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The Long Pentraxin PTX3 Promotes Fibrocyte Differentiation


Monocyte-derived, fibroblast-like cells called fibrocytes are associated with fibrotic lesions. The plasma protein serum amyloid P component (SAP; also known as pentraxin-2, PTX2) inhibits fibrocyte differentiation in vitro, and injections of SAP inhibit fibrosis in vivo. SAP is a member of the pentraxin family of proteins that includes C-reactive protein (CRP; PTX1) and pentraxin-3 (PTX3). All three pentraxins are associated with fibrosis, but only SAP and CRP have been studied for their effects on fibrocyte differentiation. We find that compared to SAP and CRP, PTX3 promotes human and murine fibrocyte differentiation. The effect of PTX3 is dependent on FcγRI. In competition studies, the fibrocyte-inhibitory activity of SAP is dominant over PTX3. Binding competition studies indicate that SAP and PTX3 bind human FcγRI at different sites. In murine models of lung fibrosis, PTX3 is present in fibrotic areas, and the PTX3 distribution is associated with collagen deposition. In lung tissue from pulmonary fibrosis patients, PTX3 has a widespread distribution, both in unaffected tissue and in fibrotic lesions, whereas SAP is restricted to areas adjacent to vessels, and absent from fibrotic areas. These data suggest that the relative levels of SAP and PTX3 present at sites of fibrosis may have a significant effect on the ability of monocytes to differentiate into fibrocytes.


During fibrosis, some monocytes leave the circulation, enter the tissue, and differentiate into fibroblast-like cells called fibrocytes [13]. Fibrocytes express markers of both hematopoietic cells (CD34, CD45, FcγR, LSP-1, MHC class II) and stromal cells (collagens and matrix metalloproteases) [26]. Fibrocytes are found in lesions associated with fibrotic diseases such as pulmonary fibrosis, congestive heart failure, cirrhosis of the liver, and nephrogenic systemic fibrosis [3,711]. In healthy tissues, there are very few fibrocytes [3]. Fibrocyte differentiation is regulated by several factors including the plasma protein Serum Amyloid P component (SAP; PTX2) [3,5,12,13].

SAP is a pentameric protein that belongs to the pentraxin family of evolutionarily conserved proteins [14]. There are three main systemic pentraxins in mammals: SAP, C-reactive protein (CRP), and the long pentraxin PTX3 [15]. SAP, CRP, and PTX3 all have regulatory roles in the immune system [16]. Pentraxins bind with different affinities to adhesion molecules, pathogens, and apoptotic cells, leading to complement activation, phagocytosis, and cytokine production [15]. In contrast to SAP and CRP, which are produced by hepatocytes, PTX3 is produced by macrophages, neutrophils, endothelial cells, epithelial cells, and fibroblasts [15]. In healthy humans, the plasma levels of CRP and PTX3 are low (< 2 μg/ml and < 25 ng/ml respectively), whereas during inflammation CRP and PTX3 levels may rise to 500 μg/ml and 1000 ng/ml respectively [1719]. In humans, plasma SAP levels are 20–60 μg/ml, and are not affected by inflammation [20].

Injections of SAP inhibit fibrosis in mouse models of pulmonary fibrosis, ischemic cardiac fibrosis, and renal fibrosis [8,2124], and in a phase 1b clinical trial, SAP injections appeared to improve lung function in pulmonary fibrosis patients [25]. In mice, overexpression of CRP strongly potentiates inflammation and fibrosis [2629]. PTX3 is associated with inflammation in humans, but in mice appears to be pro-inflammatory in some models and limits inflammation in other models [30,31].

SAP inhibits fibrocyte differentiation partly through a group of receptors called Fcγ receptors [22,3235]. In humans, there are four activating FcγR: FcγRI, FcγRIIA, FcγRIIIA, and FcγRIIIB [36,37]. In mice, there are three activating Fcγ receptors: FcγRI, FcγRIII, and FcγRIV [36,38]. The activating receptors require an accessory common gamma chain (FcRγ), for plasma membrane localization and initiating a signaling cascade [39]. Both humans and mice have an inhibitory receptor, FcγRIIb, that triggers an inhibitory signaling pathway to help modulate the immune response [40]. We have previously found that FcγRI and FcRγ are responsible for the effect of SAP on fibrocyte differentiation in both humans and mice [32]. In addition, the inhibition of cardiac or renal fibrosis in mice by injections of SAP is also dependent on FcRγ [22,33].

Although all three main pentraxins, and fibrocytes, are associated with fibrosis, only SAP and CRP have been studied for their effects on fibrocyte differentiation. In this report, we find that PTX3 promotes fibrocyte differentiation. The effect of PTX3 is dependent on FcγRI, but in competition studies, the fibrocyte-inhibitory activity of SAP is dominant over PTX3. These data suggest that the relative levels of SAP and PTX3 present at sites of fibrosis may have a significant effect on the ability of monocytes to differentiate into fibrocytes.

Materials and Methods

Human PBMC, leukocyte, and monocyte isolation, and cell culture

Human peripheral blood was collected into heparin tubes (BD Bioscience, San Jose, CA) from healthy adult volunteers who gave written consent and with specific approval from the Texas A&M University human subjects Institutional Review Board. Peripheral blood mononuclear cells (PBMC) were isolated from the blood using Ficoll-Paque Plus (GE Healthcare Biosciences, Piscataway, NJ), as described previously [5,12,41,42]. CD14+CD16- monocytes were enriched from PBMC using EasySep monocyte enrichment kits, following the manufacturer’s instructions (StemCell Technologies, Vancouver, Canada). Monocytes were checked for enrichment by flow cytometry in comparison to the un-enriched PBMC population, as described previously [12,13,41,43,44]. Monocyte preparations were 93.6% ± 2.5 (mean ± SEM, n = 3) CD14 positive and < 0.5% positive for T cells, B cells, and NK cells.

Human leukocytes were isolated from blood using Lympholyte-poly (Cedarlane Laboratories, Hornby, BC) following the manufacturer’s protocol [45]. HEK293 cells (Life Technologies, Grand Island, NY) were cultured in Freestyle (Life Technologies) medium following the manufacturer’s protocol. K562 cells (ATCC, Manassas, VA) were grown in RPMI 1640 with 10% FBS (Hyclone-GE Healthcare Life Sciences, South Logan, UT). Human dermal (PromoCell, Heidelberg, Germany) and MRC5 lung (Sigma, St Louis, MO) fibroblasts were cultured in DMEM with 10% FCS. Human lung bronchial epithelial cells (PromoCell) were incubated in epithelial cell growth medium (PromoCell) following the manufacturer’s protocol.

Cell fractionation of murine spleen cells

All experimental protocols were approved by the local ethical committees and performed in accordance with national guidelines and regulations. This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Texas A&M University Animal Use and Care Committee (TAMU AUP 2009-262 and 2013-272). Mice maintained at Leiden University Medical Center were monitored according to the rules of the Federation of European Laboratory Animal Science Associations (FELASA) and with approval of the Dierexperimentencommissie Academisch Ziekenhuis Leiden (Animal Experiments Committee Academic Hospital Leiden). Procedures at Leiden were performed under ketamine/atropine/xylazine anesthesia, and euthanasia at both institutions was achieved by CO2 asphyxiation followed by cervical dislocation. All efforts were made to minimize suffering. Spleen cells from 4–6 week male C57BL/6J mice (Jackson Laboratories, Bar Harbor, ME), and from FcγRI, FcγRIIb, FcγRIII, and FcγRI/IIb/III/IV knockout mice on the C57BL/6 background generated at Leiden University Medical Center [4648], were used in this study. Mouse spleen cells were isolated as described previously [32,4951].

Fibrocyte differentiation assay

Human PBMC were cultured in FibroLife (LifeLine Cell Technology, Walkersville, MD) defined serum-free medium (SFM), as described previously [6,42], in the presence or absence of purified human SAP (EMD Millipore, Billerica, MA), purified human CRP (EMD Millipore or Fitzgerald Industries, Acton, MA), or mammalian NSO cell-derived recombinant human PTX3 (R&D Systems, Minneapolis, MN). As commercial SAP and CRP preparations contain 0.1% azide, we buffer-exchange the SAP into 20 mM sodium phosphate, pH 7.4 and the CRP into 150 mM NaCl, 20 mM Tris, 2 mM CaCl2, pH 7.4, as described previously [5,6,12,49]. After 5 days, plates were air-dried, fixed with methanol, and stained with eosin and methylene blue, as described previously [5,12,32,41,42,49]. Fibrocytes were identified and counted based on the following criteria: an adherent cell with elongated spindle-shaped morphology and an oval nucleus, in five different 900 μm-diameter fields of view per well as described previously [5,32,41,42,49].

