A case of zootherapy with the tarantula Brachypelma vagans Ausserer, 1875 in traditional medicine of the Chol Mayan ethnic group in Mexico

Salima Machkour-M'rabet
Yann Hénaut
Peter Winterton
Roberto Rojo
A case of zootherapy with the tarantula Brachypelma vagans Ausserer, 1875 in traditional medicine of the Chol Mayan ethnic group in Mexico
R E S E A R C H Open Access
Background: In practically every human culture, the use of arthropods as medicinal resources has been reported. In Mexico, the Mayan people mainly use plants but occasionally also animals and minerals in their medicine. This article is the first to report the traditional use of the tarantula Brachypelma vagans by medicine men in the Chol community, an ancient indigenous group that inhabits the southeastern part of Mexico. We also describe the utility of such arachnids in traditional medicine. Methods: This study was carried out in different Chol communities in the states of Chiapas and Campeche (southeastern Mexico) from 2003 until 2007. We interviewed the local medicine men, patients and non-Chol people in each village visited to collect information about the rituals involved and the effectiveness of this traditional medicine and also their opinion of this traditional medicine. Results: In all independent villages, the people who present an illness called 'aire de tarantula' or tarantula wind with symptoms including chest pain, coughing and asthma, were treated by the medicine man (called 'hierbatero') with a tarantula-based beverage. From village to village, the beverage has a similar base composition but some variations occur in additional ingredients depending on the individual medicine man. Like in all traditional Mayan medicine, the ritual of the ceremony consists of drinking the tarantula-based beverage and this is principally accompanied by chants and burning of incense. Conclusions: The recipe of the tarantula-based beverage and the procedure of this ritual ceremony were fairly constant in all the villages visited. Our work shows that despite the tarantula's bad image in several cultures, in others positive use is made of these spiders, as in modern medicine.

Background

The use of plants, animals, mineral substances and other natural materials in traditional medicine by indigenous peoples, throughout the world and across time, is a well documented practice. Although plants and plant-derived materials constitute the principal source of ingredients for traditional medicine, the identification of animal resources for medical cures is also important [1,2].

Animal-based medicines can be prepared from the entire animal, from parts of the animal's body, from products of its metabolism (body secretions and excrement), or from other materials related to animals (nests and cocoons) [3]. The practice that uses animal or animal-derived products in human healing is known as zootherapy [3], according to the zootherapeutic universality hypothesis [4], zootherapy is widespread across most human cultures [5][6][7][8]. Traditional medicines use animal or animal-derived products from all taxonomic groups like echinoderms, insects, arthropods, reptiles, birds and mammals [9,8,10,11]. For example, in Sudanese traditional medicine fresh manure of a dromedary (Camelus dromedaries L. 1758) is used to alleviate arthritis [6]; in Nigeria, the hippo tusks (Hippopotamus amphibious L. 1758) are used as an aphrodisiac, the fat extracted from a manatee (Trichechus senegalensis Link 1795) is used to cure rheumatism, boils and backache [5]; in China, earthworm extract is prescribed to treat over 80 diseases like asthma, hypertension, ulcers, and epilepsy among other things [12].

The use of arthropods in traditional medicine is also widespread. In Chhattisgarh (India) over 500 insects, mites and spiders have been reported as useful to medicine to cure common and complicated ailments. For example, the red velvet mite (Trombidium grandissimum Koch 1867) is commonly used for paralysis, the bed bug (Cimex lectularius L. 1758) is used in the treatment of epilepsy, piles, alopecia and urinary disorders [9]. In different ethnic communities of India, 22% of the animals reported in traditional medicine (of a total of 109) are invertebrates used for diseases like asthma, tuberculosis, coughs and colds [10]. In northeast Brazil, the use of insects is common in medicine (14% of the listed medicinal animals in this region) principally for asthma, pneumonia, sinusitis, and coughs [11]. In the region of Chiapas (Mexico), 12 insects (16% of the animals listed) are used in the traditional medicine of the Mayan communities to cure coughs, warts and stammering [8].

A common use of spiders by man, in addition to keeping tarantulas as pets, is their consumption as food. In Cambodia, it is traditional to eat fried tarantulas (Haplopelma albostriatum Simon 1886: Aranea, Theraphosidae), and the Piaroa Indians of Amazon eat the Goliath bird-eating tarantula (Theraphosa blondi Latreille 1804) to become better hunters. The use of spiders in traditional medicine is very little documented. It was cited in India by Oudhia [9] and reported by Lev [13] with the use of crab spiders for healing in medieval times. Unfortunately, no details of spider species or their uses in traditional medicine are provided. A few other works report the use of spiders in medicine; in Brazil, Costa-Neto [14] reported the use of the chelicerates from the Goliath bird-eating tarantula to treat 'erysipelas' (or 'Holy fire'), fortification of teeth and asthma, and Costa-Neto and Resende [15] reported, in the city of Feira de Santana (Bahia State), the use of toasted bird-spider (referred to as mygalomorphs spp.) for sufferers of asthma; in Chiapas, Enríquez Vázquez [8] mention that Tzotziles and Tzetzales ethnic groups use a 'big spider' in their medicine and, Hunn [16] describes the use of a tarantula also in Chiapas (Mexico) to treat tumors, the patient being bitten in the affected zone. In this case, we may suspect that the tarantula in question is the Mexican redrump tarantula (Brachypelma vagans Ausserer 1875), as it is the only one known to occur in this area [17].

