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The role of sepiolite and palygorskite on the migration of leukocyte cells to an inflammation site
Applied Clay Science 123 (2016) 315–319
Contents lists available at ScienceDirect
Applied Clay Science journal homepage: www.elsevier.com/locate/clay
Note
The role of sepiolite and palygorskite on the migration of leukocyte cells to an inflammation site Esmeralda Juárez a, Elba Ronquillo de Jesús b, Antonio Nieto-Camacho c, Stephan Kaufhold d, Emilia García-Romero e,f, Mercedes Suárez g, Javiera Cervini-Silva b,h,i,⁎ a
Departamento de Investigación en Microbiología, Instituto Nacional de Enfermedades Respiratorias, Ismael Cosío Villegas, Mexico City, Mexico Departamento de Procesos y Tecnología, Universidad Autónoma Metropolitana Unidad Cuajimalpa, Av. Vasco de Quiroga 4871, Cuajimalpa de Morelos, Col. Santa Fe, Mexico, D.F., Mexico Laboratorio de Pruebas Biológicas, Instituto de Química, Universidad Nacional Autónoma de México, Ciudad Universitaria México City, Mexico d BGR Bundesansaltfür Geowissenschaften und Rohstoffe, Stilleweg 2, D-30655, Hannover, Germany e Departmento de Cristalografía y Mineralogía, Universidad Complutense de Madrid, Spain f Instituto de Geociencias, Universidad Complutense de Madrid — Consejo Superior de Investigaciones Científicas, Spain g Departmento de Geología. Universidad de Salamanca, Spain h Earth Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA, USA i NASA Astrobiology Institute, USA b c
a r t i c l e
i n f o
Article history: Received 20 September 2015 Received in revised form 19 January 2016 Accepted 23 January 2016 Available online 2 February 2016 Keywords: Leukocyte immunological functions Shifting from pro- to anti-inflammatory conditions
a b s t r a c t Sepiolite and palygorskite have shown beneficial health effects but understanding human cell-clay interactions has yet to become unveiled. This paper reports on the effects of sepiolite (Vallecas, Spain) and palygorskite (Torrejon El Rubio, Spain) on the infiltration of human blood leukocytes to an infiltration site. Quantification of human blood leukocyte cells under pro- and anti-inflammatory conditions was conducted, and cells visualized in an Axioscope (Carl Zeiss; Oberkochen, Germany). Images were recorded with an Axiocam Mrm monochromatic camera and ZEN Pro software (Carl Zeiss). The distribution of human blood leukocyte cells at the inflammation site varied before and after adding the clay. The relative proportion of PMN-to-monocytes(MN) (PMN/ MN) exposed to the inflammatory activity by 12-O-tetradecanoylphorbol-13-acetate (TPA) changed in the presence of sepiolite (TPA + sepiolite) or palygorskite (TPA + palygorskite) either after 4 or 24 h, namely, 0.60, 2.5, and 2.33; and 4.33, 1.53, and 2.8, respectively. PMN/MN values compared in the presence of TPA or TPA and palygorskite, however decreased sharply in the presence of TPA and sepiolite. Proposedly, decreases in PMN/ MN values caused by adding sepiolite may alter PMN and MN immunological functions, by lessening the destruction extent of invasive bacteria via phagocytosis and the conversion of MN to macrophages. Proposedly, limiting a conversion of MN to macrophages impedes resolving inflammation because of an incomplete digestion of aged cells. Evidently, shifting from pro- to anti-inflammatory conditions due to the addition of the clay altered the mechanism of infiltration of different leukocyte cells to an inflammation site. Finally, the presence of few macrophages at the inflammation site was attributed to resolution of inflammation, whereby macrophages participated in anti-inflammatory mechanisms leading to the return to homeostasis in tissues. © 2016 Elsevier B.V. All rights reserved.
1. Introduction Sepiolite and palygorskite have shown beneficial health effects but understanding human cell-clay interactions has yet to become unveiled. These clays can act as an effective anti-inflammatory, while they limit growth of human cancer cells (Cervini-Silva et al., 2015a); and inhibit
⁎ Corresponding author at: Departamento de Procesos y Tecnología, Universidad Autónoma Metropolitana, Unidad Cuajimalpa, Av Vasco de Quiroga 4871, Cuajimalpa de Morelos, Santa Fe Cuajimalpa, Mexico, D.F., C.P. 05348, Mexico. E-mail address: [email protected] (J. Cervini-Silva).
http://dx.doi.org/10.1016/j.clay.2016.01.034 0169-1317/© 2016 Elsevier B.V. All rights reserved.
oxidative stress by owing an intrinsic strong oxidant (or weak antioxidant) activity (Cervini-Silva et al., 2015b). To the authors' knowledge, however, little information has become available on how the structural and textural properties of these clays may alter the migration of leukocyte cells to an inflammation site. This paper reports on the effects of well-characterized sepiolite (Vallecas, Spain) and palygorskite (Torrejón El Rubio, Spain; García-Romero and Suarez, 2010) on the infiltration of human blood leukocytes to an infiltration site. To this end, cells were exposed to inflammatory conditions using 12-Otetradecanoylphorbol-13-acetate (TPA), then either sepiolite or palygorskite was added. After 4 or 24 h, edema in mice ears was compared.
