The sample consisted of adults aged ≥60 years old in normal macular health per grading of fundus photos using an established disease classification system. Rod-mediated DA was measured psychophysically following a photobleach using a computer-automated dark adaptometer with targets centered at 5° on the inferior vertical meridian. The speed of DA was characterized by the rod-intercept value, with abnormal DA defined as rod-intercept ≥ 12.3 minutes. We assessed several health and functional characteristics that the literature has suggested increase AMD risk (e.g., smoking, alcohol use, inflammatory markers, apolipoproteins, low luminance visual acuity, chronic medical conditions, body mass, family history). RESULTS: Among 381 participants (mean age, 68.5 years; SD, 5.5), 78% had normal and 22% had abnormal DA, with the prevalence of abnormal DA increasing with age. After age-adjustment, abnormal DA was associated with increased odds of elevated C-reactive protein (CRP), heavy use of or abstention from alcohol, high blood pressure, and drop in visual acuity under mesopic conditions. CONCLUSIONS: Despite having normal macular health according to accepted definitions of AMD presence, approximately one-quarter of older adults recruited from primary eye care clinics had abnormal DA, which was associated with known risk factors for AMD, including elevated CRP.

Estrogen effects on vascular inflammation are age dependent: role of estrogen receptors.

OBJECTIVE: 17ß-Estradiol (E2) offers cardiovascular protection in young female animals and postmenopausal women. In contrast, randomized trials of menopausal hormones performed in older women have shown harm or no cardiovascular benefit. We hypothesize that E2 effects on vascular inflammation are age dependent. APPROACH AND RESULTS: Young (10 weeks) and aged (52 weeks) female C57BL/6 mice were used as source for primary cultures of bone marrow-derived macrophages (BMMs) and vascular smooth muscle cells (VSMCs). E2 pretreatment of cells derived from young mice attenuated C-reactive protein (CRP)-induced expression of inflammatory mediators. In contrast, E2 pretreatment of cells from aged mice did not alter (BMMs) or paradoxically exaggerated (VSMCs) inflammatory mediator response to CRP. Using E2 receptor (ER) knockout mice, we demonstrated that E2 regulates inflammatory response to CRP in BMMs via ERα and in VSMCs via ERß. BMMs derived from aged (versus young) mice expressed significantly less ERα mRNA and protein. A selective ligand of the novel ER GPR30 reproduced the E2 effects in BMMs and VSMCs. Unlike in young mice, E2 did not reduce neointima formation in ligated carotid arteries of aged CRP transgenic mice. CONCLUSIONS: E2 attenuates inflammatory response to CRP in BMMs and VSMCs derived from young but not aged mice and reduces neointima formation in injured carotid arteries of young but not aged CRP transgenic mice. ERα expression in BMMs is greatly diminished with aging. These data suggest that vasoprotective effects of E2 are age dependent and may explain the vasotoxic effects of E2 seen in clinical trials of postmenopausal women.

C-reactive protein promotes acute kidney injury by impairing G1/S-dependent tubular epithelium cell regeneration.

CRP (C-reactive protein) is regarded as an inflammatory biomarker in AKI (acute kidney injury), but its exact role in AKI remains unclear. Thus we sought to investigate the role of CRP in AKI. Clinically, elevated serum CRP levels were found to associate closely with increased serum creatinine and urea levels (P<0.01) in patients with AKI, which then fell after recovery from AKI. To determine the role of CRP in AKI, an ischaemia/reperfusion mouse model of AKI was developed using Tg (transgenic) mice that express human CRP. Compared with the WT (wild-type) mice, CRP Tg mice developed more severe renal injury at 24 h after ischaemia as determined by significantly increased serum creatinine and tubular necrosis. This was associated with an impaired TEC (tubular epithelium cell) regeneration as shown by an over 60% reduction in PCNA+ (proliferating-cell nuclear antigen) and BrdU+ (bromodeoxyuridine) TECs in CRP Tg mice with AKI. In vitro, the addition of CRP to a human TEC line (HK-2) also largely suppressed the proliferation of TECs. The functional role of CRP in AKI was demonstrated further by the blocking of CRP binding to the FcγRII (Fcγ receptor II) with a neutralizing anti-CD32 antibody, which restored TEC proliferation and prevented AKI in CRP Tg mice. Moreover, we found that impaired G1/S transition by suppression of the phosphorylation of CDK2 (cyclin-dependent kinase 2) and expression of cyclin E may be a key mechanism by which CRP inhibits TEC regeneration during the AKI repair process. In conclusion, CRP plays a pathogenic role in AKI by inhibiting G1/S-dependent TEC regeneration. The results of the present study suggest that targeting CRP signalling may offer a new therapeutic potential for AKI.