The use of tarantulas throughout the world is often to treat asthma-like conditions as mentioned above. Various species of tarantulas were used to treat the diseases and many themselves provoke asthma following the inhalation of tarantula hairs [18,19]. The most likely hypothesis to explain the role of tarantula setae in the asthma reaction is the action of the chitin particles [for more details of the mechanism of action see : 20,19].

Mexican redrump tarantula

The genus Brachypelma (Mygalomorphae: Theraphosidae) comprises 21 species, registered in the CITES data base; among them, 14 occur in Mexico. The limited geographic distribution of this genus, the destruction of its habitat by landscape fragmentation, the high mortality rates in juveniles, the late age of sexual maturity (7-8 years for males, 9-10 years for females), and their high value in the pet trade, make all species of Brachypelma endangered [21,22] and therefore listed in appendix II of CITES since 1995.

Brachypelma vagans, commonly known as the Mexican redrump tarantula, has the widest distribution, being reported in the South of Mexico, Belize, El Salvador, Guatemala, Honduras and Costa Rica [17]. However, it has also been recorded in the wild in Florida [23], as trade has promoted its dispersion out of its natural range. This species is big, conspicuous and very abundant in some parts of the study area [24]. Recent ecological studies [24,25] show the close relation between the presence of B. vagans tarantulas and the traditional Mayan villages in the peninsula of Yucatan (Mexico). This relation is also common in Chiapas (Mexico) under similar conditions (unpublished data). The tarantulas are present and numerous in the center of the villages, in an open place used as a football or volley ball field and particularly for school activities, and in the backyards of the surrounding houses with 0.02 to 0.1 individuals per square meter [24,25]. A genetic study indicated that the Mexican redrump tarantulas occur in populations with a high number of related individuals. Each population is also genetically characteristic of each village where they are found [26]. These results seem, to confirm the probable relationship between the human populations of this region and the presence of B. vagans.

Chol community

The Choles are indigenous peoples living in southeastern Mexico, mainly in the highland of Chiapas (named 'Los Altos'). After their rebellion (18 th century), with other Mayan ethnic groups (Tzoltziles and Tzeltales) in opposition to the Spanish colonial invasion, the Chol community settled mainly at the borders of the rainforest in Palenque, Tila, Tumbalá and Bachajón in Chiapas and Retalhuleu in Guatemala. Now, the two major communities of Chol are in Tila and Tumbalá.

The main economic activity is agriculture especially corn and beans ('frijol'), as well as sugar cane, rice, coffee, and various fruits. The Choles call themselves 'Winik', a Mayan word that means 'man or male'. They are the 'milperos' which refers to the people whose lives and existence have revolved around the cultivation of maize, their most sacred food. The Chol culture is very rich in traditional medicine. The medicine man, or 'hierbatero', plays a fundamental role in the community, not only as a doctor but also as a friend, psychologist, confessor, and re-establisher [27].

In the unusual 1921 Mexican census, residents of each state were asked to classify themselves in several categories, including pure indigenous, indigenous mixed with white and white. Chiapas State had a total of 98105 persons (five years of age and older) speaking at least twenty-five indigenous languages, representing 27.4% of the over 5 state population. The Chol language was spoken by 10.5% (10330 speakers) of those using indigenous languages in 1921. According to the 2000 census, the population of persons, five years old and more, who spoke indigenous languages amounted to 809592 individuals in Chiapas of which the Chol language represented 17.4% (104806 speakers) [28].

Methods

Study area

The study area was located in the states of Chiapas and Campeche (southern Mexico) ( Figure 1). We investigated a total of six sites; five communities are in Chiapas: 1) Frontera Corozal (16°49'N, 90°53'W; altitude 115 m) located on the banks of the Usumacinta river that marks the border between Mexico and Guatemala and is inhabited mainly by Chol people who arrived at least 30 years ago, 2) Tila (17°17'N, 92°25'W; altitude 1063 m) is the major concentration of the Chol community and is a major religious centre, it is also inhabited by many mestizos who speak Chol language. It is an old town in Mexico that dates from the beginning of the colonial period

Data collection

In order to understand the ritual of the use of the tarantula in traditional Chol medicine, we interviewed the medicine man, patients and some other target people in the six villages. To interview the medicine man in each of the villages we established a questionnaire to understand the practice of tarantula use (Table 1). We obtained the authorization to take a few photos during the ritual (Figure 2). The interview with the patients consisted in asking them about how they felt, before and after visiting the medicine man and their symptoms. The interviews of the village people included non-Chol people living in a mixed but mainly Chol community, to record their opinion about traditional practices in Chol medicine.

Results

The use of B. vagans was prescribed for the same symptoms in all the Chol villages and villages with a mix of Chol and people of various other origins.

The patient comes to the 'hierbatero' (or herbalist) who takes the pulse at the wrist while he asks about the patient's illness and also his/her relational problems with people from the community. This first contact could be in the presence of some of the patient's relatives, to understand the relation with the good or evil powers. Once the medicine man has discarded other sicknesses like 'espanto' (fright; called 'susto' that symptoms comprise chest pressure, high blood pressure, chill, difficult to breath, general pain of legs and arms and constant fright), and reaches the conclusion that the patient is suffering from 'aire de tarantula', he asks the patient for a tarantula for the next session or gets both the animal and the necessary herbs himself. One of the 'hierbateros' explained: "This evil comes when a tarantula is hanging on the heart and eating it". The different names used in the Mayan vocabulary (or more precisely Chol vocabulary) to name spiders ('Chiwoj', 'Chiboj' [29]; or 'Chiuó') seem to be specifically used to designate the tarantula. In the Chol communities, the word 'Chiwoj' is commonly used for any tarantula-like spider but they use the name especially for Brachypelma vagans, the most conspicuous and biggest species of tarantula in the area.