Murine spleen cells were cultured in FibroLife (Lifeline Cell Technology, Frederick, MD) serum-free medium (SFM) including 50 ng/ml murine IL-13 and 25 ng/ml murine M-CSF (PeproTech, Rocky Hill, NJ), as described previously [32,42,49]. On day 3 of the incubation, wells were supplemented with IL-13 and M-CSF, as described previously [32,49]. After 5 days, plates were dried and fibrocytes counted as described above.

Bleomycin-induced lung inflammation

This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Texas A&M University Animal Use and Care Committee (TAMU AUP #2009-0265 and #2013-0007). All procedures were performed under anesthesia, and all efforts were made to minimize suffering. 4–6 week old C57BL/6 mice (Jackson Laboratory, Bar Harbor, ME) were treated with an oropharyngeal aspiration of 50 μl of 3 U/kg bleomycin (EMD Millipore, Billerica, MA) solution in 0.9% saline or saline alone, as described previously [50,52]. Mice were euthanized at 21 days after bleomycin aspiration, and the lungs were removed and inflated with pre-warmed optimal cutting temperature (OCT) compound (VWR, Radnor, PA), embedded in OCT, frozen on dry ice, and stored at −80°C as described previously [21,50,52].

Immunohistochemistry and flow cytometry

PBMC, fibroblasts, or lung epithelial cells were cultured for 5 days on eight-well glass microscope slides (Millipore), and then fixed and stained as described previously [5,6]. The cells were stained with mouse monoclonal antibodies to CD13 (WM15, BD-Biosciences, San Jose, CA), CD14 (HCD14, BioLegend, San Diego, CA), CD34 (QBend10, Beckman Coulter, Brea CA), CD45 (H130, BioLegend), CD90 (5E10, BioLegend), CXCR4 (44716, R&D Systems), S100A9 (MAC387, AbD Serotec, Raleigh, NC), MHC class II (L243, BioLegend), PM-2K (Abcam, Cambridge, MA), fibronectin (EP5, GeneTex, Irvine, CA), or rabbit polyclonal antibodies to collagen-I (Abcam), collagen VI (Novus Biologicals, Littleton, CO), PTX3 (GeneTex), SAP (EP1018Y, Abcam), or EpCAM (ab71916, Abcam). Isotype-matched irrelevant mouse monoclonal antibodies (BioLegend) or control rabbit polyclonal antibodies (anti-chicken IgY, Bethyl Laboratories, Montgomery, TX), were used as controls. Secondary F(ab’)2 biotin-conjugated donkey anti-mouse or F(ab’)2 biotin-conjugated donkey anti-rabbit antibodies were from Jackson ImmunoResearch (West Grove, PA). Staining was revealed with streptavidin-alkaline phosphatase (Invitrogen, Grand Island, NY) and Vector Red Alkaline Phosphatase Kit (Vector Laboratories, Burlingame, CA) following the manufacturers’ instructions, and slides were then counterstained with hematoxylin [5,6]. Human lung tissue sections were obtained from the National Heart Lung and Blood Institute-sponsored Lung Tissue Research Consortium (LTRC, Bethesda, MD). Murine and human lung sections were prepared and incubated with rabbit antibodies to PTX3 (GeneTex) or SAP (Abcam), as described previously [6,50]. Isotype-matched rabbit anti-chicken IgY antibodies (Bethyl) were used as controls. Images were analyzed with ImageJ (NIH, Bethesda, MD), as described previously [6,50]. For flow cytometry, PBMC were cultured for 5 days in the presence or absence of 1 μg/ml PTX3. Non-adherent cells were then removed, the adherent cells detached by treatment with trypsin-EDTA, and cells were stained for intracellular collagen-VI, as described previously [49].

PTX3 and SAP binding assays and receptor expression

Human FCGR1A, Fc common γ-chain (FCER1G), and FCGRIIIB cDNAs (PSI:Biology-materials repository, Tempe, AZ) [53] were ligated into the pCMV6-AC-His vector (OriGene, Rockville, MD) and then transfected into HEK293 cells using a 4D-Nucleofector electroporation system (Lonza, Cologne, Germany) following the manufacturer’s protocol. HEK293 cells expressing human FcγRI or FcγRIIIB and K562 cells (which express human FcγRIIA) [54], were used to measure the affinity of PTX3 and SAP for FcγR, as described previously [45]. PTX3 and SAP were labeled using Alexa Fluor 647-NHS (Life Technologies) following the manufacturer’s protocol. The binding of fluorescently labeled PTX3 or SAP was then measured as previously described using an Accuri C6 flow cytometer [45]. K562, HEK293, FcγRI+ HEK293, and FcγRIIIB+ HEK293 cells were stained for FcγRI (clone 10.1; BioLegend), FcγRII (clone FUN-2; BioLegend), and FcγRIII (clone 3G8; BioLegend) to determine the expression of the indicated receptor by flow cytometry [6,45]. Leukocytes stained for CD3 (BioLegend), CD14 (BioLegend), CD16 (BioLegend), CD19 (BioLegend), CD45 (BioLegend), FcγRI, FcγRII, and FcγRIII were assayed by flow cytometry to determine the presence of different immune cell populations as previously described [6,45]. For competition experiments, leukocytes were incubated for 30 minutes at 4°C in PBS with 2% IgG-free BSA (Jackson ImmunoResearch), containing 1 μg/ml unlabeled PTX3, SAP, or human IgG1 Fc region (Fc block, BD Biosciences). After 30 minutes, an equal volume of PBS-BSA containing 2 μg/ml (1 μg/ml final) fluorescently labelled PTX3 or SAP was added and the cells were incubated for an additional 30 minutes. For competition experiments involving FcγRI+ HEK293 cells, cells were incubated for 30 minutes at 4°C in PBS with 2% IgG-free BSA, containing the indicated concentrations of unlabeled PTX3 or SAP, and 0.5 μg/ml fluorescently labelled PTX3 or SAP. Cells were then washed twice in ice cold PBS, resuspended in PBS-BSA, and assayed by flow cytometry.

Statistical analysis

Statistical analysis was performed using Prism (GraphPad Software, San Diego, CA). Statistical significance between two groups was determined by t test, or between multiple groups using analysis of variance (ANOVA), with Tukey’s post-test. Significance was defined as p < 0.05. Data were fit to the appropriate model of binding as determined by F-tests.


PTX3 potentiates human fibrocyte differentiation

SAP and CRP bind to multiple FcγR on monocytes, but SAP inhibits monocyte to fibrocyte differentiation, whereas CRP does not [5,34]. PTX3 also binds to FcγR [34,55], and we thus examined whether PTX3 affects fibrocyte differentiation. Human PBMC were cultured for 5 days with or without PTX3. In the absence of added PTX3, we observed 420 to 1,600 fibrocytes per 105 PBMCs from the different donors, similar to what we have previously observed [5,12,56]. Because of this variability, for each donor, fibrocyte numbers were normalized to pentraxin-free controls. For all donors, 0.625 μg/ml and above PTX3 significantly increased the number of spindle-shaped cells (Fig. 1), with an EC50 of 0.59 ± 0.20 μg/ml (mean ± SEM, n = 5).

Fig 1. PTX3 promotes fibrocyte differentiation.

Human PBMC were incubated for 5 days in the absence A) or presence B) of 1 μg/ml PTX3. Bar is 50 μm. C) The effect of the indicated concentrations of PTX3 on fibrocyte numbers. For each donor, numbers were normalized to the no-PTX3 control. Values are mean ± SEM (n = 4). *p <0.05 (t-test). Line is a fit to a sigmoidal dose response curve.

To determine whether the spindle-shaped cells were fibrocytes, and if PTX3 alters the phenotype of fibrocytes, we used immunocytochemistry to stain cells for fibrocyte markers (Fig. 2). In the presence or absence of PTX3, as we previously observed for fibrocytes [6], the elongated cells were positive for markers expressed by fibrocytes including CD13, CD34, CD45, MHC class II, CXCR4, S100A9, and collagen-I, collagen VI, and fibronectin (Fig. 2C). As we observed previously [6], all monocyte-derived cells cultured in SFM had low expression of the monocyte marker CD14, and fibrocytes showed no observable staining for the fibroblast marker CD90, or the tissue macrophage marker PM-2K. These data indicate that the elongated cells are fibrocytes (Fig. 2C). By immunocytochemistry, we did not observe any obvious effect of PTX3 on the staining intensity of the fibrocyte markers, indicating that PTX3 does not appear to alter the characteristics of fibrocytes. Fibrocytes secrete collagen [57], and we observed that 1 μg/ ml PTX3 caused an increase in the levels of soluble collagens (Fig. 2D), but not the levels of collagen-VI within individual cells (Fig. 2E). These data indicate that levels of human PTX3 similar to what can be observed in human plasma during inflammation promote human fibrocyte differentiation, but do not appear to alter fibrocyte characteristics.

Fig 2. Effect of PTX3 on morphology, collagen production, and receptor expression.