Multiple lupus-associated ITGAM variants alter Mac-1 functions on neutrophils.

OBJECTIVE: Multiple studies have demonstrated that single-nucleotide polymorphisms (SNPs) in the ITGAM locus (including the nonsynonymous SNPs rs1143679, rs1143678, and rs1143683) are associated with systemic lupus erythematosus (SLE). ITGAM encodes the protein CD11b, a subunit of the ß2 integrin Mac-1. The purpose of this study was to determine the effects of ITGAM genetic variation on the biologic functions of neutrophil Mac-1. METHODS: Neutrophils from ITGAM-genotyped and -sequenced healthy donors were isolated for functional studies. The phagocytic capacity of neutrophil ITGAM variants was probed with complement-coated erythrocytes, serum-treated zymosan, heat-treated zymosan, and IgG-coated erythrocytes. The adhesion capacity of ITGAM variants, in adhering to either purified intercellular adhesion molecule 1 or tumor necrosis factor α-stimulated endothelial cells, was assessed in a flow chamber. Expression levels of total CD11b and activation of CD11b were assessed by flow cytometry. RESULTS: Mac-1-mediated neutrophil phagocytosis, determined in cultures with 2 different complement-coated particles, was significantly reduced in individuals with nonsynonymous variant alleles of ITGAM. This reduction in phagocytosis was related to variation at either rs1143679 (in the ß-propeller region) or rs1143678/rs1143683 (highly linked SNPs in the cytoplasmic/calf-1 regions). Phagocytosis mediated by Fcγ receptors was also significantly reduced in donors with variant ITGAM alleles. Similarly, firm adhesion of neutrophils was significantly reduced in individuals with variant ITGAM alleles. These functional alterations were not attributable to differences in total receptor expression or activation. CONCLUSION: The nonsynonymous ITGAM variants rs1143679 and rs1143678/rs113683 contribute to altered Mac-1 function on neutrophils. These results underscore the need to consider multiple nonsynonymous SNPs when assessing the functional consequences of ITGAM variation on immune cell processes and the risk of SLE.

C-reactive protein exacerbates renal ischemia-reperfusion injury.

Renal ischemia-reperfusion injury (IRI) is a common cause of acute kidney injury (AKI), occurring with hypotension and cardiovascular surgery and inevitably during kidney transplantation. Mortality from AKI is high due to incomplete knowledge of the pathogenesis of IRI and the lack of an effective therapy. Inflammation accompanies IRI and increases the blood level of C-reactive protein (CRP), a biomarker of worsened outcomes in AKI. To test if CRP is causal in AKI we subjected wild-type mice (WT) and human CRP transgenic mice (CRPtg) to bilateral renal IRI (both pedicles clamped for 30 min at 37°C then reperfused for 24 h). Serum human CRP level was increased approximately sixfold after IRI in CRPtg (10.62 ± 1.31 µg/ml at baseline vs. 72.01 ± 9.41 µg/ml at 24 h) but was not elevated by sham surgery wherein kidneys were manipulated but not clamped. Compared with WT, serum creatinine, urine albumin, and histological evidence of kidney damage were increased after IRI in CRPtg mice. RT-PCR analysis of mRNA isolated from whole kidneys of CRPtg and WT subjected to IRI revealed that in CRPtg kidneys 1) upregulation of markers of macrophage classical activation (M1 markers) was blunted, 2) downregulation of markers of macrophage alternative activation (M2 markers) was more robust, and 3) expression of the activating receptor FcγRI was increased. Our finding that CRP exacerbates IRI-induced AKI, perhaps by shifting the balance of macrophage activation and FcγR expression towards a detrimental portfolio, might make CRP a promising therapeutic target for the treatment of AKI.

Kidney injury accelerates cystogenesis via pathways modulated by heme oxygenase and complement.

AKI accelerates cystogenesis. Because cystogenic mutations induce strong transcriptional responses similar to those seen after AKI, these responses may accelerate the progression of cystic renal disease. Here, we modulated the severity of the AKI-like response in Cys1(cpk/cpk) mice, a model that mimics autosomal recessive polycystic kidney disease. Specifically, we induced or inhibited activity of the renoprotective enzyme heme oxygenase (HO) and determined the effects on renal cystogenesis. We found that induction of HO attenuated both renal injury and the rate of cystogenesis, whereas inhibition of HO promoted cystogenesis. HO activity mediated the response of NFκB, which is a hallmark transcriptional feature common to both cystogenesis and AKI. Among the HO-modulated effects we measured, expression of complement component 3 (C3) strongly correlated with cystogenesis, a functionally relevant association as suggested by Cys1(cpk/cpk) mice with genetically induced C3 deficiency. Because both C3 deficiency and HO induction reduce cyst number and cyst areas, these two factors define an injury-stimulated cystogenic pathway that may provide therapeutic targets to slow the formation of new renal cysts and the growth of existing cysts.