The first examination by the 'hierbatero' determines the symptoms of 'aire de tarantula'. The symptoms presented by the patients diagnosed as suffering from the condition and those mentioned by the 'hierbateros' in all sites, are concurrent and are of respiratory nature like chest pain and coughing. In Tila village, one 'hierbatero's' wife mentioned that 'aire de tarantula' is what modern doctors call asthma.

The day after the first visit, ingredients are prepared and when the patient comes for his second appointment the 'hierbatero' lights incense, sits in front of the patient and begins to pray asking the Catholic god for the health of his patient and proceeds to kill the spider by pressing the anterior central part of the body (prosoma) longitudinally, between legs II and III with the forefinger and the thumb. Even if the spider is still moving, he puts it into a mug and mashes it with a spoon before adding 96°spirit alcohol, filling the goblet ¾ full. He then generally adds tobacco (Nicotiana sp. L. 1754), garlic (Allium sativum L.), caraway (Carum carvi L.) and sometimes other plants. The contents are mixed and filtered three times with a traditional Mayan cloth (called 'paliacate').

When the 'hierbatero' has finished preparing the tarantula-based beverage, he sits in front of the patient, starts to pray, sanctifies the beverage (the Chol people have a mixed religion between Catholicism and their ancient beliefs), and then blows in the patient's face spraying the liquid. He usually drinks a little too. The patient, who until now has been motionless, has to drink all the remaining liquid and then blows it in the 'hierbatero's' face.

According to some reports, the 'hierbatero' generally puts the remains of the spider in a frying-pan until they are reduced to ash. The use of the ash is very variable: 1) it can be added to the tarantula-based beverage and drunk; 2) smeared on the patient's skin, sometimes making a cross form on the chest or on the back or; 3) tied tightly to the patient's skin with cloth.

Depending on the strength of the illness they need to visit the medicine man two or three times and when the treatment has finished, they generally pay him with hens but sometimes with food or other items.

The 'hierbateros' proceeded in almost the same way with some slight differences like the use of some herbs and the disposal of the tarantula ashes.

The community of El Limar was the only one where the 'hierbatero' did not use the tarantula to cure this illness. He used the tarantula to call the soul of the patient suffering from a condition called 'espanto' (fright) [30]. This can occur when the soul has been caught by the Earth powers like caves and rivers, for instance. To cure 'espanto', the 'hierbatero' uses a mixture of spiritual treatments and herbs (praying, offerings and threats). In this case, they put the entire tarantula in a jar with spirit and camphor, and then let the mixture marinate for a couple of days or sometimes the 'hierbatero' prepares the mixture in advance.

According to testimony, the patients are cured. People who went to see the 'hierbatero' were very sick and after treatment felt well.

Only two patients were found in the village Once de Mayo. The symptoms of both patients were pain in the chest (lungs), difficult breathing and dry cough. The patients visited the medicine men two to four times, and both were totally cured.

The opinion of most of the non-Chol people is negative; they reject this traditional medicine. However, one of the patients who was non-Chol, but was from Veracruz State, said that the tarantula-based beverage helped to cure him.

One of us (R.R.) tasted the beverage and contrary to what could be thought, did not feel any irritation in the throat which could be expected because of the urticating hairs, classified as true setae [19], many of which are likely to have passed through the rough cloth sieve.

Discussion

Many specialists consider spider danger to be overevaluated [31] and that only few species are really dangerous for humans [32]. In the study area, Brachypelma vagans is not considered as a dangerous animal, and is found in abundance within villages [24,25]. It is well accepted by the people who do not attempt to eradicate them and on the contrary use them as a medicinal resource.

Generally spiders, and especially tarantulas are thought to be dangerous, as observed in traditional zoological knowledge in Brazil where a relationship was established between traditional and academic knowledge [14]. Also, the danger of spider bites is mentioned in academic work [18]. Other works mention eye diseases caused by the urticating hairs of tarantulas kept as pets [33,34]. The bad reputation of tarantulas not only stems from the possibility of them causing human disease but also from animal-lore. In Chiapas (Mexico), as in many other parts of Central and South America, there is a widespread belief, that the tarantula bites horses which can provoke fall of the hoof and even rotten leg leading the death of the animal. For this, the tarantula has other names like 'hierba' (grass), 'mala hierba' (weed), 'mata caballo' (horse killer) or 'pica caballo' (horse stinger) [35]. This 'attack' of horses by the tarantula is also related by Chol people.

Sometimes, the Choles mention the story but it was just as an anecdote related to the spider but of a very different nature from the 'aire de tarantula'. In El Limar village, there were more stories about mortal wounds to the hoofs of livestock than in the other villages in the study. The reason could be, like the fact that they do not use the tarantulas to cure the 'aire de tarantula', a result of the transculturalization brought about by the recently opened routes of communication. Besides that, El Limar is under the influence of the neighboring town of Salto del Agua, a very important fluvial dock including a railway that goes from Merida City, in the north of the Yucatan peninsula, to Mexico City. This encourages a mixture of cultures from southeastern Mexico, although it is still a Chol region.

