Different molecular weight hyaluronic acid effects on human macrophage interleukin 1b production Larissa F. Baeva,1 Daniel B. Lyle,1 Maria Rios,2 John J. Langone,1 Marilyn M. Lightfoote1 1
Division of Biology, Office of Science and Engineering Laboratories, Center for Devices and Radiological Health, FDA, 10903 New Hampshire Ave, Silver Spring, Maryland 20993-002 2 Laboratory of Emerging Pathogens, Division of Emerging and Transfusion, Transmitted Diseases, Office of Blood Research and Review, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, Maryland 20892 Received 13 April 2012; revised 1 February 2013; accepted 19 February 2013 Published online 7 May 2013 in Wiley Online Library (wileyonlinelibrary.com). 10.1002/jbm.a.34704 ABSTRACT: This study examined the effect of hyaluronan (HA) molecular weight on immune response. HA with molecular weights ranging from the unitary disaccharide unit (400 Da) up to 1.7 3 106 Da and with very low endotoxin contamination level (less than 0.03 EU/mg) was used. Primary human monocyte/macrophage cultures were assayed for IL-1b production under a variety of inflammatory conditions with or without HA. Under the highest inflammatory states, production of interleukin 1b (IL-1b) was suppressed in the presence of high molecular weight hyaluronan (HMW-HA) and in the presence of low molecular weight hyaluronan (LMW-HA) at mg/mL concentrations. There was variability in the sensitivity of the response to HA fragments with MW below 5000 Da at micromolar
concentrations. There was variability in IL-1b cytokine productions from donor to donor in unstimulated human cell cultures. This study supplements our previous published study that investigated the immunogenic effect of HA molecular weights using murine cell line RAW264.6, rat splenocytes, and rat adherent differentiated primary macrophages. These data support the hypothesis that if the amount of endotoxin is reduced to an extremely low level, LMW-HA may not directly provoke normal tissue C 2013 Wiley macrophage-mediated inflammatory reactions. V Periodicals, Inc. J Biomed Mater Res Part A: 102A: 305–314, 2014.
Key Words: biomaterials, interleukin 1b, inflammation, macrophage, hyaluronic acid
How to cite this article: Baeva LF, Lyle DB, Rios M, Langone JJ, Lightfoote MM. 2014. Different molecular weight hyaluronic acid effects on human macrophage interleukin 1b production. J Biomed Mater Res Part A 2014:102A:305–314.
Hyaluronan (HA) has been studied for decades. Its main physiochemical characteristics were established in the 1930s. Native HA molecule has a molecular weight (MW) up to 107 Da. Naturally occurring fragmentation of the HA molecule led to the existence of the HA fragments of different MWs. Traditionally the scientists who are studying the hyaluronic acid divide the HA fragments by theirs MWs to three groups: high molecular weight hyaluronic acid (HMWHA), low molecular weight hyaluronic acid (LMW-HA), and HA oligomers or oligosaccharides. There are variations in using of the scientiﬁc terms grouping HA fragments by MWs. In the report we use HMW-HA for hyaluronic acid samples that are with MW from 132 up to 1700 kDa, LMWHA for HA samples that are from 4.77 up to 64 kDa, and HA oligomers for HA samples with MW below 5000 Da. As a result of the unique properties such as viscosity and elasticity, and wide distribution of HA among many
body tissues, hyaluronan is used in numerous areas of modern medicine. The ﬁrst formulation containing puriﬁed hyaluronic acid, Healon was approved as an ophthalmic viscosurgical device in 1980. Presently hyaluronan is used as a main component for a number of medical device applications, including treatments of osteoarthritis, ophthalmic (cataract treatment) surgery, cosmetic (eczema treatment, facial soft tissue ﬁllers), in pelvic and abdominal (postoperative adhesions) surgery. At the same time, a number of clinical reports of the adverse reaction to medical devices containing hyaluronan were published. The adverse reactions may occur in response to a procedural technique (depth and right place of injection), concentration of product used (excessive amount), protein and endotoxin contamination, and degradation of high molecular weight hyaluronan (HMW-HA) to mixture of fragments of different sizes.1–5 Reformulation and additional HA product puriﬁcation (decrease of proteins) has shown to reduce
*The hyaluronic acid products purchased from LifeCore Biomedical and Iduron are not necessarily identical to materials used in medical devices. Disclaimers: The mention of commercial products, their sources, or their use in connection with material reported herein is not to be construed as either an actual or implied endorsement of such products by the Department of Health and Human Services. The opinions and/or conclusions expressed are solely those of the authors and in no way imply a policy or position of the Food and Drug Administration. Correspondence to: L. Baeva; e-mail: [email protected]
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inﬂammatory adverse reactions.1,2 Published data support the conclusion that HMW-HA is mostly anti-inﬂammatory, whereas low molecular weight hyaluronan (LMW-HA) is inﬂammatory and works as an alarm signal to the immune system.4,6,7 We previously tested immunogenicity of panel hyaluronan samples with MW ranging from the unitary disaccharide up to 1.7 3 106 Da by quantifying the amount of nitric oxide (NO) produced by RAW 264.7, murine/macrophage cell line culture and fresh rat splenocytes, in the presence/ absence of HA. We tested LMW-HA from 4.77 up to 64 kDa and HMW-HA from 132 up to 1700 kDa. It was observed that under the highest inﬂammatory states, NO production was mildly suppressed by HMW-HA, whereas slightly augmented by LMW-HA at mg/mL concentrations. However, at micromolar concentrations (i.e. drug-like qualities) HA fragments with MW below 5000 Da, did not cause any effect.8 Our ﬁndings suggested that if endotoxin is reduced to an extremely low level, LMW-HA may not directly provoke normal tissue macrophage-mediated inﬂammatory reactions. In the present study we investigate whether similar HA effects will be observed using primary human monocytederived-macrophage (MDM). Results of this study suggest that while ongoing inﬂammatory reactions may be inﬂuenced by the MW of HA, HA formulations containing very low endotoxin contamination do not directly provoke MDM inﬂammation in in vitro system. MATERIALS AND METHODS