Biomarkers of inflammation and hemostasis associated with left ventricular mass: The Multiethnic Study of Atherosclerosis (MESA).

PURPOSE: Biomarkers of inflammation and hemostasis have been associated with left ventricular (LV) mass. We studied relationships of C-reactive protein (CRP), interleukin-6 (IL6), D-dimer, soluble intercellular adhesion molecule-1 (sICAM-1), plasminogen activator inhibitor 1 (PAI-1), soluble thrombomodulin (sTM), soluble tumor necrosis factor type 1 receptor (sTNFR1), von Willebrand factor (vWF), soluble E-selectin (sE-selectin), factor VIII, fibrinogen, matrix metalloproteinase 3 (MMP3), and matrix metalloproteinase 9 (MMP9) with LV mass in an asymptomatic population. Multi-Ethnic Study of Atherosclerosis participants underwent magnetic resonance imaging to characterize LV mass; biomarkers were measured using standardized protocols (N = 763 to 4979). Adjusted models were used to associate each biomarker with LV mass while correcting for potential confounding. FINDINGS: LV mass was associated with many biomarkers after adjustment for demographic characteristics and traditional cardiovascular risk factors. Although the demographic and risk factor adjustments attenuated the association of CRP and IL6 with LV mass, further adjustment for weight changed regression coefficients from positive to negative for CRP and IL6 for LV mass. sTM, Factor VIII, and vWF were directly associated with LV mass in fully-adjusted models. For sTNFR1, sICAM-1, D-dimer, fibrinogen, and PAI-1, adjustment for risk factors and weight rendered associations with LV mass nonsignificant. CONCLUSIONS: In this large cohort free of clinical cardiovascular disease, several hemostasis and inflammation markers were associated with LV mass. The unusual finding of a negative relationship of CRP and IL6 with LV mass only after adjustment for weight suggests that the effects of inflammation on LV mass are strongly influenced by obesity.

C-reactive protein promotes acute renal inflammation and fibrosis in unilateral ureteral obstructive nephropathy in mice.

Elevated blood level of C-reactive protein (CRP) is associated with increased risk of chronic kidney disease. However, whether this association reflects functional importance of CRP in the pathogenesis of kidney disease remains unclear. In this study, we examined the biological role of CRP in a well-characterized model of progressive kidney disease, unilateral ureteral obstruction (UUO), in mice that express the human CRP gene (CRPtg). Compared with wild-type (Wt) mice at 3 days after UUO, CRPtg mice developed more severe renal inflammation with a significant increase in tubulointerstitial T cells and macrophages, upregulation of proinflammatory cytokines (IL-1ß and TNF-α), chemokines (MCP-1), and adhesion molecules (ICAM-1). Renal fibrosis was also significantly enhanced in CRPtg mice as demonstrated by increased expression of tubulointerstitial α-smooth muscle actin and collagen types I and III compared with Wt mice. Interestingly, on days 7 and 14 after UUO, an equal severity of renal inflammation and fibrosis were observed in CRPtg and Wt mice. These findings suggested that CRP may have a role in the initiation of renal inflammation and fibrosis. Further study revealed that enhanced early renal inflammation and fibrosis on day 3 in CRPtg mice was associated with a significant upregulation of endogenous mouse CRP and FcγRI mRNA and increased activation of both NF-κB/p65 and TGF-ß/Smad2/3 signaling, while equal severity of progressive renal injury at day 7 and day 14 between CRPtg and Wt mice were attributed to equivalent levels of CRP, FcγRI, phospho-NF-κB/p65, and TGF-ß/Smad2/3 signaling. Based on these findings, we conclude that CRP may not only be a biomarker, but also a mediator in the early development of renal inflammation and fibrosis in a mouse model of UUO. Enhanced activation of both NF-κB and TGF-ß/Smad signaling pathways may be mechanisms by which CRP promotes early renal inflammation and fibrosis.

Complement receptor 1 expression on mouse erythrocytes mediates clearance of Streptococcus pneumoniae by immune adherence.