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E. Juárez et al. / Applied Clay Science 123 (2016) 315–319
Fig. 1. X-ray diffraction patterns of TOR (left) and VAL (right) samples.
2. Materials and methods 2.1. Sepiolite and palygorskite Two different clay minerals were selected in these experiments: sepiolite and palygorskite. Sepiolite and palygorskite used come from two Spanish deposits: sepiolite from Vallecas (VAL sample) and palygorskite from Torrejón el Rubio (TOR sample). VAL is a very pure sepiolite (Fig. 1) while TOR contains small amounts of quartz (~20 wt.%) and dolomite (b5 wt.%) as impurities. The mineralogical, crystal chemistry, and textural characterization of these samples have been previously reported (García-Romero and Suarez, 2010, 2013; Suárez and García-Romero, 2012). Mineralogical characterization was performed via X-ray diffraction (XRD) using a Siemens D500 XRD diffractometer equipped with a Cu-
Kα radiation source and a graphite monochromator. Textural analyses were performed from the corresponding nitrogen adsorption–desorption isotherms at −196 °C obtained from a static-volumetric apparatus (Micromeritics ASAP 2010 adsorption analyser). 2.2. 12-O-tetradecanoylphorbol-13-acetate (TPA) method Experiments were conducted in adult male CD-1 mice (20–25 g) approved by the Animal Care and Use Committee (NOM-062-ZOO-1999) provided by the Instituto de Fisiología Celular, UNAM, and maintained at 25 °C on a 12/12 h light–dark cycle with free access to food and water. Determinations of the mice ear edema using TPA as inflammatory agent were conducted according to methods described elsewhere (Merlos et al., 1991; Cervini-Silva et al., 2015a). A parallel set of experiments with commercial indomethacin as anti-inflammatory was
Fig. 2. Optical micrographs of leukocyte cells: (A) erythrocytes, (B) lymphocytes, (C) neutrophils, and (D) monocytes.
E. Juárez et al. / Applied Clay Science 123 (2016) 315–319
317
Table 1 Selected properties for sepiolite and palygorskite.a Property
Sepiolite
Palygorskite
Periodical inversions of apical oxygen in the tetrahedral sheetb Channel width (nm)c Tube length (μm)c Specific surface area (SBET, m2 g−1)c External surface area (m2 g−1) Microporous area (m2g−1) Microporous volume (cm3 g−1)
Every six atoms of Si (three tetrahedral chains) 0.37 × 1.06 10 ≤ l 322 148 174 0.0987
Every four (two tetrahedral chains) 0.37 × 0.64 1 ≤ l ≤ 10 110 50 60 0.0298
a b c
As reported in Cervini-Silva et al. (2015a). Data taken from Krekeler and Guggenheim (2008). Data taken from García-Romero and Suarez (2010).
conducted. The edematous response was determined from measured plus mass difference. The edema inhibition (EI) was calculated according to:
EI ≡
A−B 100 A
Axioscope (Carl Zeiss) A1. The image was recorded with an Axiocam Mrm monochromatic camera and ZEN Pro software (Carl Zeiss).
3. Results and discussion ð1Þ
where A and B correspond to mass determined for samples exposed to TPA only and TPA plus indomethacin or sepiolite or palygorskite. Edema inhibition values were determined at t = 24 h. 2.3. Histological cuts Histological cuts were obtained as described elsewhere (Mescher, 2013). Briefly, ear specimens were fixed in a solution of 10% formalin. Ears were dehydrated, embedded in paraffin, and sectioned. 5-μm sections were stained with haematoxylin–eosin. Infiltration of leucocytes was evaluated in selected areas (10× objective). The quantification of leukocytes found in the epidermis was conducted counting cells per field. For each experimental condition (basal, TPA, TPA + sepiolite, and TPA + palygorskite) four distinct histological sections were selected and analysed in four different fields. Cells were visualized using an Axioscope microscope (Zeiss, Oberkochen, Germany). Ten fields of random replicates of each experimental condition that covered the whole length of the ear cut were observed under 100× magnification and cells were counted based on their morphological characteristics in a blind fashion. The total number of infiltrated cells, and the percentage of lymphocytes (LM), monocytes (MN) and polymorphonuclear cells (PMN) were evaluated. 2.5. Human blood leukocytes 5 mL of buffy coats from healthy donors to the blood bank of the National Institute of Respiratory Diseases (Instituto Nacional de Enfermedades Respiratorias) were centrifuged for 20 min at 400g and the white cells layer were recovered by aspiration. The remaining erythrocytes were lysed with 10 mM NaHCO3–155 mM NH4Cl– 0.1 mM EDTA buffer. After washing with cold RPMI 1640 (Lonza, Walkersville, MD), the cells were suspended in complete medium, which consisted of RMPI 1640 supplemented with 200 mM L-glutamine (Lonza), 50 μg mL gentamycin (Lonza) and 10% human heat-inactivated serum (Valley Biomedical, Winchester, VA). Human leucocytes were incubated in chamber slides (Thermo, Rockford, IL) with 5, 10 and 20 μg/mL of palygorskite and sepiolite during 4 and 24 h at 37 °C in 5% CO2 atmosphere. After incubation, the medium was aspirated and the cells were incubated in PBS with 2 μM of Sytox Green (Life Technologies, Eugene, OR) for 10 min. The PBS was aspirated, the chambers dismantled and the slides were mounted using ProLong antifade mountant reagent. Cells were visualized in an