Reagents Lipopolysacharide (LPS) from Escherichia coli serotype O26:B6, human recombinant interferon gamma (hrIFN-g), and 2,3-diaminonaphthalene (DAN) were purchased from Sigma. Human recombinant macrophage colony stimulating factor (hrM-CSF) was purchased from Sigma. A series of HA samples with average MWs of 4770, 6550, 17,000, 35,000, 64,000, 132,000, 485,000, 1,100,000, and 1,700,000 Da and low endotoxin content (speciﬁcation at less than 0.07 EU/ mg) were purchased from LifeCore Biomedical. HA oligosaccharides (disaccharide [401 Da], hexamer [1221 Da], octamer [1607 Da], decamer [1993 Da], and dodecamer [2394 Da]) derived by digestion of Streptococcal fermentation products with mammalian hyaluronidase and certiﬁed having low in endotoxin level were purchased from Iduron. Stock solutions of the different reagents and samples were prepared as follows: HA samples were diluted in sterile RPMI 1640 (Sigma) at 10.0 (LifeCore samples) or 2.0 mg/ mL (Iduron samples) and sterilized by ﬁltration through 0.45-lm syringe-tip ﬁlter, then stored in aliquots at 220 C until use. Prior to testing, 10.0 mg/mL HA samples (LifeCore) were further diluted to 2.0 mg/mL and HA oligomers (Iduron) were diluted to 150 lg/mL. HA testing concentrations were chosen based on the results of previous studies.8 LPS was reconstituted with dimethyl sulfoxide (DMSO, Sigma) to a concentration of 250 mg/mL and frozen in aliquots at 2-70 C. hrIFN-g 10 mg/mL stock solution was prepared in 10 mL of sterile RPMI 1640 and aliquots were frozen at 220 C. To get working hrIFN-g concentration, the
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stock solution was diluted with sterile RPMI 1640 to 1 mg/ mL and aliquots were frozen at 220 C. hrM-CSF was reconstituted with 1.0 mL of sterile endotoxin-free water (Sigma) and 1.0 mL of sterile RPMI 1640 and aliquots were frozen at 5 lg/mL at 220 C. Stock solutions of DAN, at 20 mg/mL, were prepared by reconstituting 100 mg of DAN in 5 mL of DMSO, and aliquots were frozen at 220 C until further use. Human MDM culture Human peripheral blood monocytes isolated from blood donors who tested negative for all infectious agents commonly screened in blood were puriﬁed by leukapheresis countercurrent centrifugal elutriation and kindly provided by the National Institutes of Health (NIH) Blood Bank. All media and reagents were free of detectable endotoxin by Lonza Kinetic-QCL Chromogenic Limulus Amoebocyte Lysate Endotoxin Assay (Walkersville, MD). Viability of the cells was between 78% and 85%. Monocytes were seeded onto polystyrene tissue culture plates (96- or 24-well; Costar, Corning, NY 14831) at 5 3 105 to 1 3 106 cell per well and cultured in RPMI-16401GlutaMAX (GIBCO, Grand Island, NY) supplemented with 10% fetal calf serum, 2 mM glutamine, 100 U/mL penicillin, and 100 mg/mL streptomycin (CM). After 1 h (adherence step) in 5% CO2-incubator with 95% air, 100% humidity, at 37 C the cells were treated with 100 ng/mL of hrM-CSF for 7 days for the cell’s differentiation. Reagents were then added for up to 72 h, with supernatant samples removed at desired time point and frozen at 270 C for subsequent nitrite or cytokine assay. Adherence and differentiation of MDM was achieved as described previously.8 Test reagents were then added for up to 72 h, with supernatant samples removed at desired time points and frozen at 270 C for subsequent nitrite or cytokine assay. MDM prepared from frozen elutriated human monocytes Fresh elutriated human monocytes were packed by centrifugation at 800 rpm, at 14 C for 10 min and resuspended in straight fetal calf serum containing 10% of DMSO. Initially the cells were aliquoted to cryovials and transferred to a 270 C freezer for 2 days, and then to a liquid nitrogen vessel for a long-time storage. For this experiment deeply frozen human monocytes were thawed by routine method as recommended by Invitrogen Company for any type of mammalian cells. The cells were cultivated at approximately 5 3 105 cells/well in 24-well plates in complete RPMI1640 culture medium (CM) with or without hrM-CSF, 100 ng/mL for 6 days in 5% CO2/95% air, 100% humidity, at 37 C. At this time-point the cell’s conﬂuence was 80–85%. Then, the cells were exposed for 72 h to 2 mg/mL HA of MW ranging from the unitary disaccharide up to 1.7 3 106 Da plus or minus LPS, 5 lg/mL plus or minus hrIFN-g, 100 ng/mL. Assays for endotoxin and human interleukin 1b Endotoxin (LPS) was assayed according to kit instructions with the Lonza Kinetic-QCL Chromogenic Limulus
EFFECTS OF LMW1HA ON HUMAN MONOCYTES
FIGURE 1. Human Interleukin 1b (IL-1 b) production of human monocyte/macrophages stimulated by LPS, LPS 1/2 hrIFN-g, or hrIFN-g alone and effect of 4,770 Da HA on human monocyte/macrophages differentiated with hM-CSF. Human monocyte/macrophages were seeded at 5.0 3 106 cells/well, then differentiated with 100 ng/mL human recombinant macrophage colony stimulating factor (hM-CSF) and incubated in a 24well plate at 37 C for up to 72 h in the presence of medium only or 5 lg/mL LPS alone or 100 ng/mL hrIFN-g alone or 5 lg/mL LPS plus 100 ng/ mL hrIFN-g. Supernatants were removed at 72 h for IL-1 b ELISA assay. Data shown are mean 1/2 1 standard deviation of 4 replicate wells per condition. Statistical significance was calculated using the two-tailed unpaired Student’s t-test or ANOVA. A P < 0.05 was considered statistically significant.