This study revealed the similarity of the ritual associated to the use of the tarantula in traditional Chol medicine in all places investigated. In nearly all villages, the tarantula is crushed and mixed with alcohol and additional plants, and the beverage is filtered to eliminate pieces and urticating hairs. We assume that the effectiveness of the cure must come from one of the ingredients of the beverage. The ritual involves reciting prayers and singing by the medicine man. Then, the patient can drink the beverage. The similarity of protocol between very distant villages suggests that the use of the tarantula in traditional medicine is ancestral and well distributed throughout the area. Probably, this traditional use of B. vagans by the people contributed to maintaining the local population of this endangered tarantula.

We did not observe a marked difference in opinion about the use of this traditional ritual among the Chol and non-Chol peoples; both of whom use the services of Use the ashes Yes: 83% (n = 5); No: 17% (n = 1)

How many consultations

Payment

the 'hierbatero'. However, some non-Chol people in mixed villages as in 11 de Mayo, criticize the 'hierbatero' as someone who in their opinion, exploits, people's ignorance to make money. Tarantulas have been very well studied for the benefits they provide to medicine and particularly to pharmacology research like the case of the venom from the tarantula Grammostola spatulata Walckenaer 1837 (synonym G. rosea known as Chilean rose tarantula) now used as a drug to inhibit atrial fibrillation [36]. The venom peptide (known as GsMtx-4) works directly blocking the excitatory (action on stretch-activated ion channels; SACs) currents responsible for arrhythmia [36,37]. The use of GsMtx-4 as a drug, opens new clinical horizons in the diagnosis and treatment of pathologies including cardiac arrhythmia, muscular dystrophy and glioma [38,39].

Our study of this cultural tradition is a first approach to the components of the tarantula Brachypelma vagans body (including venom, urticating hairs, hemolymph and other tissues) to determine the beneficial and active substances that this tarantula may offers to pharmacology and hence to modern medicine.

Conclusions

Our study clearly shows the common use of a spider (Brachyplema vagans) in traditional Mayan medicine throughout the regions of Mexico in which the Chol communities live. Furthermore, this is the first time that the whole ritual is reported in detail. Spiders, considered negatively in most cultures, are used positively in the Mayan culture, as they are in modern medical research. Weissenberger of Ecosur for producing Figure 1. Roberto Rojo benefited from a grant given by Ecosur through the programme Pali.

, 3 )
3Tumbalá (17°16'N, 92°18W; altitude 1490 m) contains a very large Chol population, 4) Álvaro Obregón (17°26'N, 92°32'W; altitude 390 m) is a little Chol community close to Tila village, and 5) El Limar (17°27'N, 92°23'W; altitude 80 m) contains a large Chol population; the sixth community is in Campeche: 6) Once de Mayo (18°09N, 89°45'W; altitude 267 m) is a small village of around 400 inhabitants, located on the edge of the Calakmul Biosphere Reserve where we encounter Tzeltal, mestizo and Chol peoples.

Figure 1
1Locations of the Chol communities visited for this study where the tarantula is used in traditional Mayan medicine (southern Mexico).

Figure 2
2Representation of the ritual to prepare the tarantula-based beverage used in traditional Mayan medicine in Mexico. A) Medicine man kills the tarantula; B) Crushes it; C) Mixes it with spirit alcohol and some additional herbs; D) Filters the beverage to eliminate fragments and urticating hairs.

Table 1
1Synthesis of the interview with the six (two in "Once de Mayo village) medicine men in five (excluding El Limar village) Chol traditional villages in Chiapas and Campeche States of MexicoAnswers
Reponses

Take pulse
Yes: 83% (n = 5); Unknown:17% (n = 1)

Use additional plants
Garlic + Tobacco + Caraway: 67% (n = 4)
Garlic + Tobacco + pepper: 16.5% (n = 1)
Garlic + Tobacco: 16.5% (n = 1)
Mixed with alcohol
Yes: 83% (n = 5) (one used hot alcohol)
No: 17% (n = 1) (replaced by Holy water)
Ritual singing
Yes: 67% (n = 4); No: 16.5% (n = 1); Unknown:
16.5% (n = 1)

Ritual prayer
Yes: 100% (n = 6)
Patient drinks beverage
Yes: 83% (n = 5); No: 17% (n = 1)

Medicine man drinks
beverage

Yes: 50% (n = 3); No: 50% (n = 3)

Spit in the face
Yes: 33% (n = 2); No: 67% (n = 4)

Burn the spider
Yes: 83% (n = 5); No: 17% (n = 1)

Author detailsLaboratorio de Bioconservación ante el Cambio Global, El Colegio de la Frontera Sur (ECOSUR). Av. del Centenario Km. 5.5, C.P. 77900, Chetumal, Quintana Roo, Mexico. 2 Université Paul Sabatier Toulouse III, 118 route de Narbonne, 31062 Toulouse cedex, France.1

AcknowledgementsWe thank the people of 'Once de Mayo' for granting us access to their lands and for their hospitality during our stay and to the Chol people, whom we learned to admire. Thanks to Ph.D. Sophie Calmé from Ecosur (El Colegio de la Frontera Sur) for her friendship and guidance. Thanks to HolgerAuthors' contributions SMM conceived the study, participated in its design and draft final version of the manuscript. YH conceived the study, participated in its design and coordination and draft final version of the manuscript. PW participated in the design and draft of the manuscript. RR realized filed observations and draft first version of the manuscript. All authors read and approved the final manuscript.Competing interestsThe authors declare that they have no competing interests.
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BMC Immunology Ethanol inhibits LPS-induced signaling and modulates cytokine production in peritoneal macrophages in vivo in a model for binge drinking