3.1. Leukocyte infiltration vs. cell type Leukocyte cells were differentiated morphologically as follows: lymphocytes owned round nuclei and a cytoplasm barely detectable; neutrophils (PMN) were medium-size cells owning nuclei containing three lobules; monocytes (MN) were significantly larger than the other leukocyte cells, owning an oval nucleus and a notorious cytoplasm; and erythrocytes were smaller, biconcave, and lacking nuclei (Fig. 2). The distribution of leukocyte cells at the inflammation site varied before and after adding the clay. Predominant leukocyte cells during proand anti-inflammatory conditions were MN and PMN, respectively. PMN and MN levels in the blood are typically 5 × 109 and 4 × 108 cells/L, that is 12.5, or 12.5 PMN per MN. However, infiltration of leukocytes cells under pro-inflammatory (TPA) or anti-inflammatory (TPA + sepiolite or TPA + palygorskite) conditions showed lower PMN/MN values. Table 2 Reported edema inhibition (EI) and MPO content inhibition (CI) by sepiolite and palygorskite as determined by the TPA and MPO models.a TPA model t=4h
TPA Sepiolite Palygorskite Indomethacin
t = 24 h
Edema (mg)
EI (%)
Edema (mg)
EI (%)
14.54 ± 0.76 7.92 ± 1.16⁎⁎ 4.56 ± 0.97⁎⁎ 2.88 ± 0.73⁎⁎
⋅⋅⋅a 45.53 68.64 80.19
18.24 ± 1.60 8.21 ± 1.24⁎⁎ 8.20 ± 0.63⁎⁎ 3.33 ± 0.50⁎⁎,b
⋅⋅⋅ 54.98 55.04 78.29
MPO model t=4h
Basal TPA Sepiolite Palygorskite Indomethacin
t = 24 h
OD450nm biopsy−1
CI (%)
OD450nm biopsy−1
CI (%)
0.103 ± 0.006 0.598 ± 0.094 0.208 ± 0.054⁎⁎ 0.122 ± 0.044⁎⁎ 0.018 ± 0.006⁎⁎,c
⋅⋅⋅ ⋅⋅⋅ 65.19 79.57 92.53
0.364 ± 0.045 1.417 ± 0.094 1.284 ± 0.122 1.372 ± 0.140 0.056 ± 0.023⁎⁎,d
⋅⋅⋅ ⋅⋅⋅ 9.38 3.17 90.73
a Data taken from a related study (Cervini-Silva et al., 2015a). Experiments were conducted from 5 to 10 times. In all experiments, the dose corresponded to 1 mg ear−1. Data corresponded to average values ± standard error. Obtained results were analysed using the Dunnett's test. Values with p ≤ 0.5 (⁎) and p ≤ 0.01 (⁎⁎) were considered to differ statistically from the TPA group. Edema inhibition (EI) and MPO content inhibition (CI) are expressed in percent (%). b (···) = edema inhibition close 0. Edema of TPA control samples corresponded to c 15.08 ± 1.68 mg. MPO absorbance controls (OD45nm biopsy−1) corresponded to d 0.241 ± 0.22 and e 0.604 ± 0.063.
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Fig. 3. The distribution of leukocyte cells at the inflammation site varied before and after adding sepiolite and palygorskite.
3.2. Leukocyte infiltration vs. clay structure Structural differences between palygorskite and sepiolite (Table 1) appeared not to be critical for the infiltration of PMN, provided that PMN constituted 60% or more of the leukocyte cells after a 4 or 24 h exposure to either clay. Second, the relative proportion of PMN-to-MN in samples exposed to TPA, TPA and sepiolite (TPA + sepiolite) or palygorskite (TPA + palygorskite) after 4 or 24 h was 0.60, 2.5, and 2.33; and 4.33, 1.53, and 2.8, respectively. Just after a 4 h exposure, shiftings in PMN/MN values were notable, whereby the relative infiltrated PMN increased by twice as much, and MN decreased by half. In contrast, after prolonged times, the relative content of MN in samples exposed to TPA + sepiolite was higher. In summary, PMN/MN values compared in the presence of TPA or TPA and palygorskite, however decreased sharply in the presence of TPA and sepiolite. Compared to sepiolite, palygorskite owned a lower microporosity and external surfaces (Supplementary Fig. 1A). All textural parameters, specific surface area, microporous area and volume and external area, of palygorskite are lower than sepiolite (Table 1). On the other hand, an unrelated study reported on the adsorption behaviour of biogenic substances (i.e., desferrioxamine-B) on sepiolite and palygorskite showing
that the Langmuir affinity parameter (KL) was 1.909 and 0.875 L g−1, respectively (Shirvani and Nourbaksh, 2010). Therefore, the extent to which a clay surface interfered on the migration of neutrophils to the inflammation site appeared to relate to the adsorption capacity of the clay. Furthermore, decreases in PMN/MN values caused by adding sepiolite may alter PMN and MN immunological functions, by lessening the destruction extent of invasive bacteria via phagocytosis and the conversion of MN to macrophages. Proposedly, limiting a conversion of MN to macrophages impedes resolving inflammation because of an incomplete digestion of aged cells. 3.3. Leukocyte infiltration vs. edema and MPO content inhibition A direct comparison between reported edema inhibition for TPA, TPA + palygorskite / TPA + sepiolite (Table 2), and PMN/MN values (Fig. 3) was conducted next. Edema after 4 and 24 h was 14.3, 4.5, and 8.5 mg, and 18.2, 8.5, and 9.0 mg. Therefore, infiltration of inflammation cells coincided albeit in part with edema inhibition solely after prolonged exposure. Next, a comparison between myeloperoxidase (MPO) content inhibition (contained mostly in PMN) and corresponding PMN/MN values after 4 and 24 h were 0.6, 2.33, and 2.5, and 0.6,
Fig. 4. Histological sections from mice ears exposed to TPA (4 h) and stained with haematoxylin–eosin treated with 1 mg ear−1 sepiolite or palygorskite for 4 and 24 h. (20 × Scale = 100 μm). Arrows indicate the infiltrated leukocytes. (A) Basal, (B) TPA, (C) TPA + palygorskite, and (D) TPA + sepiolite (after Cervini-Silva et al., 2015a).