Amoebocyte Lysate Endotoxin Assay (Walkersville, MD) with a sensitivity range of 0.005–50.0 EU/mL. Human interleukin 1b (IL-1b) ELISA kit was purchased from Invitrogen and used according to directions (Carlsbad, CA). Statistical analysis Data are expressed as mean61 standard deviation of n independent observations. Statistical signiﬁcance was calculated using the two-tailed unpaired Student’s t test or ANOVA. RESULTS
We previously tested a panel of HA samples with MW ranging from the unitary disaccharide of 400 Da up to 1.7 3 106 Da in culture of RAW264.7 murine/macrophage cell line and fresh neonate rat splenocytes, and found that if endotoxin is reduced to an extremely low level, LMW-HA may not directly provoke normal tissue macrophage-mediated inﬂammatory reactions.8 In this study we investigated whether these ﬁndings would hold true in human primary macrophage (MDM) cultures, using the same reagents sources and treatment regimen. Optimal cell density and hrM-CSF estimation Human elutriated monocytes were cultured in 3 3 96-well plates (the cells were seeded at 5 3 104, 1 3 105, 5 3 105/well) for 7 days and exposed to hrM-CSF of different concentrations, such as 0, 10, 50, 100 ng/mL. The best yield of differentiated human monocytes attached to well surface with 75–85% of conﬂuence was obtained at 5 3 105/well concentration of cells and treatment with hrM-CSF at 100 ng/mL (data not shown).
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IL-1b production by MDM prepared from frozen elutriated human monocytes and cultivated with inﬂammatory agents Cell-free supernatants were obtained and assayed via DAN ﬂuorescence for nitrite concentration9 and via IL-1b ELISA for IL-1b production. No detectable NO synthesis activity was found in supernatants of human macrophages (data not shown). The lack of detection by nitrite accumulation in vitro agrees with previously published data.10,11 The lack of detectable NO-production by MDM under the inﬂammatory conditions forced us to use IL-1b ELISA method. Figure 1 shows the amount of IL-1b produced by MDM in the absence (basal state) and in the presence of LPS (5 lg/mL) alone (high inﬂammatory state), hrIFN-g (100 ng/mL) alone and in combination of LPS and hrIFN-g (highest inﬂammatory state), with and without HA of 4770 Da MW. Data demonstrate that under experimental conditions the MDM exhibits a response to the inﬂammatory agents, LPS (5 lg/mL) alone, or hrIFN-g (100 ng/mL) alone. For the highest inﬂammatory condition of wells containing 5 lg/mL of LPS plus 100 ng/mL of hrIFN-g there was a signiﬁcant increase of IL-1b production. At the same time IL1b production of human monocytes under highest inﬂammatory condition (LPS plus hrIFN-g) was signiﬁcantly (25– 35%) suppressed by the presence of 2 mg/mL HA with MW 4770 Da. The data suggest that experimental conditions used allow detecting the difference in response between the cells in an unstimulated and inﬂammatory conditions and that the presence of HA signiﬁcantly decreases the cells inﬂammatory reaction.
FIGURE 2. Effect of HA of different molecular weights on IL-1 b production of human monocyte/macrophages 1/2 hrIFN-g. Human monocyte/ macrophages were seeded at 53106 cells/well and differentiated with hM-CSF in 24-well plate for 7 days at 37 C, then the cells were incubated in the presence or absence of 100 ng/mL hrIFN-g plus 2 mg/mL HA samples of different molecular weights, as compared to vehicle only control. Supernatants were removed at 72 h for IL-1 b assay. Data shown are mean 1/2 1 standard deviation of 4 replicate wells per condition. Statistical significance was calculated using the two-tailed unpaired Student’s t-test or ANOVA. A P < 0.05 was considered statistically significant.
The same experimental conditions were chosen to test MDM cell response in the presence of HA with MWs ranging from disaccharide up to 1.7 3 106 Da. Effect of HA of different MWs on IL-1b production by MDM in the presence of inﬂammatory agents Our previous data8 showed that culturing RAW264.7 cells or rat splenocytes in the presence of HA of different MWs from 4770 up to 1,700,000 Da resulted in mild suppression of NO production by HMW-HA and slight augmentation by LMW-HA at 2 mg/mL concentrations. In this work we extend the investigation to primary human MDM using the same panel of HA samples and testing conditions, including the same HA testing concentration. We run three independent experiments using MDM from three donors. MDM were cultivated in 8 3 24- well plates (the cells were seeded at 5 3 105 cells/well or 5 3 106 cells/well) for 3 days and exposed to 2 mg/mL HA of different MWs for 72 h with and without 5 lg /mL LPS plus or minus 100 ng/mL mrIFN-g. Cell-free supernatants were obtained and assayed via Human IL-1b ELISA for IL-1b concentration. Figures 2 and 3 demonstrate the IL-1b cytokine production from MDM under different inﬂammatory conditions. As shown in Figure 2, the Vehicle only controls the concentration of IL-1b cytokine, which was not signiﬁcantly changed by the hrIFN-g only presence. Also the cytokine production was not signiﬁcantly changed by the presence of HA samples with MW in the range of 6550–1,700,000 Da. However, the IL-1 b cytokine production decreased compare to the no HA (Vehicle only) control by the presence of 4770 Da HA sample. For a basal state of MDM (no any inﬂammatory agents), only the 4770 and 254,000 Da samples produced a small, but statistically signiﬁcant, increase in IL-1b cytokine production versus the no HA (Vehicle only) control.