After 5 days incubation with 1 μg/ml PTX3, PBMC were air-dried, fixed, and stained with A) anti-collagen-I antibodies or B) control antibodies. Positive staining was identified by red staining, and nuclei are counterstained blue. Bar is 50 μm. C After 5 days, PBMC were air-dried, fixed, and stained with antibodies. Following immunocytochemical staining, at least 100 elongated cells with oval nuclei were examined from at least 10 randomly selected fields, and the percentage of positive cells is expressed as the mean ± SEM (n = 3–5 separate donors). ND – none detected. D) Supernatants from PBMC incubated in the absence (-) or presence (+) of 1 μg/ml PTX3 were assessed by western blotting, using anti-collagen I-V antibodies. Blot is representative of three separate experiments. E) After 5 days, adherent cells (macrophages and fibrocytes) were stained with collagen-VI (teal line SFM, red +PTX3) or control IgY (black SFM, orange +PTX3) antibodies. The data are representative of three separate experiments.

PTX3 acts directly on monocytes

To determine whether the potentiation of fibrocyte differentiation by PTX3 is a direct effect on monocytes, or due to an indirect effect on the T cells, B cells, or NK cells present within the PBMC preparation, we incubated purified CD14+ CD16- monocytes with PTX3 (Fig. 3). As previously observed, SAP inhibited, but CRP had no effect, on monocyte to fibrocyte differentiation [5,12]. For all donors, 1 μg/ml PTX3 significantly potentiated fibrocyte differentiation (Fig. 3). These data suggest that PTX3 acts directly on monocytes to potentiate fibrocyte differentiation.

Fig 3. PTX3 acts directly on CD14+ CD16- (FcγRIIIA) monocytes.

PBMC and isolated CD14+ CD16- monocytes were incubated for 5 days in the presence or absence of 1 μg/ml SAP, CRP, or PTX3. After 5 days, cells were air-dried, fixed, and stained, and the number of fibrocytes was counted. Values are mean ± SEM (n = 3). *p <0.05; **p <0.01; *** <0.001 (ANOVA).

SAP inhibits PTX3-induced fibrocyte differentiation

As fibrotic environments rarely, if ever, contain just one type of pentraxin, we examined how SAP and PTX3 might compete with each other. PBMC were cultured in SFM with increasing concentrations of SAP in the absence or presence of 1 μg/ml PTX3 (Fig. 4). In the presence of PTX3, the inhibitory activity of SAP was maintained (Fig. 4). The SAP IC50 for inhibiting fibrocyte differentiation was 0.14 ± 0.05 μg/ml in the absence of PTX3, and 0.10 ± 0.02 μg/ml (mean ± SEM, n = 3, difference not significant by t-test) in the presence of PTX3. There was no significant difference in the dose-response curve Hill coefficient for SAP in the absence (1.6 ± 0.5) or presence of PTX3 (2.2 ± 0.8). These data indicate that PTX3 does not significantly affect the ability of SAP to inhibit fibrocyte differentiation.

Fig 4. SAP inhibits fibrocyte differentiation in the presence of PTX3.

PBMC were incubated with increasing concentrations of SAP in the presence or absence of 1 μg/ml PTX3. After 5 days, cells were air-dried, fixed, and stained, and the number of fibrocytes was counted. Values are mean ± SEM (n = 3). **p <0.01 (t-test). Lines are fits to sigmoidal dose response curves with variable Hill coefficients.

FcγRI mediates the effect of PTX3 on murine fibrocyte differentiation

To determine if PTX3 also promotes murine fibrocyte differentiation, spleen cells from C57BL/6 mice were cultured for 5 days in SFM in the presence or absence of PTX3. Compared to the control, 1 μg/ml PTX3 significantly potentiated murine fibrocyte differentiation (Fig. 5). PTX3 binds to multiple FcγR [34,55]. To determine whether one or more FcγR mediate the potentiating effect of PTX3 on fibrocyte differentiation, we cultured spleen cells from different FcγR knockout mice in the presence or absence of PTX3. PTX3 potentiated fibrocyte differentiation in cultures of spleen cells from FcγRIIb and FcγRIII KO mice (Fig. 5). However, PTX3 did not potentiate fibrocyte differentiation in cultures of spleen cells from FcγRI knockout and FcγRI/IIb/III/IV quadruple knockout mice (Fig. 5). In addition, compared to C57BL/6 spleen cells cultured in the presence of PTX3, there were significantly less fibrocytes in spleen cells from FcγRI knockout and FcγRI/IIb/III/IV quadruple knockout mice cultured with PTX3. By t-test, there were significantly less fibrocytes in cultures of spleen cells from FcγRI and FcγR quad KO when cultured with PTX3, compared to the SFM control, suggesting that in the absence of FcγRI or FcγRVI PTX3 may bind to an additional receptor(s) to generate an inhibitory signal (Fig. 5). These data suggest that FcγRI is required for the potentiating effect of PTX3 on murine fibrocyte differentiation, and that in the absence of FcγR additional unknown receptors may also regulate the effect of PTX3.

Fig 5. FcγRI KO and quad FcγR KO spleen cells are less sensitive to PTX3 induced fibrocyte differentiation.

Spleen cells were cultured for 5 days in the A-D) absence or E-H) presence of PTX3, from A and E) wildtype C57BL/6 mice, B and F) quadruple FcγRI/IIb/III/IV, C and G) FcγRI, and D and H) FcγRIIb knockout mice. After 5 days, cells were air-dried, fixed, and stained, and the number of fibrocytes was counted. Bar is 100 μm. I) For each strain, the count in the presence of PTX3 was normalized to the count in the absence of PTX3. Values are mean ± SEM (n = 3). *p <0.05, **p <0.01 (ANOVA), compared to SFM. ## p<0.01 (ANOVA) compared to C57BL/6.

PTX3 binds to human leukocytes and human FcγRI and FcγRIIa on cell lines

Both PTX3 and SAP require FcγRI to regulate fibrocyte differentiation ([32,45,51] and Fig. 5). To determine if the opposing effects of PTX3 and SAP on fibrocyte differentiation were due to these pentraxins binding to distinct or overlapping sites on FcγR, or other pentraxin receptors, we examined the binding of PTX3 and SAP to human leukocytes and to human FcγR expressed on the human-derived cell lines HEK293 and K562.

We determined the binding of Alexa647-labelled PTX3 and SAP to human lymphocytes, monocytes, and neutrophils, as identified by their forward and size scatter characteristics and receptor expression using flow cytometry (Fig. 6A). When PTX3-647 was incubated with leukocytes, we observed minimal binding to the lymphocyte population (Fig. 6A). Because B cells (∼5% of lymphocytes) express FcγRIIB [36,45], and NK cells (∼10% of lymphocytes) express FcγRIIIA [36,45], this suggests that PTX3 does not bind to these receptors under our experimental conditions. However, PTX3-647 did bind to monocytes and neutrophils (Fig. 6A). All monocytes express FcγRI, FcγRIIA, and some monocytes also express FcγRIIIA [6,45]. This indicates that PTX3 could be binding to any or all of the FcγRs on monocytes. Because NK cells express FcγRIIIA, and we did not detect binding of PTX3-647 to NK cells, this suggests that PTX3 binds to FcγRI and/or FcγRIIA on monocytes. Neutrophils express FcγRIIA and FcγRIIIB [6,45]. This then suggests that PTX3 binds to FcγRIIA or possibly FcγRIIIB on neutrophils. PTX3 bound to lymphocytes with a single binding site and a KD of 0.5 ± 1.6 ng/ml. However, both monocytes and neutrophils bound PTX3 with two-site binding characteristics. Monocytes had a high affinity binding site with a KD of 1.3 ± 4.3 ng/ml, and a low affinity binding site with a KD of 3400 ± 6500 ng/ml. Neutrophils had a high affinity binding site of 0.2 ± 2.9 ng/ml, and a low affinity binding site of 2200 ± 6600 ng/ml. By 2-way ANOVA, the differences in both the binding of PTX3 and cell type are significant, p < 0.0001 and p = 0.0296 respectively.

Fig 6. Alexa Fluor 647–labeled PTX3 binds to FcγRI and FcγRIIA.

A) PTX3-647 was incubated with isolated leukocytes and then subjected to flow cytometry. Neutrophils, monocytes, and lymphocytes were identified based on forward scatter and side scatter. Curves are fits of the resulting data to models of one or two-site binding with variable Hill coefficient. B) HEK293 cells expressing FcγRI, or K562 cells, which express FcγRIIA and C) HEK293 cells expressing FcγRIIIB were incubated with PTX3-647. The cells were then washed, and the binding of the labeled PTX3 to the cells was measured by flow cytometry. Autofluorescence values were subtracted from the total binding values. Median fluorescence intensity values were normalized to the intensity value of 1 μg/ml concentration. Values are normalized mean ± SEM, n = 3–6. Curves are fits to models of one-site binding with variable Hill coefficient. The absence of error bars indicates that the error was smaller than the plot symbol. *p <0.05, **p <0.01 (1-way ANOVA, Dunnett's test), compared to no PTX3-647 binding.