E. Juárez et al. / Applied Clay Science 123 (2016) 315–319
0.15, and 0.2; and 4.33, 2.91, and 1.63, and 1.43, 1.35, and 1.3, respectively. Even though an inverse relation between infiltrating PMN and MPO content was found to be true, no relation between PMN/MN values and MPO content was registered. Apparently, shifting from pro- to antiinflammatory conditions due to the addition of the clay altered the mechanism of infiltration of leukocyte cells to an inflammation site, yet appeared not to affect the production of MPO.
3.4. Additional considerations Infiltration inhibition by sepiolite and palygorskite may be caused either by decreases in the recruitment of leukocytes, decrease or removal of inflammation mediators, or stimulation of anti-inflammatory mediators such as resolvins, protectins, or lipoxins (Ortega-Gómez et al., 2013). At the same time evidence showing a reduction in infiltrate numbers, restoration of histology, and presence of few macrophages at the inflammation site (Fig. 4) were best accounted for by resolution of inflammation, leading to subsequent anti-inflammatory mechanisms whereby macrophages could be responsible for the return to homeostasis in tissues (Fig. 4 after Cervini-Silva et al., 2015a).
4. Conclusions The presence of sepiolite or palygorskite caused shifting from pro- to anti-inflammatory conditions, it altered the mechanism of infiltration of leukocyte cells and, consequently, the distribution of different leukocyte cells at an inflammation site. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.clay.2016.01.034.
319
Acknowledgements The authors thank Jaime Ortega Lechuga (Universidad Autónoma Metropolitana -Cuajimalpa), Daniela Rodríguez Montaño (Unidad de Histología, Instituto de Fisiología Celular, Universidad Nacional Autónoma de México), and Natascha Schleuning (Bundesansaltfür Geowissenschaften und Rohstoffe, BGR) for technical assistance; and Universidad Autónoma Metropolitana for support (Grant No. UAM-C 33678). References Cervini-Silva, J., Nieto-Camacho, A., Ramírez-Apan, M.T., Gómez-Vidales, V., Palacios, E., Montoya, A., Ronquillo de Jesus, E., 2015a. Anti-inflammatory, anti-bacterial, and cytotoxic activity of fibrous clays. Colloids Surf. B 123, 1–6. Cervini-Silva, J., Nieto-Camacho, A., Gómez-Vidales, V., 2015b. Oxidative stress inhibition and oxidant activity by fibrous clays. Colloids Surf. B 133, 32–35. García-Romero, E., Suarez, M., 2010. On the chemical composition of sepiolite and palygorskite. Clay Clay Miner. 58 (1), 1–20. García-Romero, E., Suarez, M., 2013. Sepiolite-palygorskite: textural study and genetic considerations. Appl. Clay Sci. 86, 129–144. Krekeler, M.P.S., Guggenheim, S., 2008. Defects in microstructure in palygorskite-sepiolite minerals: a transmission electron microscopy (TEM) study. Appl. Clay Sci. 39, 98–105. Merlos, M., Gómez, L.A., Giral, M., Vericat, M.L., Garcia-Rafarell, J., Form, J., 1991. Effect of PAF-antagonist in mouse ear oedema induced by several inflammatory agents. Br. J. Pharmacol. 104, 990–994. Mescher, A., 2013. Junqueira's Basic Histology: Text and Atlas. Thirteenth ed. Mg Graw Hill Lange, New York (13th Edition, 13th Edition, 520 pp.). Ortega-Gómez, A., Perretti, M., Soehnlein, O., 2013. Resolution of inflammation: an integrated view. EMBO Mol. Med. 5, 661–674. Shirvani, M., Nourbaksh, F., 2010. Desferrioxamine-B adsorption to and iron dissolution from palygorskite and sepiolite. Appl. Clay Sci. 48, 393–397. Suárez, M., García-Romero, E., 2012. Variability of the surface properties of sepiolite. Appl. Clay Sci. 67, 72–82.