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No statistical changes were noted in the presence of any other MW HA samples. As seen in Figure 3, for the higher inﬂammatory condition of wells containing 5 lg /mL LPS the IL-1b concentration was not signiﬁcantly increased in the presence of the HA samples with MW of 17,000, 35,000, and 64,000 Da versus the no HA (Vehicle only) control. However, it was signiﬁcantly decreased by the presence of HA samples with MW in the range of 132,000–1,700,000 Da, and by 4770–6550 Da, as well. As seen in Figure 3, for the highest inﬂammatory condition of wells containing 5 lg /mL LPS plus 100 ng/mL mrIFN-g the IL-1b concentration was not signiﬁcantly increased versus the no HA (Vehicle only) control by any HA samples at 2 mg/mL, but in fact was signiﬁcantly decreased in the presence of HA with MW in the range of 132,000–1,700,000 Da. These data suggest that under our experimental conditions hrIFN-g alone do not provoke an inﬂammatory response and, also when endotoxin is reduced to below 0.03 EU/mg, no increase on Il-1b production by monocyte/ macrophages in the presence of HA occurs. LPS-mediated inﬂammatory reaction (alone or with hrIFN-g) decreased by the presence of HMW-HA. In summary, no overall effect was observed on the IL-1b production by MDM either in a basal or in inﬂammatory states, that is, in presence of LPS alone, hrIFN-g alone, or LPS plus hrIFN-g. Effect of HA oligosaccharides (from disaccharide to dodecamer) on IL-1b production by MDM exposed to inﬂammatory agents The effect of panel of HA oligosaccharides composed of disaccharide (0.401 kDa), hexamer (1.221 kDa), octamer
EFFECTS OF LMW1HA ON HUMAN MONOCYTES
FIGURE 3. Effect of HA of different molecular weights on IL-1 b production of human monocyte/macrophages to endotoxin 1/2 hrIFN-g. Human monocyte/macrophages were seeded at 5 3 106 cells/well and differentiated with hM-CSF in 24-well plate for 7 days at 37 C, then the cells were incubated in the presence or absence of 5 lg/mL LPS 1/2 100 ng/mL hrIFN-g plus 2 mg/mL HA samples of different molecular weights, as compared to vehicle only control. Supernatants were removed at 72 h for IL-1 b assay. Data shown are mean 1/2 1 standard deviation of 4 replicate wells per condition. Statistical significance was calculated using the two-tailed unpaired Student’s t-test or ANOVA. A P < 0.05 was considered statistically significant.
(1.607 kDa), decamer (1.993 kDa), and dodecamer (2.394 kDa) on MDM response was examined. For these experiments we increased the HA samples concentration in six times over our previous study8 from 25 to 150 lg/mL. MDM were cultured in 96-well plate and exposed for 72 h to 150 lg/mL HA of extremely small MWs in the absence of inﬂammatory agents, in the presence of LPS (5 lg /mL) alone, and in the presence of both LPS (5 lg /mL) and mrIFN-g (100 ng/mL). The control condition (no HA) in absence of HA was included in order to exclude any effects because of the frozen RPMI 1640 medium present in the HA samples. Cell-free supernatants were obtained and assayed via ELISA for IL-1b concentration as measure of an inﬂammatory response. Figure 4 shows these experiments data. There was 10fold variability in the sensitivity and response to these inﬂammatory agents by monocytes received from different donors. Thus, we used different scale for IL-1b concentration to illustrate the response of the monocyte/macrophage cell cultures from the three human donors. Figure 4(A): ﬁrst donor—Y scale from 0 to 600 pg/mL of IL-1b; Figure 4(B): second donor—Y scale from 0 to 200 pg/mL of IL1b; Figure 4(C): third donor—Y scale from 0 to 80 pg/mL of IL-1b). As seen in Figure 4(A), for all the cells states, basal or with the inﬂammatory agents there was no signiﬁcant difference in IL-1b production in the presence of the HA oligosaccharides compare to no HA vehicle control. Except the clear small decrease in the presence of HA disaccharide (0.401 kDa) at a basal state, the small decrease
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in the presence of HA octamer (1607 kDa) at a high inﬂammatory state (LPS, 5 lg /mL), and the small increase in the presence of HA disaccharide (0.401 kDa) at a highest inﬂammatory state [LPS (5 lg /mL) 1 mrIFN-g (100 ng/ mL)]. Figure 4(B), (second donor data) shows similar ﬁnding: for all the cells states there was no signiﬁcant difference in IL-1b production, except that is in the absence of inﬂammatory agents (basal state), the presence of HA disaccharide (0.401 kDa) caused a clear signiﬁcant decrease (also as ﬁrst donor data); and, only HA dodecamer (2.394 kDa) HA sample produced small, but signiﬁcant decrease in the IL-1b production in highest LPS 1 hrIFN-g induced inﬂammatory state. Figure 4(C) summarizing the results for monocytes received from third donor, shows that HA disaccharide (0.401 kDa) or hexamer (1.221 kDa), octamer (1.607 kDa), or decamer (1.993 kDa) produced signiﬁcant decrease of IL-1b production in a basal or LPS 1 hrIFN-g inﬂammatory state. HA dodecamer (2.394 kDa) did not produce signiﬁcant changes to IL-1b production in all, basal or activated states. The third donor monocyte/macrophage IL-1b production was not signiﬁcantly changed by the presence of 150 lg/mL HA oligosaccharides in LPS inﬂammatory state. These data suggest that under our experimental conditions when endotoxin in HA is reduced to below 0.03 EU/ mg, HA oligomers do not increase Il-1b production by MDM. Donors macrophage’s response to LPS mediated inﬂammation and presence of HA oligosaccharides, may vary greatly, similar to common human allergic reaction.