In addition, we assessed the binding of PTX3-647 to K562 cells, which, with respect to FcγR, only express FcγRIIA [54], and HEK293 cells transfected with either human FcγRI or FcγRIIIB (Fig. 6B and C). We observed significant PTX3-647 binding to HEK293 cells expressing FcγRI and PTX3-647 binding to K562 cells (Fig. 6B). We saw no significant PTX3 binding to HEK293 cells expressing FcγRIIIB (Fig. 6C). Together, our data indicate that PTX3 binds to FcγRI and FcγRIIA on human monocytes, neutrophils, and cell lines, but not to FcγRIIB, FcγRIIIA, and FcγRIIIB (Fig. 6).

To determine if PTX3, SAP, and IgG bind to similar or distinct sites on human leukocytes and on HEK293 cells expressing human FcγRI, we pre-incubated cells with unlabeled PTX3, SAP, or human IgG1 Fc, and then Alexa 647-labelled PTX3. We observed that on monocytes, PTX3-647 binding was inhibited by unlabeled PTX3, but not SAP or IgG Fc (Fig. 7A). On HEK293 cells transfected with human FcγRI, we observed that unlabeled PTX3 could inhibit PTX3-647 binding (Fig. 7B). In similar assays, unlabeled SAP inhibits SAP binding to FcγRI expressing macrophages [22], and we observed that unlabeled SAP could not inhibit PTX3-647 binding to FcγRI (Fig. 7C). In addition, unlabeled PTX3 could not inhibit SAP-647 binding to FcγRI (Fig. 7B). These data suggest that PTX3 and SAP bind to distinct sites on FcγRI, and that PTX3 may also binds to sites that are distinct from IgG.

Fig 7. PTX3 and SAP bind to overlapping sites on leukocytes.

A) Human Leukocytes were incubated with 1 μg/ml unlabeled PTX3, SAP, or human IgG Fc for 30 minutes at 4°C. Cells were then incubated with 1 μg/ml labelled PTX3, for an additional 30 minutes at 4°C. The cells were then washed, and the binding of the labeled PTX3 to the cells was measured by flow cytometry. Values are mean ± SEM, n = 5. * p <0.05, ** p <0.01 (ANOVA), compared to cells incubated with PTX3-647. HEK293 cells expressing FcγRI were incubated with B) PTX3-647 or SAP-647 in the presence of unlabeled PTX3, or C) PTX3-647 and unlabeled SAP. The cells were then washed, and the binding of the labeled pentraxin to the cells was measured by flow cytometry. Autofluorescence values were subtracted from the total binding values. Values are normalized mean ± SEM, n = 3. Lines are fits to models of one-site binding with variable Hill coefficient or linear regression. ** p <0.01 (t-test), compared to cells incubated with PTX3-647 alone.

Expression of PTX3 in lung cells, murine models of fibrosis, and pulmonary fibrosis patients

Fibrocytes are found in pulmonary fibrosis lesions, and PTX3 accumulation is associated with fibrotic lesions in asthma and myocardial infarction [3,5860]. In addition, PTX3 is expressed by dermal fibroblasts and renal epithelial cells and upregulated by TNF-α [6163]. We observed that human lung fibroblasts and bronchial epithelial cells also produce PTX3, and as with the dermal fibroblasts, TNF-α upregulated PTX3 in lung fibroblasts (Fig. 8). We did not detect the expression of SAP by human lung fibroblasts and epithelial cells (Fig. 8). These data indicate that the local production of PTX3 by fibroblasts and epithelial cells may augment the differentiation of monocytes into fibrocytes in fibrotic lesions.

Fig 8. Human lung fibroblasts and epithelial cells express PTX3.

Human A-C) lung fibroblasts, D-F) lung fibroblasts incubated with 50 ng/ml TNF-α, or G-I) lung epithelial cells were cultured for 2 days in 8-well glass slides. Cells were then air-dried, fixed, and labeled with A, D, and G) anti-PTX3 antibodies, B, E, and H) anti-SAP antibodies, C and F) anti-collagen I antibodies, or I) anti-EpCAM antibodies. Positive staining was identified by red staining, and nuclei are counterstained blue. Bar is 100 μm.

To determine if PTX3 was also upregulated in pulmonary fibrosis, we stained lung tissue from mice that aspirated bleomycin or saline. At 21 days after bleomycin aspiration, compared to mice that received saline, mice that aspirated bleomycin had more PTX3 staining, and this was associated with areas of increased collagen-I staining (Fig. 9). These data suggest that bleomycin may lead to the increased local production of PTX3 and this may promote increased fibrocyte differentiation.

Fig 9. Distribution of PTX3 in mouse lungs following bleomycin aspiration.

Cryosections of mouse lungs 21 days after A-C) saline or D-F) bleomycin aspiration were incubated with antibodies against A and D) PTX3, B and E) collagen-I, or C and F) control rabbit antibodies Sections were counterstained with hematoxylin. Bar is 0.2 mm. Images are representative of 3 independent experiments.

To determine if PTX3 is associated with human lung fibrosis, we examined the distribution of PTX3 in lung tissue from chronic obstructive pulmonary disease (COPD) patients with relatively normal lungs (> 80% FVC) and idiopathic pulmonary fibrosis (IPF) patients (<50% FVC) (Table 1) (Fig. 10). Lung tissue from COPD patients showed a widespread distribution of PTX3, especially in the alveolar septa (Fig. 10B). In the lung tissue from pulmonary fibrosis patients, PTX3 was distributed throughout the tissue, including the lung epithelium, alveolar leukocytes, and fibrotic areas, but PTX3 distribution was reduced within fibroblastic foci (Fig. 10D-H). As with PTX3, there was a widespread distribution of SAP throughout the lung tissue from COPD patients (Fig. 10C). However, in fibrotic lesions from pulmonary fibrosis patients, SAP was restricted to areas adjacent to vessels, and apparently absent from the fibrotic areas (Fig. 10F). However, when we quantified the staining levels for the expression of PTX3 to the total lung tissue area, there were no significant differences between COPD and IPF patients (Fig. 11). These data suggest that the expression of PTX3 by the lung tissue is not due to increased expression of PTX3 by cells, but that the increased number of cells leads to increased PTX3 expression. These data also indicate that PTX3 and SAP are present throughout lung tissue with a relatively normal architecture, while PTX3 and a relatively small amount of SAP are present in fibrotic lesions.

Fig 10. The distribution of PTX3 and SAP in COPD and pulmonary fibrosis lung tissue.

Human lung tissue from A-C) COPD patients or D-H) pulmonary fibrosis patients was stained with A) control rabbit antibodies, B, D, E, G and H) anti-PTX3 or C and F) anti-SAP antibodies. Tissues were counterstained with hematoxylin. Positive staining is identified by red color, and nuclei are counterstained blue. Bar is 0.2 mm. Long arrow indicate PTX3 staining in an alveolar septum, short arrow indicates PTX3 staining in lung epithelium, and asterisks indicate fibroblastic foci.

Fig 11. Quantification of PTX3 staining in COPD and pulmonary fibrosis lung tissue.

A) The percentage of total area of image containing lung tissue, B) the percentage area of lung tissue stained by PTX3 antibodies, and C) the percentage of lung tissue stained by PTX3 antibodies as a percentage of the total area of the lung. Values are mean ± SEM, n = 7–9 patients per group. *** indicates p < 0.0001 (t-test).


We found that PTX3 promotes fibrocyte differentiation by a FcγRI dependent mechanism. However, the fibrocyte-inhibitory activity of SAP is dominant over PTX3. In fibrotic lung tissue, we found that the distribution of PTX3 was widespread, and present in alveolar macrophages, lung epithelial cells, and fibroblasts. However, SAP had a restricted distribution and was apparently absent from the fibrotic areas. These data suggest that the relative levels of SAP and PTX3 present at sites of fibrosis may have a significant effect on the ability of monocytes to differentiate into fibrocytes.

Fibrocytes have been detected in human pathological conditions including pulmonary fibrosis [3,10,6468], keloid scars [69], asthma [64], chronic kidney disease [11], and nephrogenic systemic fibrosis [70]. Fibrocytes are also present in the fibrotic lesions in animal models of pulmonary fibrosis [7,9,65,7177], liver fibrosis [9] and renal fibrosis [76,78]. In addition to contributing to the mass of fibrotic lesions, fibrocytes promote angiogenesis [79,80], which can then promote the growth of the lesion, and secrete TGF-β [81], which activates resident fibroblasts. Therefore, in situations where PTX3 is abundant and therefore available to promote fibrocyte differentiation, this may also regulate angiogenesis. Finally, the injection of mature fibrocytes into mice potentiate lung fibrosis, suggesting that the increased recruitment of monocyte-derived fibrocytes may potentiate an ongoing fibrotic response [72]. Whether the main role of fibrocytes is to directly drive fibrosis, through the production of extracellular matrix proteins, or to act in a paracrine manner to activate stromal cells (fibroblasts) to produce more extracellular matrix proteins, or regulate angiogenesis, is still unclear [3,80,82].