Contents lists available at ScienceDirect
Applied Clay Science journal homepage: www.elsevier.com/locate/clay
Note
The role of sepiolite and palygorskite on the migration of leukocyte cells to an inflammation site Esmeralda Juárez a, Elba Ronquillo de Jesús b, Antonio Nieto-Camacho c, Stephan Kaufhold d, Emilia García-Romero e,f, Mercedes Suárez g, Javiera Cervini-Silva b,h,i,⁎ a
Departamento de Investigación en Microbiología, Instituto Nacional de Enfermedades Respiratorias, Ismael Cosío Villegas, Mexico City, Mexico Departamento de Procesos y Tecnología, Universidad Autónoma Metropolitana Unidad Cuajimalpa, Av. Vasco de Quiroga 4871, Cuajimalpa de Morelos, Col. Santa Fe, Mexico, D.F., Mexico Laboratorio de Pruebas Biológicas, Instituto de Química, Universidad Nacional Autónoma de México, Ciudad Universitaria México City, Mexico d BGR Bundesansaltfür Geowissenschaften und Rohstoffe, Stilleweg 2, D-30655, Hannover, Germany e Departmento de Cristalografía y Mineralogía, Universidad Complutense de Madrid, Spain f Instituto de Geociencias, Universidad Complutense de Madrid — Consejo Superior de Investigaciones Científicas, Spain g Departmento de Geología. Universidad de Salamanca, Spain h Earth Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA, USA i NASA Astrobiology Institute, USA b c
a r t i c l e
i n f o
Article history: Received 20 September 2015 Received in revised form 19 January 2016 Accepted 23 January 2016 Available online 2 February 2016 Keywords: Leukocyte immunological functions Shifting from pro- to anti-inflammatory conditions
a b s t r a c t Sepiolite and palygorskite have shown beneficial health effects but understanding human cell-clay interactions has yet to become unveiled. This paper reports on the effects of sepiolite (Vallecas, Spain) and palygorskite (Torrejon El Rubio, Spain) on the infiltration of human blood leukocytes to an infiltration site. Quantification of human blood leukocyte cells under pro- and anti-inflammatory conditions was conducted, and cells visualized in an Axioscope (Carl Zeiss; Oberkochen, Germany). Images were recorded with an Axiocam Mrm monochromatic camera and ZEN Pro software (Carl Zeiss). The distribution of human blood leukocyte cells at the inflammation site varied before and after adding the clay. The relative proportion of PMN-to-monocytes(MN) (PMN/ MN) exposed to the inflammatory activity by 12-O-tetradecanoylphorbol-13-acetate (TPA) changed in the presence of sepiolite (TPA + sepiolite) or palygorskite (TPA + palygorskite) either after 4 or 24 h, namely, 0.60, 2.5, and 2.33; and 4.33, 1.53, and 2.8, respectively. PMN/MN values compared in the presence of TPA or TPA and palygorskite, however decreased sharply in the presence of TPA and sepiolite. Proposedly, decreases in PMN/ MN values caused by adding sepiolite may alter PMN and MN immunological functions, by lessening the destruction extent of invasive bacteria via phagocytosis and the conversion of MN to macrophages. Proposedly, limiting a conversion of MN to macrophages impedes resolving inflammation because of an incomplete digestion of aged cells. Evidently, shifting from pro- to anti-inflammatory conditions due to the addition of the clay altered the mechanism of infiltration of different leukocyte cells to an inflammation site. Finally, the presence of few macrophages at the inflammation site was attributed to resolution of inflammation, whereby macrophages participated in anti-inflammatory mechanisms leading to the return to homeostasis in tissues. © 2016 Elsevier B.V. All rights reserved.
1. Introduction Sepiolite and palygorskite have shown beneficial health effects but understanding human cell-clay interactions has yet to become unveiled. These clays can act as an effective anti-inflammatory, while they limit growth of human cancer cells (Cervini-Silva et al., 2015a); and inhibit
⁎ Corresponding author at: Departamento de Procesos y Tecnología, Universidad Autónoma Metropolitana, Unidad Cuajimalpa, Av Vasco de Quiroga 4871, Cuajimalpa de Morelos, Santa Fe Cuajimalpa, Mexico, D.F., C.P. 05348, Mexico. E-mail address: [email protected] (J. Cervini-Silva).
http://dx.doi.org/10.1016/j.clay.2016.01.034 0169-1317/© 2016 Elsevier B.V. All rights reserved.
oxidative stress by owing an intrinsic strong oxidant (or weak antioxidant) activity (Cervini-Silva et al., 2015b). To the authors' knowledge, however, little information has become available on how the structural and textural properties of these clays may alter the migration of leukocyte cells to an inflammation site. This paper reports on the effects of well-characterized sepiolite (Vallecas, Spain) and palygorskite (Torrejón El Rubio, Spain; García-Romero and Suarez, 2010) on the infiltration of human blood leukocytes to an infiltration site. To this end, cells were exposed to inflammatory conditions using 12-Otetradecanoylphorbol-13-acetate (TPA), then either sepiolite or palygorskite was added. After 4 or 24 h, edema in mice ears was compared.