FIGURE 4. Effect of HA oligosaccharides on IL-1 b production of human monocyte/macrophages in the presence or absence of endotoxin 1/2 hrIFN-g. Human monocyte/macrophages were seeded at 5 3 106 cells/well and differentiated with hM-CSF in 96-well plate for 7 days at 37 C, then the cells were incubated in the presence or absence of 5 lg/mL LPS 1/2 100 ng/mL hrIFN-g plus 150 lg/mL HA oligomers of different molecular weights, as compared to vehicle only control. Supernatants were removed at 72 h for IL-1 b assay. Data shown are mean 1/2 1 standard deviation of 4 replicate wells per condition. Statistical significance was calculated using the two-tailed unpaired Student’s t-test or ANOVA. A P < 0.05 was considered statistically significant. Figures 4A, 4B, and 4C demonstrate effect of HA oligosaccharides on IL-1 b Production of human monocyte/macrophages from each donor alone.
The current literature suggests two mechanisms for HA as a possible inﬂammatory agent, either by direct interaction of small fragments with speciﬁc cellular receptors or via inﬂammatory contaminants such as endotoxin. We tested LMW-HA with MW ranged from 4.77 up to 64 kDa and HMW-HA with MW ranged from 132 kDa up to 1,700 kDa at 2 mg/mL. This concentration was chosen to be consistent with our previous investigations.8 In our previous
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experiments with RAW cells we found that a minimum of 2 mg/mL HA concentration was required to observe any changes in cell response. Concentrations higher than 2 mg/ mL were not investigated because of the changes in the consistency of HA solutions to gel-like structures. Our data support the idea that HMW-HA with low endotoxin contamination (below 0.03 EU/mg) is not inﬂammatory. Human MDM were assayed for IL-1b production under a variety of inﬂammatory conditions in the
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FIGURE 4. (Continued).
presence or absence of HA. For the basal, unstimulated state of MDM small but signiﬁcant increase of IL-1b concentration was observed in the presence of 254.4 kDa HA sample only. HA samples of other MW did not have any signiﬁcant effects compare to Vehicle only control (no-HA). For MDM stimulated by hrIFN-g alone, a signiﬁcant decrease in IL-1b production was observed in the presence of 4.77 kDa HA only, with all other HA samples not altering IL-1b concentrations. For MDM stimulated by LPS alone the IL-1b production was not increased by LMW-HA samples and was signiﬁcantly decreased by the presence of HMW-HA samples. For MDM stimulated by LPS 1 hrIFN-g, the IL-1b production did not increase in the presence of LMW-HA, though a signiﬁcant decrease was observed in presence of HMW-HA. HA fragments with MW below 5000 Da that we used in the previous study8 were tested at micromolar concentrations (i.e. drug-like qualities), how it has been suggested in earlier literature.7 However, at these concentrations HA oligomers did not cause any effects.8 Within the study we used the HA oligomers at six times increased concentration, 150 mg/mL using MDM harvested from three different human donors. Combined, the three sets of data suggest that the HA oligosaccharides did not cause a signiﬁcant increase of IL-1b production. One donor’s cells produced a signiﬁcant decrease of this cytokine production in the presence of the HA oligosaccharides. Previously we compared the NO production by of RAW 264.7, a murine macrophage cell line, and by primary rat macrophages in response to HA fragments of distinct MWs (i.e., from the unitary disaccharide up to 1.7 3 106 Da), and under the same varying inﬂammatory conditions.8 Brieﬂy, these data from primary and commercial available cell line
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rodent macrophages suggest that when contaminating endotoxin is at a very low level (less than 0.03 EU/mg HA), little or no direct inﬂammatory effect may result from the presence of HA independent of its MW. However, we observed small, but signiﬁcant increases of nitrite concentration (indicative of NO production) for the highest (LPS1 IFN-g) inﬂammatory conditions. A signiﬁcant increase of nitrite concentration was observed by the presence of 4.7 and 6.55 kDa HA (LMW-HA) for the cell line RAW 264.7 and not for primary rat macrophages. For the low (mrIFN-g only) inﬂammatory condition only 6.55 and 17 kDa HA (LMWHA) samples showed clear increase in nitrite for the RAW 264.7 cell line and not for primary rat macrophages. Our data for all the cell types tested suggest that LMW-HA may have a small inﬂammatory potential, whereas HMW-HA may be inhibitory. The data are in agreement with the other published reports, which suggest that HMW-HA can be anti-inﬂammatory, whereas LMW-HA can be proinﬂammatory.3,4,6 Other investigators have reported that HMW-HA(2–6 3 103 kDa) speciﬁcally inhibits TLR2 signaling,6 HMW-HA (100–300 kDa) was found to signiﬁcantly reduce colorectal carcinoma growth in vitro (splenocytes) and in vivo (mice).12 HMW-HA has direct suppressive effects on regulatory T cells (Human PBMC),13 and HMW-HA prevents T-cellmediated liver injury by reducing proinﬂammatory cytokines in mice.14 Naturally HA (HMW-HA) reveals nonsigniﬁcant cytotoxic effects to the tumor cell types (HEp-2, human laryngeal carcinoma, Daoy—human medulloblastoma, MCF7—human breast adenocarcinoma, and WiDr—human colon adenocarcinoma).15 Both naturally occurring HA (HMW-HA) and unsaturated HA oligomers (< 40 mers) exhibit a reasonable antilipid oxidation activity,15 HMW-HA signiﬁcantly reduced the increment in inﬂammatory cytokines and