Compared to human lung tissue, the lungs of saline-treated mice expressed little PTX3. Whether this is a difference in PTX3 expression between human and murine lungs, or due to elevated PTX3 expression even in COPD patients compared to healthy controls, is unclear. In both LPS and ventilator-induced lung injury models in rats, PTX3 was detectable in sham/control lung samples [83,84], but in a smoke inhalation model in mice PTX3 was not expressed by lung cells in control animals [85]. In humans, some studies have detected PTX3 in normal lung tissue [86], whereas in samples from invasive pulmonary aspergillosis, only alveolar macrophages and not the lung tissue expressed PTX3 [87]. These data suggest that not only the severity and length of the insult may regulate the expression of PTX3 by lung cells, but that the expression of PTX3 in lung cells may also vary between species [88].

Although the concentration of PTX3 found to promote fibrocyte differentiation is higher than detected in circulating plasma, the concentration used is 10 fold lower than used in most other in vitro systems [8992]. In addition, the plasma concentration of PTX3 is unlikely to represent the concentration present in the tissues, especially during an immune response. Although it is difficult to determine the actual concentration of PTX3 in tissues, using data from cells cultured in vitro may provide information to permit an approximation for the levels of PTX3 in tissues. Many cells present at sites of inflammation or fibrosis, including endothelial cells, fibroblasts, neutrophils, and macrophages secrete PTX3, with levels ranging from 20–100 ng per 106 cells [17,9395]. In murine models of lung inflammation and fibrosis, there can be 10 x 106 leukocytes present in the vascular, interstitial, and alveolar compartments of the lung [96,97]. As a model for lung fibrosis, bleomycin instillation into the lung generates patchy inflammation and fibrosis with typically 10–20% of the lung tissue affected [98,99]. Therefore, if we assume that the majority of the infiltrating cells associate with sites of tissue injury [99], this would suggest that approximately 10 x 106 cells would be confined to specific areas of lung tissue. As 106 cells secrete 50–100 ng PTX3, and 106 cells occupy approximately 100 mm3 (100 mm3 is 100 μl) this suggests that the local concentration of PTX3 within tissues could reach 1 μg/ml, and be even higher within the lung interstitium.

Surface plasmon resonance experiments show that SAP and CRP, bind to all of the human FcγR, whereas PTX3 only binds to human FcγRIII and weakly to human FcγRIIA [22,34]. However, we observed that PTX3 binds to FcγRI and FcγRIIA on leukocytes, K562 cells, and transfected HEK293 cells. This inconsistency with the previously published data may be explained by the differences in the glycosylation state of the receptors and/or the lack of some intracellular signaling components that promote receptor binding [37]. As FcγRI and FcγRIIIA lack an intrinsic motif that binds to intracellular signaling components, they interact with the intracellular protein FcRγ. The absence of FcRγ reduces the affinity of FcγRI and FcγRIIIA for IgG in humans [37,100]. This can potentially alter PTX3 binding to FcγRI and FcγRIIA. Together, this suggests that the PTX3 affinity for FcγRs is dependent on the modification of these FcγRs and the interactions they make before binding PTX3.

There are several possible explanations for the observation that SAP appears to signal through FcγRI on monocytes to inhibit fibrocyte differentiation, while PTX3 appears to signal through the same receptor on the same cells to promote fibrocyte differentiation [32]. At first glance, it would appear that SAP might be an agonist, and PTX3 an inverse agonist (or vice versa) of FcγRI, but the observation that in serum-free medium, mouse cells lacking FcγRI show only slightly reduced levels of fibrocyte differentiation, suggests that there is little constitutive signaling from FcγRI with respect to promoting or inhibiting fibrocyte differentiation [32,51].

In humans, the main activating FcγR on monocytes are FcγRI, FcγRIIA, and FcγRIIIA, whereas in mice the main activating FcγR on monocytes are FcγRI, FcγRIII, and FcγRIV [38,101]. Human FcγRI is orthologous to mouse FcγRI, human FcγRIIA is most closely related to mouse FcγRIII, and human FcγRIIIA is most closely related to mouse FcγRIV [38,102]. We found that the FcγRI knockout and FcγRI/IIb/III/IV quadruple knockout mice spleen cells were insensitive to PTX3. We did not have access to the single FcγRIV knockout mouse spleen cells, but the PTX3 effect on human CD14+CD16- (FcγRIIIA-) monocytes and the lack of PTX3 binding to FcγRIII on leukocytes and HEK293 cells, suggests that human FcγRIIIA or murine FcγRIV are not essential for PTX3 activity. Combined with our previous observation that SAP inhibits human and murine fibrocyte differentiation mainly through FcγRI, these results suggest that FcγRI plays a major role in regulating fibrocyte differentiation [32,41].

Fc receptors can differentially activate signaling cascades depending on the affinity or avidity of their ligands [103,104]. Therefore, the differential effect of SAP and PTX3 on fibrocyte differentiation may be due to the pentameric structure of SAP, compared to the decameric structure of PTX3, or the affinity of these pentraxins for FcγR [5,34,105,106]. FcγRI may thus exhibit functional selectivity/biased agonism, with SAP preferentially activating one downstream pathway, and PTX3 activating a different pathway [107109]. We have previously observed that although SAP and aggregated IgG both inhibit fibrocyte differentiation, aggregated IgG inhibits fibrocyte differentiation through a pathway involving Syk (a non-receptor cytoplasmic tyrosine kinase) but that SAP appears to signal through a Syk-independent pathway [41]. A second possibility is that a different receptor modulates the signal from FcγRI. Mouse cells lacking FcγRI, lacking the common FcRγ chain, or lacking all four FcγR receptors still show inhibition of fibrocyte differentiation by SAP, albeit with an increased IC50 [32]. This then suggests that a different receptor binds SAP, and this receptor could thus modulate the signal from FcγRI so that SAP binding to FcγRI and the unknown receptor would inhibit fibrocyte differentiation, and PTX3, binding to FcγRI and not binding to the unknown receptor, or additionally binding to a third receptor, would increase fibrocyte differentiation. SAP and CRP, but not PTX3, also bind to the IgA receptor (CD89) [110]. However, we have previously shown that IgA does not regulate human fibrocyte differentiation [41], and in mice the IgA receptor is absent [111]. Although mice do not have CD89, they express Fcα/μR, a dual IgA/IgM receptor, which is independent of FcRγ [112], however we have previously shown that IgM does not regulate human fibrocyte differentiation [41]. Therefore, additional unknown receptor(s) may well be involved in the regulation of fibrocyte differentiation by pentraxins. Since fibrocytes play a key role in fibrosis, and FcγRI plays a key role in regulating fibrocyte differentiation, our results on the effects of SAP and PTX3 indicate that FcγRI signaling represents an interesting new therapeutic target.


This work was supported in part by U.S. National Institutes of Health (NIH) grants R01 HL083029 and R01 HL118507. We thank Jill Claassens at Leiden University Medical Center for the generous assistance with the Fc receptor knockout spleens, and Dr. Bryce Binstadt and Jennifer Auger at the University of Minnesota for additional spleens.

Author Contributions

Conceived and designed the experiments: DP NC JSV RHG. Performed the experiments: DP NC VV. Analyzed the data: DP NC VV RHG. Wrote the paper: DP NC JSV RHG.