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E. Juárez et al. / Applied Clay Science 123 (2016) 315–319
Fig. 1. X-ray diffraction patterns of TOR (left) and VAL (right) samples.
2. Materials and methods 2.1. Sepiolite and palygorskite Two different clay minerals were selected in these experiments: sepiolite and palygorskite. Sepiolite and palygorskite used come from two Spanish deposits: sepiolite from Vallecas (VAL sample) and palygorskite from Torrejón el Rubio (TOR sample). VAL is a very pure sepiolite (Fig. 1) while TOR contains small amounts of quartz (~20 wt.%) and dolomite (b5 wt.%) as impurities. The mineralogical, crystal chemistry, and textural characterization of these samples have been previously reported (García-Romero and Suarez, 2010, 2013; Suárez and García-Romero, 2012). Mineralogical characterization was performed via X-ray diffraction (XRD) using a Siemens D500 XRD diffractometer equipped with a Cu-
Kα radiation source and a graphite monochromator. Textural analyses were performed from the corresponding nitrogen adsorption–desorption isotherms at −196 °C obtained from a static-volumetric apparatus (Micromeritics ASAP 2010 adsorption analyser). 2.2. 12-O-tetradecanoylphorbol-13-acetate (TPA) method Experiments were conducted in adult male CD-1 mice (20–25 g) approved by the Animal Care and Use Committee (NOM-062-ZOO-1999) provided by the Instituto de Fisiología Celular, UNAM, and maintained at 25 °C on a 12/12 h light–dark cycle with free access to food and water. Determinations of the mice ear edema using TPA as inflammatory agent were conducted according to methods described elsewhere (Merlos et al., 1991; Cervini-Silva et al., 2015a). A parallel set of experiments with commercial indomethacin as anti-inflammatory was
Fig. 2. Optical micrographs of leukocyte cells: (A) erythrocytes, (B) lymphocytes, (C) neutrophils, and (D) monocytes.
E. Juárez et al. / Applied Clay Science 123 (2016) 315–319
317
Table 1 Selected properties for sepiolite and palygorskite.a Property
Sepiolite
Palygorskite
Periodical inversions of apical oxygen in the tetrahedral sheetb Channel width (nm)c Tube length (μm)c Specific surface area (SBET, m2 g−1)c External surface area (m2 g−1) Microporous area (m2g−1) Microporous volume (cm3 g−1)
Every six atoms of Si (three tetrahedral chains) 0.37 × 1.06 10 ≤ l 322 148 174 0.0987
Every four (two tetrahedral chains) 0.37 × 0.64 1 ≤ l ≤ 10 110 50 60 0.0298
a b c
As reported in Cervini-Silva et al. (2015a). Data taken from Krekeler and Guggenheim (2008). Data taken from García-Romero and Suarez (2010).
conducted. The edematous response was determined from measured plus mass difference. The edema inhibition (EI) was calculated according to:
EI ≡
A−B 100 A
Axioscope (Carl Zeiss) A1. The image was recorded with an Axiocam Mrm monochromatic camera and ZEN Pro software (Carl Zeiss).
3. Results and discussion ð1Þ
where A and B correspond to mass determined for samples exposed to TPA only and TPA plus indomethacin or sepiolite or palygorskite. Edema inhibition values were determined at t = 24 h. 2.3. Histological cuts Histological cuts were obtained as described elsewhere (Mescher, 2013). Briefly, ear specimens were fixed in a solution of 10% formalin. Ears were dehydrated, embedded in paraffin, and sectioned. 5-μm sections were stained with haematoxylin–eosin. Infiltration of leucocytes was evaluated in selected areas (10× objective). The quantification of leukocytes found in the epidermis was conducted counting cells per field. For each experimental condition (basal, TPA, TPA + sepiolite, and TPA + palygorskite) four distinct histological sections were selected and analysed in four different fields. Cells were visualized using an Axioscope microscope (Zeiss, Oberkochen, Germany). Ten fields of random replicates of each experimental condition that covered the whole length of the ear cut were observed under 100× magnification and cells were counted based on their morphological characteristics in a blind fashion. The total number of infiltrated cells, and the percentage of lymphocytes (LM), monocytes (MN) and polymorphonuclear cells (PMN) were evaluated. 2.5. Human blood leukocytes 5 mL of buffy coats from healthy donors to the blood bank of the National Institute of Respiratory Diseases (Instituto Nacional de Enfermedades Respiratorias) were centrifuged for 20 min at 400g and the white cells layer were recovered by aspiration. The remaining erythrocytes were lysed with 10 mM NaHCO3–155 mM NH4Cl– 0.1 mM EDTA buffer. After washing with cold RPMI 1640 (Lonza, Walkersville, MD), the cells were suspended in complete medium, which consisted of RMPI 1640 supplemented with 200 mM L-glutamine (Lonza), 50 μg mL gentamycin (Lonza) and 10% human heat-inactivated serum (Valley Biomedical, Winchester, VA). Human leucocytes were incubated in chamber slides (Thermo, Rockford, IL) with 5, 10 and 20 μg/mL of palygorskite and sepiolite during 4 and 24 h at 37 °C in 5% CO2 atmosphere. After incubation, the medium was aspirated and the cells were incubated in PBS with 2 μM of Sytox Green (Life Technologies, Eugene, OR) for 10 min. The PBS was aspirated, the chambers dismantled and the slides were mounted using ProLong antifade mountant reagent. Cells were visualized in an