Stephen B Pruett email@example.com
Department of Cellular Biology and Anatomy
Louisiana State University Health Sciences Center
71130ShreveportLAUSA

Department of Basic Sciences
College of Veterinary Medicine
Mississippi State University
Mississippi State39762MSUSA

Ruping Fan email@example.com
Department of Cellular Biology and Anatomy
Louisiana State University Health Sciences Center
71130ShreveportLAUSA

BMC Immunology Ethanol inhibits LPS-induced signaling and modulates cytokine production in peritoneal macrophages in vivo in a model for binge drinking
10.1186/1471-2172-10-49BioMed Central Page 1 of 10 (page number not for citation purposes) Research article * Corresponding author
Background:Previous reports indicate that ethanol, in a binge drinking model in mice, inhibits the production of pro-inflammatory cytokines in vivo. However, the inhibition of signaling through TLR4 has not been investigated in this experimental model in vivo. Considering evidence that signaling can be very different in vitro and in vivo, the present study was conducted to determine if effects of ethanol on TLR4 signaling reported for cells in culture or cells removed from ethanol treated mice and stimulated in culture also occur when ethanol treatment and TLR4 activation occur in vivo.Results: Phosphorylated p38, ERK, and c-Jun (nuclear) were quantified with kits or by western blot using samples taken 15, 30, and 60 min after stimulation of peritoneal macrophages with lipopolysaccharide in vivo. Effects of ethanol were assessed by administering ethanol by gavage at 6 g/kg 30 min before administration of lipopolysaccharide (LPS). Cytokine concentrations in the samples of peritoneal lavage fluid and in serum were determined at 1, 2, and 6 hr after lipopolysaccharide administration. All of these data were used to measure the area under the concentration vs time curve, which provided an indication of the overall effects of ethanol in this system. Ethanol suppressed production of most pro-inflammatory cytokines to a similar degree as it inhibited key TLR4 signaling events. However, NF-B (p65) translocation to the nucleus was not inhibited by ethanol. To determine if NF-B composed of other subunits was inhibited, transgenic mice with a luciferase reporter were used. This revealed a reproducible inhibition of NF-B activity, which is consistent with the observed inhibition of cytokines whose expression is known to be NF-B dependent.Conclusion:Overall, the effects of ethanol on signalling in vivo were similar to those reported for in vitro exposure to ethanol and/or lipopolysaccharide. However, inhibition of the activation of NF-B was not detected as translocation of p65 to the nucleus but was detected using transgenic reporter mice. The observation that ethanol given 24 hr before dosing with LPS modulated production of some cytokines indicates a persistent effect which does not require continued presence of ethanol.

Background

Acute ethanol exposure can suppress signaling through toll-like receptors (TLR). This signaling normally leads to the production of pro-inflammatory cytokines [1][2][3]. However, studies of this matter have been performed using macrophage-like cell lines or human monocytes following in vitro exposure to ethanol and an inflammatory stimulus, or using macrophages from ethanol-treated mice stimulated ex vivo. A number of studies in the past several years demonstrate that signaling can be remarkably different in vitro (or ex vivo) and in vivo, even when using the same stimulus [4][5][6]. In addition, we found that the pattern of IL-10 and IL-12 production was very different in a macrophage-like cell line in vitro and in the peritoneal cavity or serum in vivo [7]. Therefore, it is important to confirm that the inhibition of TLR4-mediated signaling by ethanol that has been reported in vitro and ex vivo also occurs when both ethanol and the TLR4 agonist are administered in vivo and the signaling events occur in vivo. In most cases, signaling cannot be evaluated using freshly isolated cells because of the transient nature of signaling and the time required to purify the desired cell type from the complex mixture of cells found in lymphoid organs, tissues, or blood. However, we reported previously that B6C3F1 mice have a population of resident cells in the peritoneal cavity that are >85% macrophages [8]. These cells can be obtained very quickly, placed on ice and extracted within a few minutes, thus allowing cellular signaling studies to be conducted using cells in which cellular signaling and any inhibition of that signaling actually occurred in the experimental animal. This approach is utilized here to quantitatively assess the effects of ethanol on signaling over time and cytokine production over time.

The mitogen activated protein (MAP) kinase pathway seems particularly important in TLR4 signaling [9], so key members of this group, ERK and p38, were evaluated. Among several transcription factors activated by TLR4 signaling, AP-1 and NF-B are required for expression of several of the cytokines that are inhibited by ethanol (e.g., IL-6, TNF-, and IL-12) [10][11][12]. Therefore, activation of these transcription factors was evaluated. Assessment of immunological parameters at only one point in time can yield results that are not at all representative of the overall effects over time. In the present study phoshphorylated (activated) ERK and p38 as well as phosphorylated c-Jun (a component of the AP-1 transcription factor) and nuclear p65 (as an indicator of NF-B activation) were evaluated over the course of time and the area under the concentration vs time curve was calculated as an indicator of the overall difference in signaling over time. Similarly, cytokine concentrations in the peritoneal cavity were quantified over time. We have previously shown that IL-6 (as a representative pro-inflammatory cytokine) obtained by peritoneal lavage is not derived primarily from blood but produced locally [13], probably by the peritoneal macrophages in which signaling was evaluated.