Complement-containing immune complexes can be presented to phagocytes by human erythrocytes bearing complement receptor 1 (CR1). Although this has long been assumed to be a mechanism by which humans are able to protect themselves from "extracellular" bacteria such as pneumococci, there is little direct evidence. In these studies we have investigated this question by comparing results for erythrocytes from transgenic mice expressing human CR1 on their erythrocytes to the results for wild-type mouse erythrocytes that do not express CR1. We demonstrate that human CR1 expression on murine erythrocytes allows immune adherence to beads opsonized with either mouse or human serum as a source of complement. The role of CR1 in immune adherence was supported by studies showing that it was blocked by the addition of antibody to human CR1. Furthermore, human CR1 expression enhances the immune adherence of opsonized pneumococci to erythrocytes in vitro, and the pneumococci attached to erythrocytes via CR1 can be transferred in vitro to live macrophages. Even more importantly, we observed that if complement-opsonized pneumococci are injected intravenously with CR1(+) mouse erythrocytes into wild-type mice (after a short in vitro incubation), they are cleared faster than opsonized pneumococci similarly injected with wild-type mouse erythrocytes. Finally, we have shown that the intravenous (i.v.) injection of pneumococci into CR1(+) mice also results in more rapid blood clearance than in wild-type mice. These data support that immune adherence via CR1 on erythrocytes likely plays an important role in the clearance of opsonized bacteria from human blood.

C-reactive protein-mediated vascular injury requires complement.

BACKGROUND: We previously demonstrated that vascular injury-induced neointima formation is exaggerated in human C-reactive protein (CRP) transgenic (CRPtg) compared to nontransgenic (NTG) mice. We now test the hypothesis that complement is required for this effect. METHODS AND RESULTS: CRPtg and NTG with a normal complement system versus their counterparts lacking expression of complement component 3 (C3) protein (CRPtg/C3(-/-) and NTG/C3(-/-)) underwent carotid artery ligation. Twenty-eight days later, the injured vessels in CRPtg had thicker neointimas and more immunoreactive C3 in the surrounding adventitia compared with NTG. In CRPtg/C3(-/-), there was no increase in neointimal thickness compared with NTG or NTG/C3(-/-). Decreasing human CRP blood levels through administration of a selective antisense oligonucleotide eliminated the depletion of serum C3 associated with vascular injury and reduced immunoreactive C3 in the resultant lesions. In injured vessels, C3 colocalized with F4/80 (macrophage marker), and in vitro, human CRP elicited increased expression of C3 by bone marrow-derived macrophages. CONCLUSIONS: Human CRP exaggeration of neointima formation in injured mouse carotid arteries associates with decreased circulating C3 and increased tissue-localized C3. C3 elimination or pharmacological reduction of human CRP prevents CRP-driven exacerbation of the injury response. In the CRPtg model system, mouse C3 is essential for the effect of human CRP.

C-reactive protein promotes cardiac fibrosis and inflammation in angiotensin II-induced hypertensive cardiac disease.

C-reactive protein (CRP) is a risk factor or biomarker for cardiovascular diseases, including hypertension. The present study investigated the functional importance of human CRP in hypertensive cardiac remodeling by a chronic infusion of angiotensin II (Ang II) into mice that express human CRP. Compared with the wild-type mice, although Ang II infusion caused an equally high systolic blood pressure, levels of human CRP were further elevated, and cardiac remodeling was markedly exacerbated in mice that express human CRP, resulting in a significant reduction in the left ventricular ejection fraction and fractional shortening and an increase in cardiac fibrosis (collagen I and III and alpha-smooth muscle actin) and inflammation (interleukin 1beta and tumor necrosis factor-alpha). The enhancement in cardiac remodeling in mice that express human CRP was associated with further upregulation of the Ang II type I receptor and transforming growth factor-beta1 and overactivation of both transforming growth factor-beta/Smad and nuclear factor-kappaB signaling pathways. Furthermore, in vitro studies in cardiac fibroblasts revealed that CRP alone was able to significantly induce expression of the Ang II type I receptor, collagen I/III, and alpha-smooth muscle actin, as well as proinflammation cytokines (interleukin 1beta and tumor necrosis factor-alpha), which was further enhanced by addition of Ang II. In conclusion, CRP is not only a biomarker but also a mediator in Ang II-mediated cardiac remodeling. Enhanced upregulation of the Ang II type I receptor and activation of the transforming growth factor-beta/Smad and nuclear factor-kappaB signaling pathways may be the mechanisms by which CRP promotes cardiac fibrosis and inflammation under high Ang II conditions.