3.1. Leukocyte infiltration vs. cell type Leukocyte cells were differentiated morphologically as follows: lymphocytes owned round nuclei and a cytoplasm barely detectable; neutrophils (PMN) were medium-size cells owning nuclei containing three lobules; monocytes (MN) were significantly larger than the other leukocyte cells, owning an oval nucleus and a notorious cytoplasm; and erythrocytes were smaller, biconcave, and lacking nuclei (Fig. 2). The distribution of leukocyte cells at the inflammation site varied before and after adding the clay. Predominant leukocyte cells during proand anti-inflammatory conditions were MN and PMN, respectively. PMN and MN levels in the blood are typically 5 × 109 and 4 × 108 cells/L, that is 12.5, or 12.5 PMN per MN. However, infiltration of leukocytes cells under pro-inflammatory (TPA) or anti-inflammatory (TPA + sepiolite or TPA + palygorskite) conditions showed lower PMN/MN values. Table 2 Reported edema inhibition (EI) and MPO content inhibition (CI) by sepiolite and palygorskite as determined by the TPA and MPO models.a TPA model t=4h
TPA Sepiolite Palygorskite Indomethacin
t = 24 h
Edema (mg)
EI (%)
Edema (mg)
EI (%)
14.54 ± 0.76 7.92 ± 1.16⁎⁎ 4.56 ± 0.97⁎⁎ 2.88 ± 0.73⁎⁎
⋅⋅⋅a 45.53 68.64 80.19
18.24 ± 1.60 8.21 ± 1.24⁎⁎ 8.20 ± 0.63⁎⁎ 3.33 ± 0.50⁎⁎,b
⋅⋅⋅ 54.98 55.04 78.29
MPO model t=4h
Basal TPA Sepiolite Palygorskite Indomethacin
t = 24 h
OD450nm biopsy−1
CI (%)
OD450nm biopsy−1
CI (%)
0.103 ± 0.006 0.598 ± 0.094 0.208 ± 0.054⁎⁎ 0.122 ± 0.044⁎⁎ 0.018 ± 0.006⁎⁎,c
⋅⋅⋅ ⋅⋅⋅ 65.19 79.57 92.53
0.364 ± 0.045 1.417 ± 0.094 1.284 ± 0.122 1.372 ± 0.140 0.056 ± 0.023⁎⁎,d
⋅⋅⋅ ⋅⋅⋅ 9.38 3.17 90.73
a Data taken from a related study (Cervini-Silva et al., 2015a). Experiments were conducted from 5 to 10 times. In all experiments, the dose corresponded to 1 mg ear−1. Data corresponded to average values ± standard error. Obtained results were analysed using the Dunnett's test. Values with p ≤ 0.5 (⁎) and p ≤ 0.01 (⁎⁎) were considered to differ statistically from the TPA group. Edema inhibition (EI) and MPO content inhibition (CI) are expressed in percent (%). b (···) = edema inhibition close 0. Edema of TPA control samples corresponded to c 15.08 ± 1.68 mg. MPO absorbance controls (OD45nm biopsy−1) corresponded to d 0.241 ± 0.22 and e 0.604 ± 0.063.
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Fig. 3. The distribution of leukocyte cells at the inflammation site varied before and after adding sepiolite and palygorskite.
3.2. Leukocyte infiltration vs. clay structure Structural differences between palygorskite and sepiolite (Table 1) appeared not to be critical for the infiltration of PMN, provided that PMN constituted 60% or more of the leukocyte cells after a 4 or 24 h exposure to either clay. Second, the relative proportion of PMN-to-MN in samples exposed to TPA, TPA and sepiolite (TPA + sepiolite) or palygorskite (TPA + palygorskite) after 4 or 24 h was 0.60, 2.5, and 2.33; and 4.33, 1.53, and 2.8, respectively. Just after a 4 h exposure, shiftings in PMN/MN values were notable, whereby the relative infiltrated PMN increased by twice as much, and MN decreased by half. In contrast, after prolonged times, the relative content of MN in samples exposed to TPA + sepiolite was higher. In summary, PMN/MN values compared in the presence of TPA or TPA and palygorskite, however decreased sharply in the presence of TPA and sepiolite. Compared to sepiolite, palygorskite owned a lower microporosity and external surfaces (Supplementary Fig. 1A). All textural parameters, specific surface area, microporous area and volume and external area, of palygorskite are lower than sepiolite (Table 1). On the other hand, an unrelated study reported on the adsorption behaviour of biogenic substances (i.e., desferrioxamine-B) on sepiolite and palygorskite showing
that the Langmuir affinity parameter (KL) was 1.909 and 0.875 L g−1, respectively (Shirvani and Nourbaksh, 2010). Therefore, the extent to which a clay surface interfered on the migration of neutrophils to the inflammation site appeared to relate to the adsorption capacity of the clay. Furthermore, decreases in PMN/MN values caused by adding sepiolite may alter PMN and MN immunological functions, by lessening the destruction extent of invasive bacteria via phagocytosis and the conversion of MN to macrophages. Proposedly, limiting a conversion of MN to macrophages impedes resolving inflammation because of an incomplete digestion of aged cells. 3.3. Leukocyte infiltration vs. edema and MPO content inhibition A direct comparison between reported edema inhibition for TPA, TPA + palygorskite / TPA + sepiolite (Table 2), and PMN/MN values (Fig. 3) was conducted next. Edema after 4 and 24 h was 14.3, 4.5, and 8.5 mg, and 18.2, 8.5, and 9.0 mg. Therefore, infiltration of inflammation cells coincided albeit in part with edema inhibition solely after prolonged exposure. Next, a comparison between myeloperoxidase (MPO) content inhibition (contained mostly in PMN) and corresponding PMN/MN values after 4 and 24 h were 0.6, 2.33, and 2.5, and 0.6,
Fig. 4. Histological sections from mice ears exposed to TPA (4 h) and stained with haematoxylin–eosin treated with 1 mg ear−1 sepiolite or palygorskite for 4 and 24 h. (20 × Scale = 100 μm). Arrows indicate the infiltrated leukocytes. (A) Basal, (B) TPA, (C) TPA + palygorskite, and (D) TPA + sepiolite (after Cervini-Silva et al., 2015a).