The results generally confirmed previous reports of inhibition of signaling beginning early in the TLR4 signaling pathway and extending to the ends of the pathway, the activated transcription factors. However, there was no indication of inhibition of p65 translocation into the nucleus, suggesting that ethanol does not inhibit NF-B activation. However, to insure that activation mediated by NF-B proteins that do not contain p65 was also not inhibited, transgenic mice with an NK-B luciferase reporter construct were evaluated by luminescence imaging in live anesthetized mice.

Results

Effects of ethanol on activation of MAP kinases, AP-1, and NF-B

In a previous study, we reported that ethanol alone at the same dosage and timing as used in the present study does not induce increases in the production of IL-6, IL-10, or IL-12 [13]. Thus, it seemed likely that "ethanol only" controls would not be informative, so they were not included. Data shown in Figure 1 indicate that activation of AP-1 (as indicated by phosphorylation and nuclear translocation of c-Jun, one of the components of AP-1) was suppressed by ethanol, and suppression was maximal at 30 min. Results shown in Figure 2 indicate that activation (as indicated by phosphorylation) of kinases upstream of these transcription factors were significantly inhibited and that the inhibition of p-c-Jun shown in Figure 1 was also significant at 30 min. Suppression was significant (by Student's t test) for at least one time point for all kinases, and suppression of p-p38 was almost significant at 30 min. Phosphorylation had decreased to unstimulated levels within 1 hr, indicating that the role of these signaling mediators was completed in that time and the area under the concentration vs. time curve in this study reflects the complete period of signaling. The area under the concentration vs. time curve values are shown in Table 1. As would be expected on the basis of the graphs in Figure 2, ethanol suppressed the AUC values in all cases. The acti- vation of p-c-Jun was inhibited to a greater extent than p-p38 or p-ERK, and peritoneal cytokines were inhibited to varying degrees, but the ratio of LPS + ethanol to LPS groups was within the range of the values noted for signaling parameters, except that p-ERK was inhibited less than any of the cytokines in which ERK is involved in signaling. Assessing signaling in peritoneal macrophages and cytokines produced by these cells in the peritoneal cavity allows assessment of these parameters following a representative in vivo innate inflammatory response. The results are consistent with the idea that inhibition of signaling contributes to inhibition of cytokine production.

Results shown in Figure 3 indicate that LPS causes substantial activation of NF-B, as indicated by translocation of p65 to the nucleus. Ethanol did not significantly inhibit this activation. However, it remained possible that other forms of NF-B were inhibited by ethanol, so that overall inhibition of NF-B could contribute to inhibition of the induction of cytokines. Therefore, transgenic mice with an NF-B responsive luciferase reporter construct were used to detect activation of all transcription activating forms of NF-B. Results shown in Figure 4 demonstrate significant inhibition of NF-B activation by ethanol at 4 or 5 g/kg, which we have previously reported to produce peak blood ethanol levels of 200 and 300 mg% (43.5 and 65.2 mM), respectively [14].

Inhibition of nuclear translocation of transcription factors by ethanol Figure 1 Inhibition of nuclear translocation of transcription factors by ethanol. Mice were treated with ethanol at 6 g/ kg (by gavage with a 32%, v/v solution). Thirty minutes later, LPS (60 g/mouse) was administered intravenously. Then at the indicated times after LPS administration, peritoneal macrophages were isolated by lavage using ice cold PBS. Nuclear extracts were prepared and the indicated proteins assessed by western blot. Each lane represents a sample from one mouse (n = 2 for naive; n = 4 for most groups; n = 3 for LPS + ethanol at 60 min). Quantitation and statistical analysis of these data are shown in Figures 2 and 3. Figure 2 Activation of signaling molecules induced by LPS in vivo is inhibited by ethanol. Mice were treated with ethanol at 6 g/kg (by oral gavage with a 32%, v/v solution). Thirty minutes later, LPS (60 g/mouse) was administered intravenously. Then at the indicated times after LPS administration, peritoneal macrophages were isolated by gavage using ice cold PBS. Cell pellets were immediately extracted and analyzed for the indicated phosphorylated proteins by western blot. Values shown are means ± SEM (n = 5 mice per group). Values at each time point for LPS-treated and LPS plus ethanol-treated animals were compared by Student's t test and significant differences are indicated by ** (p < 0.01).

Activation of signaling molecules induced by LPS in vivo is inhibited by ethanol

Effects of ethanol on cytokines in serum over time and dependence on time of ethanol administration

Results shown in Figure 5 demonstrate that ethanol inhibits the production of most pro-inflammatory cytokines throughout the time that they are produced in response to LPS. The dosage used in this study (6 g/kg) yields a peak blood ethanol concentration of 400 mg% (87 mM) [15]. Such concentrations have been reported in binge drinkers, but more importantly, inhibition of cytokine production is dose responsive with effects noted at dosages as low as 3 g/kg, by gavage [16]. Because the duration of the ethanol action has not previously been determined in this model, ethanol was administered to different groups 24, 2, and 0.5 hr before administration of LPS ( Figures 5 and 6). The concentration of IL-6 and IL-12 p70 in the serum was significantly decreased (p < 0.05) when ethanol was administered 24, 2, and 0.5 hr before LPS. This 24 hr duration of effects has not been reported previously and is relevant with regard to potential increased risk of infection not just on the day of binge drinking but possibly also the next Ethanol does not alter the translocation of the p65 compo-nent of NF-B to the nucleus Ethanol inhibits NF-B reporter construct activation by LPS in vivo Figure 4 Ethanol inhibits NF-B reporter construct activation by LPS in vivo. Transgenic mice with an NF-B reporter driving expression of luciferase were imaged using an IVIS luminescence imager. Below each image of light emission profiles for the mice is a graph in which the data for those mice were analyzed using the IVIS software for integrated photon emission. Values shown are means ± SEM for the indicated group sizes. Values significantly different from the LPS control are indicated by ** (p < 0.01).