  1. 1. Auffray C, Sieweke MH, Geissmann F (2009) Blood Monocytes: Development, Heterogeneity, and Relationship with Dendritic Cells. Annual Review of Immunology 27: 669–692. pmid:19132917
  2. 2. Bucala R, Spiegel LA, Chesney J, Hogan M, Cerami A (1994) Circulating fibrocytes define a new leukocyte subpopulation that mediates tissue repair. Molecular Medicine 1: 71–81. pmid:8790603
  3. 3. Reilkoff RA, Bucala R, Herzog EL (2011) Fibrocytes: emerging effector cells in chronic inflammation. Nat Rev Immunol 11: 427–435. pmid:21597472
  4. 4. Abe R, Donnelly SC, Peng T, Bucala R, Metz CN (2001) Peripheral blood fibrocytes: differentiation pathway and migration to wound sites. Journal of Immunology 166: 7556–7562. pmid:11390511
  5. 5. Pilling D, Buckley CD, Salmon M, Gomer RH (2003) Inhibition of fibrocyte differentiation by serum amyloid P. Journal Of Immunology 17: 5537–5546.
  6. 6. Pilling D, Fan T, Huang D, Kaul B, Gomer RH (2009) Identification of markers that distinguish monocyte-derived fibrocytes from monocytes, macrophages, and fibroblasts. PLOS ONE 4: e7475. pmid:19834619
  7. 7. Phillips RJ, Burdick MD, Hong K, Lutz MA, Murray LA, et al. (2004) Circulating fibrocytes traffic to the lungs in response to CXCL12 and mediate fibrosis. J ClinInvest 114: 438–446.
  8. 8. Haudek SB, Xia Y, Huebener P, Lee JM, Carlson S, et al. (2006) Bone Marrow-derived Fibroblast Precursors Mediate Ischemic Cardiomyopathy in Mice. Proceedings of the National Academy of Sciences 103: 18284–18289. pmid:17114286
  9. 9. Kisseleva T, Uchinami H, Feirt N, Quintana-Bustamante O, Segovia JC, et al. (2006) Bone marrow-derived fibrocytes participate in pathogenesis of liver fibrosis. J Hepatol 45: 429–438. pmid:16846660
  10. 10. Mehrad B, Burdick MD, Zisman DA, Keane MP, Belperio JA, et al. (2007) Circulating peripheral blood fibrocytes in human fibrotic interstitial lung disease. Biochem Biophys Res Commun 353: 104–108. pmid:17174272
  11. 11. Sakai N, Furuichi K, Shinozaki Y, Yamauchi H, Toyama T, et al. (2010) Fibrocytes are involved in the pathogenesis of human chronic kidney disease. Human pathology 41: 672–678. pmid:20040395
  12. 12. Shao DD, Suresh R, Vakil V, Gomer RH, Pilling D (2008) Pivotal Advance: Th-1 cytokines inhibit, and Th-2 cytokines promote fibrocyte differentiation. Journal of Leukocyte Biology 83: 1323–1333. pmid:18332234
  13. 13. Maharjan AS, Pilling D, Gomer RH (2011) High and Low Molecular Weight Hyaluronic Acid Differentially Regulate Human Fibrocyte Differentiation. PLOS One 6: e26078. pmid:22022512
  14. 14. Lu J, Marjon KD, Mold C, Du Clos TW, Sun PD (2012) Pentraxins and Fc receptors. Immunological Reviews 250: 230–238. pmid:23046133
  15. 15. Mantovani A, Biswas SK, Galdiero MR, Sica A, Locati M (2013) Macrophage plasticity and polarization in tissue repair and remodelling. The Journal of Pathology 229: 176–185. pmid:23096265
  16. 16. Deban L, Jaillon S, Garlanda C, Bottazzi B, Mantovani A (2011) Pentraxins in innate immunity: lessons from PTX3. Cell Tissue Res 343: 237–249. pmid:20683616
  17. 17. Bottazzi B, Doni A, Garlanda C, Mantovani A (2010) An Integrated View of Humoral Innate Immunity: Pentraxins as a Paradigm. Annual Review of Immunology 28: 157–183. pmid:19968561
  18. 18. Wagenaar JF, Goris MG, Gasem MH, Isbandrio B, Moalli F, et al. (2009) Long pentraxin PTX3 is associated with mortality and disease severity in severe Leptospirosis. J Infect 58: 425–432. pmid:19443038
  19. 19. Sprong T, Peri G, Neeleman C, Mantovani A, Signorini S, et al. (2009) Pentraxin 3 and C-reactive protein in severe meningococcal disease. Shock 31: 28–32. pmid:18650775
  20. 20. Hawkins PN, Tennent GA, Woo P, Pepys MB (1991) Studies in vivo and in vitro of serum amyloid P component in normals and in a patient with AA amyloidosis. ClinExpImmunol 84: 308–316.
  21. 21. Pilling D, Roife D, Wang M, Ronkainen SD, Crawford JR, et al. (2007) Reduction of bleomycin-induced pulmonary fibrosis by serum amyloid P. The Journal of Immunology 179: 4035–4044. pmid:17785842
  22. 22. Castano AP, Lin SL, Surowy T, Nowlin BT, Turlapati SA, et al. (2009) Serum amyloid P inhibits fibrosis through Fc gamma R-dependent monocyte-macrophage regulation in vivo. Science translational medicine 1: 5ra13. pmid:20368175
  23. 23. Maharjan AS, Roife D, Brazill D, Gomer RH (2013) Serum amyloid P inhibits granulocyte adhesion. Fibrogenesis Tissue Repair 6: 2. pmid:23324174
  24. 24. Murray LA, Rosada R, Moreira AP, Joshi A, Kramer MS, et al. (2010) Serum Amyloid P Therapeutically Attenuates Murine Bleomycin-Induced Pulmonary Fibrosis via Its Effects on Macrophages. PLOS ONE 5: e9683. pmid:20300636
  25. 25. Dillingh MR, van den Blink B, Moerland M, van Dongen MGJ, Levi M, et al. (2013) Recombinant human serum amyloid P in healthy volunteers and patients with pulmonary fibrosis. Pulmonary Pharmacology & Therapeutics 26: 672–676
  26. 26. Li ZI, Chung AC, Zhou L, Huang XR, Liu F, et al. (2011) C-reactive protein promotes acute renal inflammation and fibrosis in unilateral ureteral obstructive nephropathy in mice. Lab Invest 91: 837–851. pmid:21383672
  27. 27. Teoh H, Quan A, Lovren F, Wang G, Tirgari S, et al. (2008) Impaired endothelial function in C-reactive protein overexpressing mice. Atherosclerosis 201: 318–325. pmid:18433756
  28. 28. Liu F, Chen HY, Huang XR, Chung AC, Zhou L, et al. (2011) C-reactive protein promotes diabetic kidney disease in a mouse model of type 1 diabetes. Diabetologia 54: 2713–2723. pmid:21744073
  29. 29. Pegues MA, McCrory MA, Zarjou A, Szalai AJ (2013) C-reactive protein exacerbates renal ischemia-reperfusion injury. American Journal of Physiology—Renal Physiology 304: F1358–F1365.
  30. 30. Mantovani A, Valentino S, Gentile S, Inforzato A, Bottazzi B, et al. (2013) The long pentraxin PTX3: a paradigm for humoral pattern recognition molecules. Annals of the New York Academy of Sciences 1285: 1–14. pmid:23527487
  31. 31. Bonacina F, Baragetti A, Catapano AL, Norata GD (2013) Long Pentraxin 3: Experimental and Clinical Relevance in Cardiovascular Diseases. Mediators of Inflammation 2013: 10.
  32. 32. Crawford JR, Pilling D, Gomer RH (2012) FcγRI mediates serum amyloid P inhibition of fibrocyte differentiation. Journal of Leukocyte Biology 92: 699–711. pmid:22493081
  33. 33. Haudek SB, Trial J, Xia Y, Gupta D, Pilling D, et al. (2008) Fc Receptor Engagement Mediates Differentiation of Cardiac Fibroblast Precursor Cells. Proceedings of the National Academy of Sciences 105: 10179–10184 pmid:18632582
  34. 34. Lu J, Marnell LL, Marjon KD, Mold C, Du Clos TW, et al. (2008) Structural recognition and functional activation of FcgR by innate pentraxins. Nature 456: 989–992. pmid:19011614
  35. 35. Bharadwaj D, Mold C, Markham E, Du Clos TW (2001) Serum amyloid P component binds to Fc gamma receptors and opsonizes particles for phagocytosis. JImmunol 166: 6735–6741. pmid:11359830
  36. 36. Nimmerjahn F, Ravetch JV (2008) Fcgamma receptors as regulators of immune responses. Nat Rev Immunol 8: 34–47. pmid:18064051
  37. 37. Bruhns P, Iannascoli B, England P, Mancardi DA, Fernandez N, et al. (2009) Specificity and affinity of human Fcgamma receptors and their polymorphic variants for human IgG subclasses. Blood 113: 3716–3725. pmid:19018092
  38. 38. Bruhns P (2012) Properties of mouse and human IgG receptors and their contribution to disease models. Blood 119: 5640–5649. pmid:22535666
  39. 39. Nimmerjahn F, Ravetch JV (2006) Fcgamma receptors: old friends and new family members. Immunity 24: 19–28. pmid:16413920