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0.15, and 0.2; and 4.33, 2.91, and 1.63, and 1.43, 1.35, and 1.3, respectively. Even though an inverse relation between infiltrating PMN and MPO content was found to be true, no relation between PMN/MN values and MPO content was registered. Apparently, shifting from pro- to antiinflammatory conditions due to the addition of the clay altered the mechanism of infiltration of leukocyte cells to an inflammation site, yet appeared not to affect the production of MPO.
3.4. Additional considerations Infiltration inhibition by sepiolite and palygorskite may be caused either by decreases in the recruitment of leukocytes, decrease or removal of inflammation mediators, or stimulation of anti-inflammatory mediators such as resolvins, protectins, or lipoxins (Ortega-Gómez et al., 2013). At the same time evidence showing a reduction in infiltrate numbers, restoration of histology, and presence of few macrophages at the inflammation site (Fig. 4) were best accounted for by resolution of inflammation, leading to subsequent anti-inflammatory mechanisms whereby macrophages could be responsible for the return to homeostasis in tissues (Fig. 4 after Cervini-Silva et al., 2015a).
4. Conclusions The presence of sepiolite or palygorskite caused shifting from pro- to anti-inflammatory conditions, it altered the mechanism of infiltration of leukocyte cells and, consequently, the distribution of different leukocyte cells at an inflammation site. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.clay.2016.01.034.
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Acknowledgements The authors thank Jaime Ortega Lechuga (Universidad Autónoma Metropolitana -Cuajimalpa), Daniela Rodríguez Montaño (Unidad de Histología, Instituto de Fisiología Celular, Universidad Nacional Autónoma de México), and Natascha Schleuning (Bundesansaltfür Geowissenschaften und Rohstoffe, BGR) for technical assistance; and Universidad Autónoma Metropolitana for support (Grant No. UAM-C 33678). References Cervini-Silva, J., Nieto-Camacho, A., Ramírez-Apan, M.T., Gómez-Vidales, V., Palacios, E., Montoya, A., Ronquillo de Jesus, E., 2015a. Anti-inflammatory, anti-bacterial, and cytotoxic activity of fibrous clays. Colloids Surf. B 123, 1–6. Cervini-Silva, J., Nieto-Camacho, A., Gómez-Vidales, V., 2015b. Oxidative stress inhibition and oxidant activity by fibrous clays. Colloids Surf. B 133, 32–35. García-Romero, E., Suarez, M., 2010. On the chemical composition of sepiolite and palygorskite. Clay Clay Miner. 58 (1), 1–20. García-Romero, E., Suarez, M., 2013. Sepiolite-palygorskite: textural study and genetic considerations. Appl. Clay Sci. 86, 129–144. Krekeler, M.P.S., Guggenheim, S., 2008. Defects in microstructure in palygorskite-sepiolite minerals: a transmission electron microscopy (TEM) study. Appl. Clay Sci. 39, 98–105. Merlos, M., Gómez, L.A., Giral, M., Vericat, M.L., Garcia-Rafarell, J., Form, J., 1991. Effect of PAF-antagonist in mouse ear oedema induced by several inflammatory agents. Br. J. Pharmacol. 104, 990–994. Mescher, A., 2013. Junqueira's Basic Histology: Text and Atlas. Thirteenth ed. Mg Graw Hill Lange, New York (13th Edition, 13th Edition, 520 pp.). Ortega-Gómez, A., Perretti, M., Soehnlein, O., 2013. Resolution of inflammation: an integrated view. EMBO Mol. Med. 5, 661–674. Shirvani, M., Nourbaksh, F., 2010. Desferrioxamine-B adsorption to and iron dissolution from palygorskite and sepiolite. Appl. Clay Sci. 48, 393–397. Suárez, M., García-Romero, E., 2012. Variability of the surface properties of sepiolite. Appl. Clay Sci. 67, 72–82.