day. The cytokines significantly suppressed by ethanol at one or more time points were IL-6, IL-12 p70, IL-12 p40, and TNF-. The IL-1 response was also decreased, but the decrease was primarily at 1 hr after LPS. The concentration of IL-10 was significantly enhanced by treatment with ethanol 0.5 or 2 hr before LPS. A similar effect was noted in mice treated with polyinosinic polycytidylic acid to activate TLR3 [13]. One unexpected observation was the enhancement of IL-12 production by ethanol administration 24 hr before LPS. Previously such an increase had only been noted for IL-10 [13]. This increase was not observed for IL-12 p40, but it has been established that expression of the genes for IL-12 p40 and p35 are regulated independently and both of these proteins are required for the formation of the p70 heterodimer of IL-12 [17]. Thus, IL-12 p35 may be the limiting factor in IL-12 p70 production, and upregulation of IL-12 p35 could cause increased IL-12 p70 without an increase in IL-12 p40.

Ethanol modulates production of some cytokines in serum, even when administered 24 hr before LPS Figure 5 Ethanol modulates production of some cytokines in serum, even when administered 24 hr before LPS. Mice were treated with ethanol (6 g/kg by gavage) at the indicated times before LPS, and blood and peritoneal fluid samples were obtained for analysis 2 hr after LPS. Values shown are means ± SEM (n = 5). Because of overlapping values, showing statistical results on the graphs is not practical. Major statistically significant outcomes are identified in the text of the results section.

Effects of ethanol on cytokines in the peritoneal cavity over time and dependence on time of ethanol administration

Previous studies indicate that serum and peritoneal cytokines are derived from different sources [13,18]. Because peritoneal macrophages were used in the cellular signaling studies reported here, it seemed appropriate to also assess cytokine production in the peritoneal cavity. Results of this analysis are shown in Figure 6. There were some clear differences in the pattern of ethanol effects on cytokine production as compared to effects on serum cytokines. For example, the peritoneal IL-1 concentration was decreased by ethanol administration at all three time points before LPS. The concentration of IL-6 was decreased less by administration of ethanol 0.5 hr before LPS than by administration 2 or 24 hr before. In contrast, all three times of administration yielded similar inhibition in the serum. The concentration of IL-12 p40 in the peritoneal cavity was decreased less when ethanol was administered 24 hr before LPS than at the other times of administration. In contrast, the concentrations in serum were similar for all three times of administration. The effects of ethanol at all times of administration were similar for TNF- in serum and peritoneal fluid. It should be noted that the much lower concentration of cytokines in peritoneal lavage fluid than in serum may simply reflect the unavoidable dilution of the peritoneal fluid during the lavage process. The amount of dilution is not known, but it may be sufficient that the actual concentrations in the peritoneal cavity would be similar to those in serum.

Discussion

Results shown in this study demonstrate that LPS-induced signaling through TLR4 is inhibited by ethanol in vivo in a mouse model for binge drinking. Rapid isolation of peritoneal macrophages allowed detection of the activation of all tested signaling parameters by LPS and any inhibition caused by ethanol. The mechanism by which ethanol disrupts signaling is not completely understood, but studies conducted primarily in vitro suggest that ethanol disrupts movement of components of the TLR4 receptor complex into lipid rafts and inhibits associated Ethanol modulates production of some cytokines in peritoneal fluid even when administered 24 hr before LPS Figure 6 Ethanol modulates production of some cytokines in peritoneal fluid even when administered 24 hr before LPS. Mice were treated with ethanol (6 g/kg by gavage) at the indicated times before LPS, and blood and peritoneal fluid samples were obtained for analysis 2 hr after LPS. Values shown are means ± SEM (n = 5). Because of overlapping values, showing statistical results on the graphs is not practical. Major statistically significant outcomes are identified in the text of the results section.

receptor clustering, rearrangement of the actin cytoskeleton, and activation of the signaling cascade [19][20][21][22]. The results presented here demonstrate that later signaling events are also inhibited by ethanol and that this occurs in vivo. Similar results were obtained by others following acute ethanol administration to mice in vivo and stimulation of cells with LPS ex vivo [23] and following acute ethanol exposure of human monocytes in culture [19]. Together, these results and the results reported here suggest that inhibition of production of pro-inflammatory cytokines by acute exposure to ethanol is a robust phenomenon that is probably mediated directly by ethanol's effect on ligand binding or events normally induced just after ligand binding to the TLR4 receptor complex.