  40. 40. Ravetch JV, Lanier LL (2000) Immune inhibitory receptors. Science 290: 84–89. pmid:11021804
  41. 41. Pilling D, Tucker NM, Gomer RH (2006) Aggregated IgG inhibits the differentiation of human fibrocytes. Journal Of Leukocyte Biology 79: 1242–1251. pmid:16543402
  42. 42. Pilling D, Vakil V, Gomer RH (2009) Improved serum-free culture conditions for the differentiation of human and murine fibrocytes. Journal of Immunological Methods 351: 62–70. pmid:19818792
  43. 43. Maharjan AS, Pilling D, Gomer RH (2010) Toll-like receptor 2 agonists inhibit human fibrocyte differentiation. Fibrogenesis Tissue Repair 3: 23. pmid:21106092
  44. 44. White MJV, Glenn M, Gomer RH (2013) Trypsin Potentiates Human Fibrocyte Differentiation. PLOS ONE 8: e70795. pmid:23951012
  45. 45. Cox N, Pilling D, Gomer RH (2014) Distinct Fcγ Receptors Mediate the Effect of Serum Amyloid P on Neutrophil Adhesion and Fibrocyte Differentiation. The Journal of Immunology 193: 1701–1708. pmid:25024390
  46. 46. Ioan-Facsinay A, de Kimpe SJ, Hellwig SM, van Lent PL, Hofhuis FM, et al. (2002) FcgammaRI (CD64) contributes substantially to severity of arthritis, hypersensitivity responses, and protection from bacterial infection. Immunity 16: 391–402. pmid:11911824
  47. 47. Hazenbos WL, Gessner JE, Hofhuis FM, Kuipers H, Meyer D, et al. (1996) Impaired IgG-dependent anaphylaxis and Arthus reaction in Fc gamma RIII (CD16) deficient mice. Immunity 5: 181–188. pmid:8769481
  48. 48. Boross P, Arandhara VL, Martin-Ramirez J, Santiago-Raber M-L, Carlucci F, et al. (2011) The Inhibiting Fc Receptor for IgG, FcγRIIB, Is a Modifier of Autoimmune Susceptibility. The Journal of Immunology 187: 1304–1313. pmid:21724994
  49. 49. Crawford JR, Pilling D, Gomer RH (2010) Improved serum-free culture conditions for spleen-derived murine fibrocytes. Journal Of Immunological Methods 363: 9–20. pmid:20888336
  50. 50. Pilling D, Gomer RH (2014) Persistent Lung Inflammation and Fibrosis in Serum Amyloid P Component (Apcs-/-) Knockout Mice. PLOS ONE 9: e93730. pmid:24695531
  51. 51. Pilling D, Crawford JR, Verbeek JS, Gomer RH (2014) Inhibition of murine fibrocyte differentiation by cross-linked IgG is dependent on FcγRI. Journal of Leukocyte Biology 96.
  52. 52. Herlihy SE, Pilling D, Maharjan AS, Gomer RH (2013) Dipeptidyl Peptidase IV Is a Human and Murine Neutrophil Chemorepellent. The Journal of Immunology 190: 6468–6477. pmid:23677473
  53. 53. Seiler CY, Park JG, Sharma A, Hunter P, Surapaneni P, et al. (2014) DNASU plasmid and PSI:Biology-Materials repositories: resources to accelerate biological research. Nucleic Acids Res 42: D1253–1260. pmid:24225319
  54. 54. Boonnak K, Slike BM, Donofrio GC, Marovich MA (2013) Human FcγRII Cytoplasmic Domains Differentially Influence Antibody-Mediated Dengue Virus Infection. The Journal of Immunology 190: 5659–5665. pmid:23616574
  55. 55. Moalli F, Doni A, Deban L, Zelante T, Zagarella S, et al. (2010) Role of complement and Fc{gamma} receptors in the protective activity of the long pentraxin PTX3 against Aspergillus fumigatus. Blood 116: 5170–5180. pmid:20829368
  56. 56. Cox N, Pilling D, Gomer RH (2012) NaCl Potentiates Human Fibrocyte Differentiation. PLOS One 7: e45674. pmid:23029177
  57. 57. Bianchetti L, Barczyk M, Cardoso J, Schmidt M, Bellini A, et al. (2012) Extracellular matrix remodelling properties of human fibrocytes. Journal of Cellular and Molecular Medicine 16: 483–495. pmid:21595824
  58. 58. Nebuloni M, Pasqualini F, Zerbi P, Lauri E, Mantovani A, et al. (2011) PTX3 expression in the heart tissues of patients with myocardial infarction and infectious myocarditis. Cardiovasc Pathol 20: e27–35. pmid:20356766
  59. 59. Zhang J, Shan L, Koussih L, Redhu NS, Halayko AJ, et al. (2012) Pentraxin 3 (PTX3) Expression in Allergic Asthmatic Airways: Role in Airway Smooth Muscle Migration and Chemokine Production. PLOS ONE 7: e34965. pmid:22529962
  60. 60. Balhara J, Koussih L, Zhang J, Gounni AS (2013) Pentraxin 3: An Immuno-regulator in the Lungs. Frontiers in Immunology 4.
  61. 61. Nauta AJ, de Haij S, Bottazzi B, Mantovani A, Borrias MC, et al. (2005) Human renal epithelial cells produce the long pentraxin PTX3. Kidney Int 67: 543–553. pmid:15673302
  62. 62. Introna M, Alles VV, Castellano M, Picardi G, De Gioia L, et al. (1996) Cloning of mouse ptx3, a new member of the pentraxin gene family expressed at extrahepatic sites. Blood 87: 1862–1872. pmid:8634434
  63. 63. Lee GW, Lee TH, Vilcek J (1993) TSG-14, a tumor necrosis factor- and IL-1-inducible protein, is a novel member of the pentaxin family of acute phase proteins. The Journal of Immunology 150: 1804–1812. pmid:7679696
  64. 64. Schmidt M, Sun G, Stacey MA, Mori L, Mattoli S (2003) Identification of Circulating Fibrocytes as Precursors of Bronchial Myofibroblasts in Asthma. The Journal of Immunology 171: 380–389. pmid:12817021
  65. 65. Quan TE, Cowper S, Wu SP, Bockenstedt LK, Bucala R (2004) Circulating fibrocytes: collagen-secreting cells of the peripheral blood. The International Journal of Biochemistry & Cell Biology 36: 598–606.
  66. 66. Yang L, Scott PG, Dodd C, Medina A, Jiao H, et al. (2005) Identification of fibrocytes in postburn hypertrophic scar. Wound Repair and Regeneration 13: 398–404. pmid:16008729
  67. 67. Mori L, Bellini A, Stacey MA, Schmidt M, Mattoli S (2005) Fibrocytes contribute to the myofibroblast population in wounded skin and originate from the bone marrow. Experimental Cell Research 304: 81–90. pmid:15707576
  68. 68. Andersson-Sjöland A, de Alba CG, Nihlberg K, Becerril C, Ramírez R, et al. (2008) Fibrocytes are a potential source of lung fibroblasts in idiopathic pulmonary fibrosis. The International Journal of Biochemistry & Cell Biology 40: 2129–2140.
  69. 69. Aiba S, Tagami H (1997) Inverse correlation between CD34 expression and proline-4-hydroxylase immunoreactivity on spindle cells noted in hypertrophic scars and keloids. J Cutan Pathol 24: 65–69. pmid:9162737
  70. 70. Cowper SE (2003) Nephrogenic fibrosing dermopathy: the first 6 years. CurrOpinRheumatol 15: 785–790.
  71. 71. Hashimoto N, Jin H, Liu T, Chensue SW, Phan SH (2004) Bone marrow-derived progenitor cells in pulmonary fibrosis. Journal Of Clinical Investigation 113: 243–252. pmid:14722616
  72. 72. Moore BB, Murray L, Das A, Wilke CA, Herrygers AB, et al. (2006) The Role of CCL12 in the Recruitment of Fibrocytes and Lung Fibrosis. American Journal Of Respiratory Cell And Molecular Biology 35: 175–181. pmid:16543609
  73. 73. Direkze NC, Forbes SJ, Brittan M, Hunt T, Jeffery R, et al. (2003) Multiple Organ Engraftment by Bone-Marrow-Derived Myofibroblasts and Fibroblasts in Bone-Marrow-Transplanted Mice. Stem Cells 21: 514–520. pmid:12968105
  74. 74. Epperly MW, Guo H, Gretton JE, Greenberger JS (2003) Bone Marrow Origin of Myofibroblasts in Irradiation Pulmonary Fibrosis. American Journal Of Respiratory Cell And Molecular Biology 29: 213–224. pmid:12649121
  75. 75. Varcoe RL, Mikhail M, Guiffre AK, Pennings G, Vicaretti M, et al. (2006) The role of the fibrocyte in intimal hyperplasia. J ThrombHaemost 4: 1125–1133. pmid:16689767
  76. 76. Sakai N, Wada T, Yokoyama H, Lipp M, Ueha S, et al. (2006) Secondary lymphoid tissue chemokine (SLC/CCL21)/CCR7 signaling regulates fibrocytes in renal fibrosis. Proceedings of the National Academy of Sciences 103: 14098–14103. pmid:16966615
  77. 77. Santhiago MR, Singh V, Barbosa FL, Agrawal V, Wilson SE (2011) Monocyte development inhibitor PRM-151 decreases corneal myofibroblast generation in rabbits. Experimental Eye Research 93: 810–817. pmid:21978952