apoptosis induced by LPS in chondrocytes.16 In the cited references HMW-HA was tested between 1200 and 5000 kDa. Several studies have previously suggested that LMWHA is proinﬂammatory.4,6,9,16,17 According to other authors, LMW-HA was found to be involved in inﬂammatory processes by different types of cells, such as: mouse T lymphocytes,6 murine tumor cell line,12 mouse articular chondrocytes,16 murine, and human keratinocytes.17 In our study, we observed small enhancements of IL-1b production by MDM under stimulus by LPS in the absence of interferon-gamma and in the presence of the LMW-HA fractions 17.0 and 35.0 kDa. However, there was no increase or even a slight decrease of the IL-1b production by MDM under even the highest inﬂammatory conditions, LPS 1 hrIFN-g. Our previous results showed that LMW-HA fractions from 4.77 to 17.0 kDa can slightly increase the NO-production by RAW264.7 cells in the presence of both LPS and mrIFN-g. These results are in general agreement with studies reporting the proinﬂammatory effects by LMW-HA on murine and human macrophages, such as: increase of TNF-a, IL-1b, IL-6 and IFN-g16; and stimulate production of DEFB2 (a peptide with a good bacteriostatic activity produced by epithelial cells).17 It is known that HA oligomers modulate inﬂammation. A large body of literature supports the idea that HA oligosaccharides can be proinﬂammatory.18–22 The only small HA fragments of tetra- and hexa-saccharide size induce differentiation of human monocytes to dendritic cells and increase human monocyte-derived dendritic cell production of the cytokines IL-1b, TNF-a, and IL-12.18 Exposure to only small HA fragments (2–3 disaccharide units in size) induce metalloproteases expression in tumor cells.19 Other authors have reported that small HA fragments (4, 6 mers) activate NFkB factor, induce metalloprotease and cytokine expression.20 Then, small HA fragments (HA oligosaccharides) activate human monocyte-derived dendritic cells,21 induce expression of inﬂammatory cytokine gene in a mouse alveolar macrophage cell line.22 Positive effects of HA oligomers with MWs of less than 10 kDa (2–20 mers) include a very strong antioxidant and cytotoxic activities shown at least at four tumor cell lines.15 Together TGF-b1 (transforming growth factor beta 1) and HA oligomers of size 0.756 kDa dramatically improve elastin matrix protein regeneration by adult vascular smooth muscle cells.23 HA oligosaccharides inhibit murine melanoma tumor growth in vivo,24 anchorage-independent growth of tumor cells, such as human lung carcinoma, human colon carcinoma, or murine mammary carcinoma cells25; hyaluronan octasaccharides inhibit tumorigenicity of osteosarcoma cells in vitro and in vivo26; HA oligosaccharides (4–14 mers) sensitize MDR (multidrug resistance) of breast cancer cells to cytotoxic drugs and after the HA oligosaccharides treatment higher apoptosis level was observed in the resistant murine lymphoma cells than in the sensitive cells27; treatment with small hyaluronan oligosaccharides (2–10 mers) inhibit tumorigenesis in primary human ovarian carcinoma cells.28 Therefore, our present and previous data (from RAW264.7 cell line, rat splenocytes/ macrophages and
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human monocyte/macrophages) agrees with previously cited reports and demonstrate that HA oligosaccharides in concentrations not greater than 150 lg/mL may be involved in anti-inﬂammatory processes or do not induce an inﬂammatory reaction. In summary, the present results support the tentative hypothesis that, if endotoxin is at extremely low level, LMW-HA by itself may not provoke inﬂammatory reaction. The conditions of our experiments were limited by two factors: only one inﬂammatory mediator was investigated and HA sample’s concentration at 2 mg/mL for HA with 5–1700 kDa MW or 150 mg/mL for oligosaccharides below 5 kDa MW. Testing outside of these limited parameters might reveal additional effects. The study also showed variability in the sensitivity of the response to the inﬂammatory agents between cells from different donors. In agreement with our results, previously published studies29–33 also report donordepended variability in production of various cytokines in cell cultures of different types of human cells, such as human monocyte and monocyte-derived macrophage30–33 or mesenchymal stem cells29 stimulated with broad range of inﬂammatory agents of different concentrations (LPS, PMA, TNF-a, IL-1b, ET-1) donor depended variability in production of various cytokines—IL-2, IL-1a, IL-5, IL-17,29 IL-1,33 IL-1b, TNF-a,31,32 IL-6.32 Other authors have found a wide variation in the investigated glucocorticoid sensitivity in human peripheral blood samples from healthy volunteers that vary depending on time of day (morning or evening hours) when the samples were collected.30 As LMW-HA usually is prepared by enzymatic digestion of HMW-HA, there is a possibility to have more impurities from the enzyme in addition to already existing impurities. Impurities, such