Antibody to the type 3 capsule facilitates immune adherence of pneumococci to erythrocytes and augments their transfer to macrophages.

Streptococcus pneumoniae has been shown to bind to erythrocytes via a process called immune adherence. This adherence and the subsequent transfer of pneumococci from erythrocytes to macrophages are both dependent on complement C3 deposition onto the pneumococcal surface. The observation that anti-capsule antibody increases C3 deposition on the pneumococcal capsule indicated that anti-capsule antibody may also facilitate the clearance of pneumococci through immune adherence. Using pneumococcal strain WU2 (capsule type 3) and its nonencapsulated mutant JD908, we found that monoclonal antibody (MAb) to type 3 capsule increases complement C3, C1q, and C4 deposition on WU2 and enhanced the immune adherence of WU2 to erythrocytes. The MAb to type 3 capsule also enhanced the transfer of WU2 from erythrocytes to macrophages. Moreover, the transfer reaction was inhibited by preincubating macrophages with anti-CR3 or anti-Fc gammaRIII/II MAb, indicating that CR3 and Fc gammaRIII/II on macrophages mediate this process. The transfer reactions of JD908 (opsonized with complement) and WU2 (opsonized with complement plus MAb to type 3 capsule) were similarly inhibited by anti-CR3 MAb, but only the latter was inhibited by anti-Fc gammaRIII/II MAb. This finding indicates that although complement and the macrophage receptor CR3 are essential for the transfer reaction, if antibody is present it can further enhance the transfer reaction through a process dependent on Fc gammaRIII/II. Using pre- and postvaccination sera of people immunized with the 23-valent pneumococcal polysaccharide vaccine, we confirmed that human anti-capsule antibodies are also able to increase the immune adherence of pneumococci and their transfer to macrophages.

Immune opsonins modulate BLyS/BAFF release in a receptor-specific fashion.

TNF ligand superfamily member 13B (B lymphocyte stimulator (BLyS), B cell activating factor (BAFF)) promotes primary B cell proliferation and Ig production. While the soluble form of BLyS/BAFF is thought to be the primary biologically active form, little is known about the regulation of its cleavage and processing. We provide evidence that Fcgamma receptor cross-linking triggers a rapid release of soluble, biologically active BLyS/BAFF from myeloid cells. Surprisingly, this function is primarily mediated by FcgammaRI, but not FcgammaRIIa as defined by specific mAb, and can be initiated by both IgG and C reactive protein as ligands. The generation of a B cell proliferation and survival factor by both innate and adaptive immune opsonins through engagement of an Fcgamma receptor, which can also enhance Ag uptake and presentation, provides a unique opportunity to facilitate Ab production. These results provide a mechanism by which Fcgamma receptors can elevate circulating BLyS levels and promote autoantibody production in immune complex-mediated autoimmune diseases.

Impaired endothelial function in C-reactive protein overexpressing mice.

Increasing evidence suggests that the inflammatory biomarker, C-reactive protein (CRP), may play a causal role in the development and progression of atherothrombosis. Since endothelial dysfunction is an early and integral component of atherosclerosis, we hypothesized that endothelial homeostasis would be impaired in CRP-overexpressing CRP transgenic (CRPtg) mice. Male CRPtg and wild-type mice were injected thrice over 2 weeks with vehicle or turpentine to induce the inflammation-sensitive CRP transgene. Serum human CRP levels in turpentine-treated CRPtg mice was 276.28 +/- 95.7 microg/ml. Human CRP was undetectable in the sera of wild-type mice and present at only low levels (1.41 +/- 0.2 microg/ml) in vehicle-treated CRPtg mice (n=6-8 mice/group). Aortic segments from turpentine-induced CRP-overexpressing CRPtg mice demonstrated impaired endothelium-dependent responses to acetylcholine vs. those from vehicle-treated CRPtg controls (57.1 +/- 9.5% vs. 85.0 +/- 5.0%, P<0.05, n=6). Nitric oxide release as well as phosphorylated eNOS protein expression from isolated aortic segments of CRPtg mice overexpressing CRP were markedly reduced compared to that from vehicle-treated controls. Masson's trichrome staining revealed increased perivascular fibrosis in CRP-overexpressing CRPtg mice. CRP overexpression was also associated with augmented aortic endothelial staining for VCAM-1 and MCP-1 and enhanced macrophage infiltration. Mice overexpressing the human CRP gene exhibit endothelial dysfunction, possibly via reduced NO bioavailability, with resultant changes in vascular structure. These data further support a role for CRP in mediating endothelial dysfunction.