  78. 78. Sakai N, Wada T, Matsushima K, Bucala R, Iwai M, et al. (2008) The renin-angiotensin system contributes to renal fibrosis through regulation of fibrocytes. Journal of Hypertension 26: 780–790 710.1097/HJH.1090b1013e3282f1093e1099e1096. pmid:18327089
  79. 79. Hartlapp I, Abe R, Saeed RW, Peng T, Voelter W, et al. (2001) Fibrocytes induce an angiogenic phenotype in cultured endothelial cells and promote angiogenesis in vivo. The FASEB Journal 15: 2215–2224. pmid:11641248
  80. 80. Smadja DM, Dorfmüller P, Guerin CL, Bieche I, Badoual C, et al. (2014) Cooperation between human fibrocytes and endothelial colony-forming cells increases angiogenesis via the CXCR4 pathway. Thrombosis and Haemostasis 112: 1002–1013. pmid:25103869
  81. 81. Wang JF, Jiao H, Stewart TL, Shankowsky HA, Scott PG, et al. (2007) Fibrocytes from burn patients regulate the activities of fibroblasts. Wound Repair Regen 15: 113–121. pmid:17244327
  82. 82. Kleaveland KR, Moore BB, Kim KK (2014) Paracrine functions of fibrocytes to promote lung fibrosis. Expert Review of Respiratory Medicine 8: 163–172. pmid:24451025
  83. 83. Han B, Mura M, Andrade CF, Okutani D, Lodyga M, et al. (2005) TNFα-Induced Long Pentraxin PTX3 Expression in Human Lung Epithelial Cells via JNK. The Journal of Immunology 175: 8303–8311. pmid:16339571
  84. 84. Okutani D, Han B, Mura M, Waddell TK, Keshavjee S, et al. (2007) High-volume ventilation induces pentraxin 3 expression in multiple acute lung injury models in rats. American Journal of Physiology—Lung Cellular and Molecular Physiology 292: L144–L153.
  85. 85. Pauwels NS, Bracke KR, Maes T, Van Pottelberge GR, Garlanda C, et al. (2010) Cigarette smoke induces PTX3 expression in pulmonary veins of mice in an IL-1 dependent manner. Respir Res 11: 134. pmid:20920344
  86. 86. Jaillon S, Mancuso G, Hamon Y, Beauvillain C, Cotici V, et al. (2013) Prototypic Long Pentraxin PTX3 Is Present in Breast Milk, Spreads in Tissues, and Protects Neonate Mice from Pseudomonas aeruginosa Lung Infection. The Journal of Immunology 191: 1873–1882. pmid:23863905
  87. 87. Garlanda C, Hirsch E, Bozza S, Salustri A, De Acetis M, et al. (2002) Non-redundant role of the long pentraxin PTX3 in anti-fungal innate immune response. Nature 420: 182–186. pmid:12432394
  88. 88. He X, Han B, Liu M (2007) Long pentraxin 3 in pulmonary infection and acute lung injury. American Journal of Physiology—Lung Cellular and Molecular Physiology 292: L1039–L1049.
  89. 89. van Rossum AP, Fazzini F, Limburg PC, Manfredi AA, Rovere-Querini P, et al. (2004) The prototypic tissue pentraxin PTX3, in contrast to the short pentraxin serum amyloid P, inhibits phagocytosis of late apoptotic neutrophils by macrophages. Arthritis Rheum 50: 2667–2674. pmid:15334483
  90. 90. Job ER, Bottazzi B, Gilbertson B, Edenborough KM, Brown LE, et al. (2013) Serum Amyloid P Is a Sialylated Glycoprotein Inhibitor of Influenza A Viruses. PLOS ONE 8: e59623. pmid:23544079
  91. 91. Ma YJ, Doni A, Skjoedt MO, Honore C, Arendrup M, et al. (2011) Heterocomplexes of mannose-binding lectin and the pentraxins PTX3 or serum amyloid P component trigger cross-activation of the complement system. J Biol Chem 286: 3405–3417. pmid:21106539
  92. 92. Daigo K, Nakakido M, Ohashi R, Fukuda R, Matsubara K, et al. (2014) Protective effect of the long pentraxin PTX3 against histone-mediated endothelial cell cytotoxicity in sepsis. ra88-ra88 p.
  93. 93. Jaillon S, Peri G, Delneste Y, Fremaux I, Doni A, et al. (2007) The humoral pattern recognition receptor PTX3 is stored in neutrophil granules and localizes in extracellular traps. J Exp Med 204: 793–804. pmid:17389238
  94. 94. Doni A, Mantovani G, Porta C, Tuckermann J, Reichardt HM, et al. (2008) Cell-specific Regulation of PTX3 by Glucocorticoid Hormones in Hematopoietic and Nonhematopoietic Cells. Journal of Biological Chemistry 283: 29983–29992. pmid:18703503
  95. 95. Doni A, Michela M, Bottazzi B, Peri G, Valentino S, et al. (2006) Regulation of PTX3, a key component of humoral innate immunity in human dendritic cells: stimulation by IL-10 and inhibition by IFN-gamma. J Leukoc Biol 79: 797–802. pmid:16461742
  96. 96. Banerjee E, Henderson W (2012) Characterization of lung stem cell niches in a mouse model of bleomycin-induced fibrosis. Stem Cell Research & Therapy 3: 21.
  97. 97. Misharin AV, Morales-Nebreda L, Mutlu GM, Budinger GRS, Perlman H (2013) Flow Cytometric Analysis of Macrophages and Dendritic Cell Subsets in the Mouse Lung. American Journal of Respiratory Cell and Molecular Biology 49: 503–510. pmid:23672262
  98. 98. Degryse AL, Tanjore H, Xu XC, Polosukhin VV, Jones BR, et al. (2010) Repetitive intratracheal bleomycin models several features of idiopathic pulmonary fibrosis. L442-L452 p.
  99. 99. Moore BB, Lawson WE, Oury TD, Sisson TH, Raghavendran K, et al. (2013) Animal Models of Fibrotic Lung Disease. American Journal of Respiratory Cell and Molecular Biology 49: 167–179. pmid:23526222
  100. 100. van Vugt MJ, Heijnen AF, Capel PJ, Park SY, Ra C, et al. (1996) FcR gamma-chain is essential for both surface expression and function of human Fc gamma RI (CD64) in vivo. Blood 87: 3593–3599. pmid:8611682
  101. 101. Boross P, Verbeek JS (2006) The complex role of Fcgamma receptors in the pathology of arthritis. Springer Semin Immunopathol 28: 339–350. pmid:17043867
  102. 102. Hughes AL (1996) Gene duplication and recombination in the evolution of mammalian Fc receptors. Journal of molecular evolution 43: 4–10. pmid:8660423
  103. 103. Daeron M (1997) Fc Receptor Biology. Annual Review Of Immunology 15: 203–234. pmid:9143687
  104. 104. Kitaura J, Xiao W, Maeda-Yamamoto M, Kawakami Y, Lowell CA, et al. (2004) Early Divergence of Fcε Receptor I Signals for Receptor Up-Regulation and Internalization from Degranulation, Cytokine Production, and Survival. The Journal of Immunology 173: 4317–4323. pmid:15383560
  105. 105. Hutchinson WL, Hohenester E, Pepys MB (2000) Human serum amyloid P component is a single uncomplexed pentamer in whole serum. MolMed 6: 482–493. pmid:10972085
  106. 106. Bottazzi B, Vouret-Craviari V, Bastone A, De Gioia L, Matteucci C, et al. (1997) Multimer formation and ligand recognition by the long pentraxin PTX3. Similarities and differences with the short pentraxins C-reactive protein and serum amyloid P component. Journal of Biological Chemistry 272: 32817–32823. pmid:9407058
  107. 107. Kenakin T (2011) Functional Selectivity and Biased Receptor Signaling. Journal of Pharmacology and Experimental Therapeutics 336: 296–302. pmid:21030484
  108. 108. Verzijl D, Ijzerman A (2011) Functional selectivity of adenosine receptor ligands. Purinergic Signalling 7: 171–192. pmid:21544511
  109. 109. Rajagopal S, Rajagopal K, Lefkowitz RJ (2010) Teaching old receptors new tricks: biasing seven-transmembrane receptors. Nat Rev Drug Discov 9: 373–386. pmid:20431569
  110. 110. Lu J, Marjon KD, Marnell LL, Wang R, Mold C, et al. (2011) Recognition and functional activation of the human IgA receptor (FcalphaRI) by C-reactive protein. Proc Natl Acad Sci U S A 108: 4974–4979. pmid:21383176
  111. 111. van Egmond M, van Vuuren AJ, Morton HC, van Spriel AB, Shen L, et al. (1999) Human immunoglobulin A receptor (FcalphaRI, CD89) function in transgenic mice requires both FcR gamma chain and CR3 (CD11b/CD18). Blood 93: 4387–4394. pmid:10361137
  112. 112. Monteiro RC, van de Winkel JGJ (2003) IgA Fc Receptors. Annual Review Of Immunology 21: 177–204. pmid:12524384