as DNA, proteins, and endotoxin even in very low, nano-molar, concentrations have a well-documented proinﬂammatory effect.2,34,35 It was reported2 that if the protein impurities of the HA product (dermal ﬁller) were decreased the incidence of hypersensitivity reactions to the product was also decreased. Clinical experience has demonstrated that many HA products exist, such as dermal soft tissue ﬁllers or a number of viscosupplementation products (hyaluronan and its derivatives) for treatment of osteoarthritis of the knee, could lead to immediate or delayed adverse reactions, small or severe complications: skin redness, pain, swelling, and erythema.1,2,36–38 Widely distributed hyaluronan products, such as Synvisc, Hyalgan and Supartz were investigated by BALB/c mice. These results demonstrated that all three hyaluronan products can cause an inﬂammatory soft-tissue reaction.39 The adverse reactions relate to three main causes: impurities, concentration, and excessive amount of product injected, and injection technique.1,2,39–41 The three main causes of the inﬂammatory reactions require additional research that ﬁnally will lead to improvement of treatments with these products. Many studies have suggested that biological activities of HA depend on its MW.3,4,16 The mechanisms determining the suppressive or enhancing characteristics of HA still have not entirely investigated.42 The biological properties of HA oligosaccharides (of lengths 2–6 units) could be dependent on
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different molecular conformations and dynamics occurred at their ends have been reported.43 This study using three donors conﬁrms the well-known variability in immune responses seen in human donor tissue. Further research is necessary to conﬁrm the variability documented in this study. In the future, we plan to investigate the effect of HA oligosaccharides on the response of different cell types, including human primary cells, under variable inﬂammatory conditions and at higher HA concentrations( > 150 mg /mL) than have been studied. REFERENCES 1. Lowe NJ, MD, FRCS, Maxwell CA, MB, ChB, Patnaik R, MD. Adverse reactions to dermal fillers: Review. J Dermatol Surg 2005;31:1616–1625. 2. Andre P, Lowe NJ, Parc A, Clerici TH, Zimmermann U. Adverse reactions to dermal fillers: A review of European experiences. J Cosmet Laser Ther 2005;7:171–176. 3. Jiang D, Liang J, Noble PW. Hyaluronan in tissue injury and repair. Annu Rev Cell Dev Biol 2007;23:435–461. 4. Stern R, Asari AA, Sugahara KN. Hyaluronan fragments: An information-rich system. European J Cell Biol 2006;85:699–715. 5. Adams ME, Lussier AJ, Peyron JG. A risk-benefit assessment of injections of hyaluronan and its derivatives in the treatment of osteoarthritis of the knee. Drug Saf 2000;23:115–130. 6. Scheibner KA, Lutz MA, Boodoo S, Fenton MJ, Powell JD, Horton MR. Hyaluronan fragments act as an endogenous danger signal by engaging TLR2. J Immunol 2006;177:1272–1281. 7. Powell JD, Horton MR. Threat matrix: low-molecular-weight hyaluronan as a danger signal. Immunol Res 2005;32:207–218. 8. Lyle DB, Breger JC, Baeva LF, Shallcross JC, Durfor CN, Wang NS, Langone JJ. Low molecular weight hyaluronic acid effects on murine macrophage nitric oxide production. J Biomed Mater Res A 2010;94:893–904. 9. Misko TP, Schilling RJ, Salvemini D, Moore WM, Currie MG. A fluorometric assay for the measurement of nitrite in biological samples. Anal Biochem 1993;214:11–16. 10. Schneemann M, Schedon G, Hofer S, Blau N, Guerrero L, Schaffner A. Nitric oxide synthase is not a constituent of the antimicrobial armature of human mononuclear phagocytes. J Infect Dis 1993;167:1358–1363. 11. Panaro MA, Acquafredda A, Lisi S, Lofrumento DD, Trotta T, Satalino R, Saccia M, Mitolo V, Brandonisio O. Inducible nitric oxide synthase and nitric oxide production in Leishmania infantuminfected human macrophages stimulated with interferon-g and bacterial lipopolysaccharide. Int J Clin Lab Res 1999;29:122–127. 12. Alaniz L, Rizzo M, Malvicini M, Jaunarena J, Avella D, Atorrasagasti C, Aquino JB, Garcia M, Matar P, Silva M, Mazzolini G. Low molecular weight hyaluronan inhibits colorrectal carcinoma growth by decreasing tumor cell proliferation and stimulating immune response. Cancer Lett 2009;278:9–16. 13. Bollyky PL, Lord JD, Masewicz SA, Evanko SP, Buckner JH, Wight TN, Nepom GT. High molecular weight hyaluronan promotes the suppressive effects of CD41CD251 regulatory T cells. J Immunol 2007;179:744–747. 14. Nakamura K, Yokohama S, Yoneda M, Okamoto S, Tamaki Y, Ito T, Okada M, Aso K, Makino I. High, but not low, molecular weight hyaluronan prevents T-cell-mediated liver injury by reducing proinflammatory cytokines in mice. J Gastroenterol 2004;39:346– 354. 15. El-Safory NS, Lee C-K. Cytotoxic and antioxidant effects of unsaturated hyaluronic acid oligomers. Carbohydr Polym. Forthcoming. 16. Campo GM, Avenoso A, Campo S, D’Ascola A, Traina P, Rugolo CA, Calatroni A. Differential effect of molecular mass hyaluronan on lipopolysaccharide-induced damage in chondrocytes. Innate Immun 2010;16:48–63. 17. Gariboldi S, Palazzo M, Zanobbio L, Selleri S, Sommariva M, Sfondrini L, Cavicchini S, Balsari A, Rumio C. Low molecular weight hyaluronic acid increases the self-defense of skin
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epithelium by induction of b-defensin 2 via TLR2 and TLR4. J Immunol 2008;181:2103–2110. Termeer CC, Hennies J, Voith U, Ahrens T, Weiss JM, Prehm P, Simon JC. Oligosaccharides of hyaluronan are potent activators of dendritic cells. J Immunol 2000;165:1863–1870. Fieber C, Baumann P, Vallon R, Termeer, C, Simon JC, Hofmann M, Angel P, Herrlich P, Sleeman JP. Hyaluronan-oligosaccharideinduced transcription of metalloproteases. J Cell Sci 2004;117:359–367. Voelcker V, Gebhardt C, Averbeck M, Saalbach A, Wolf V, Weih F, Sleeman J, Anderegg U, Simon J. Hyaluronan fragments induce cytokine and metalloprotease upregulation in human melanoma cells in part by signaling via TLR4. Exp Dermatol 2007;17:100– 107. Termeer C, Benedix F, Sleeman J, Fieber C, Voith U, Ahrens T, Miyake K, Freudenberg M, Galanos C, Simon JC. Oligosaccharides of hyaluronan activate dendritic cells via toll-like receptor 4. J Exp Med 2002;195:99–111. McKee CM, Penno MB, Cowman M, Burdick MD, Strieter RM, Bao C, Noble PW. Hyaluronan (HA) fragments induce chemokine gene expression in alveolar macrophages. J Clin Invest 1996;98:2403– 2413. Kothapalli C, Taylor PM, Smolenski R, Yacoub MH, Ramamurthi A. Transforming growth factor beta 1 and hyaluronan oligomers synergistically enhance elastin matrix regeneration by vascular smooth muscle cells. Tissue Eng Part A. 2009;15:501–511. Zeng C, Toole BP, Kinney SD, Kuo J-w, Stamenkovic I. Inhibition of tumor growth in vivo by hyaluronan oligomers. J Cancer 1998;77:396–401. Ghatak S, Misra S, Toole BP. Hyaluronan oligosaccharides inhibit anchorage-independent growth of tumor cells by suppressing the phosphoinositide 3-kinase/akt cell survival pathway. J Biol Chem 2002;277:38013–38020. Hosono K, Nishida Y, Knudson W, Knudson CB, Naruse T, Suzuki Y, Ishiguro N. Hyaluronan oligosaccharides inhibit tumorigenicity of osteosarcoma cell lines MG-63 and LM-8 in vitro and in vivo via perturbation of hyaluronan-rich pericellular matrix of the cells. Am J Pathol 2007;171:274–286. Cordo Russo RI, Garcia MG, Alaniz L, Blanco G, Alvarez E, Hajos SE. Hyaluronan oligosaccharides sensitize lymphoma resistant cell lines to vincristine by modulating P-glycoprotein activity and PI3K/Akt pathway. Int J Cancer 2008;122:1012–1018. Slomiany MG, Dai L, Tolliver LB, Grass GD, Zeng Y, Toole BP. Inhibition of functional hyaluronan-CD44 interactions in CD133-positive primary human ovarian carcinoma cells by small hyaluronan oligosaccharides. Clin Cancer Res 2009;15:7593–7601. Zhukareva V, Obrocka M, Houle JD, Fischer I, Neuhuber B. Secretion profile of human bone marrow stromal cells: Donor variability and response to inflammatory stimili. Cytokine 2010;50:317– 321. Gratsias Y, Moutsatsou P, Chrysanthopoulou G, Tsagarakis S, Thalassinos N, Sekeris C. Diurnal changes in glucocorticoid sensitivity in human peripheral blood samples. Steroids 2000;65:851– 856. Molnar-Kimber KL, Yonno L, Heaslip RJ, Weichman BM. Differential regulation of TNF-a and IL-1b production from endotoxin stimulated human monocytes by phosphodiesterase inhibitors. Mediators Inflamm 1992;1:411–417. Helset E, Sildnes T, Seljelid R, Konopski ZS. Endothelin-1 stimulates human monocytes in vitro to release TNF-a, IL-1b and IL-6. J Mediators Inflamm 1993;2:417–422. Elias JA, Schreiber AD, Gustilo K, Chien P, Rossman MD, Lammie PJ, Daniele RP. Differential interleukin 1 elaboration by unfractionated and density fractionated human alveolar macrophages and blood monocytes: Relationship to cell maturity. J Immunol 1985;135;5:3198–3204. Filion MC, Philips NC. Pro-inflammatory activity of contaminating DNA in hyaluronic acid preparations. J Pharm Pharmacol 2001;53:555–561. Raetz CR, Whitfield C. Lipopolysaccharide endotoxins. Annu Rev Biochem 2002;71:635–700.
36. Roos J, Epaulard O, Juvin R, Chen C, Pavese P, Brion JP. Acute pseudoseptic arthritis after intraarticular sodium hyaluronan. Joint Bone Spine 2004;71:352–354. 37. Morton AH, Shannon P, Chen AL, Cesare PE, Adler EM, Desai P, Leopold SS, Warme WJ, Pettis PD, Shott S. Increased frequency of acute reaction to intra-articular hylan G-F20 (Synvisc) in patients receiving more than one course of treatment. J Bone Joint Surg Am 2003;85:2050–2051. 38. Patel VJ, Bruck MC, Katz BE. Hypersensitivity reaction to hyaluronic acid with negative skin testing. Plast Reconstr Surg 2006;117: 92e–94e. 39. Ottaviani RA, Wooley P, Song Z, Markel DC. Inflammatory and immunological responses to hyaluronan preparations. J Bone Joint Surg 2007;89:148–157.
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40. Humphrey CD, Arkins JP, Dayan SH. Soft tissue fillers in the nose. Aesthet Surg J 2009;29:477–484. 41. Glogau RG, Kane MAC. Effect of injection techniques on the rate of local adverse events in patients implanted with nonanimal hyaluronic acid gel dermal fillers. Dermatol Surg 2008;34:S105– S109. 42. Alaniz L, Garcia M, Rizzo M, Piccioni , Mazzolini G. Altered hyaluronan biosynthesis and cancer progression: an immunological perspective. Med Chem 2009;9:1538–1546. 43. Blundell CD, DeAngelis PL, Day AJ, Almond A. Use of 15NNMR to resolve molecular details in isotopically-enriched carbohydrates: Sequence-specific observations in hyaluronan oligomers up to decasaccharides. Glycobiology 2004;14:999– 1009.
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