The inhibition of signaling was greatest 30 min after LPS administration for ERK, p38, and c-Jun. This was the case even though the kinetics of activation were not all the same for these signaling molecules. This suggests that ethanol is most active with regard to signal disruption 1 hr after administration (30 min after LPS). This is near the time at which ethanol reaches maximum blood levels after intragastric administration of the dosage used in this study [14]. It was surprising that the LPS-induced translocation of the p65 component of NF-B to the nucleus was not affected by ethanol. Most of the cytokines affected by ethanol require NF-B for activation, so it would be surprising if inhibition of NF-B activation could not be detected. Inhibition of LPS-induced p65 translocation has been reported in human monocytes treated with ethanol in vitro, but decreased phosphorylation of p65 (which was not assessed in the present study) was found to be more important [24]. It is not clear why no change in p65 translocation was observed here, but we have previously reported inhibition of p65 translocation by acute ethanol exposure in mice treated with polyinosinic polycytidylic acid to activate TLR3 [8]. The same methodology and materials were used in the present study, so it is unlikely the current negative result can be explained as a technical problem. However, the use of NF-B reporter mice clearly demonstrated suppression of NF-B activation. It is possible that p65 enters the nucleus normally in ethanol treated mice but decreased phosphorylation decreases its transactivation function. It is also possible that NF-B composed of subunits other than p65 is responsible for activation of NF-B mediated gene expression and that entry of these subunits into the nucleus is inhibited by ethanol. These issues are under investigation, and results will be reported in a separate publication.

The combination of decreased activation of AP-1 (as indicated by decreased phosphorylation of c-Jun) and NF-B would be expected to inhibit LPS-induced expression of cytokines whose expression is dependent on these transcription factors. This includes TNF- [12], IL-12 [25], IL-1 [26], and IL-6 [27]. All of these cytokines were significantly suppressed at one or more time points in the peritoneal cavity in ethanol-treated mice. The basis for the increased production of IL-10 is not clear, but it does not seem to involve a mechanism reported in human monocytes in which ethanol increases LPS-induced p38 activation, leading to increased heme oxygenase 1, which increases IL-10 gene expression [28]. In the present study, enhanced IL-10 production was associated with suppressed p38 activation. The basis for this difference is not clear, but this could either represent a species difference (human vs. mouse) or a difference in effects of LPS and/ or ethanol in vivo (present study) vs. in vitro [28].

The decreased pro-inflammatory cytokines and increased IL-10 observed here would be expected to decrease resistance to infection, and we have previously reported such a decrease in mice treated by acute administration of ethanol [13]. Cytokines inhibited by ethanol in the present study, such as IL-12, IL-6, and TNF- have been shown to be important in innate resistance to infections [29][30][31].

Increased IL-10 concentrations would be expected to decrease innate resistance to infections [32]. Furthermore, this decrease in some pro-inflammatory cytokines is observed even when ethanol is administered as much as 24 hr before immunological challenge. Thus, the time frame during which the risk of infection is increased might be at least 24 hr.

Differences were observed in the pattern of cytokine expression and effects of ethanol when comparing concentrations of cytokines in the serum and peritoneal cavity. For example, serum IL-1 was not inhibited significantly at 2 hr following LPS by ethanol administered 0.5, 2, or 24 hr before LPS. However, in the peritoneal cavity there was significant inhibition 2 hr after LPS caused by administration of ethanol at 0.5, 2, or 24 hr. Other differences were also noted, which are consistent with our previous conclusion that cytokines in the peritoneal cavity and in the serum are derived predominantly from different cells types which are affected differently both by LPS and ethanol [33]. Some of these differences could also be due to differences in the concentration, rate of clearance, and concentrations of active metabolites of ethanol in liver or spleen (sources for a substantial portion of pro-inflammatory cytokines in serum) as compared to peritoneal cavity. These issues are currently under investigation. In any case, the changes in signaling in peritoneal macrophages and changes in cytokine production in the peritoneal cavity are comparable, which is consistent with the idea that decreased signaling is a major cause of altered cytokine production.

The effects of ethanol at 0.5 or 2.0 hr would be consistent with our previous findings suggesting direct action of eth-anol on macrophages, which requires that the ethanol is present during stimulation with LPS to produce the effect [21,22]. However, inhibition of the production of some cytokines by ethanol administered 24 hr before LPS demonstrates persistent effects as well. Ethanol is completely cleared in this model within 12 hr [14], so effects that persist 24 hr after dosing cannot represent direct action of ethanol, but must represent relatively persistent effects on cells. The mechanism for these effects is under investigation.

The results presented here indicate that activation of cellular signaling induced by LPS in peritoneal macrophages is inhibited by ethanol in vivo. The magnitude of changes in AUC values for signaling molecule activation and changes in AUC values for cytokine concentration were generally similar. Disparate amounts of change would not have supported a cause-effect relationship between inhibition of signaling and inhibition of cytokine production. However, the results shown here as well as abundant results from published studies indicating that signaling inhibitors decrease cytokine production [34][35][36], suggest that a cause-effect relationship between inhibition of signaling and inhibition of cytokine production is feasible. These results were obtained using an animal model in which ethanol has been demonstrated to decrease innate immunity to infection [13], suggesting that the observed changes are functionally relevant with regard to host resistance as well.

Conclusion

The results shown here indicate that acute ethanol exposure inhibits signalling through TLR4 in vivo in a manner similar to that which has been demonstrated in vitro and ex vivo. However, detecting inhibition of NF-B required the use of reporter mice and was not successful when p65 (the most common subunit of activating NF-B heterodimers) was evaluated. The observation that ethanol given 24 hr before LPS inhibits production of some cytokines indicates a persistent effect of ethanol on cells such that inhibition of the response occurs even after ethanol is cleared.

Methods

Mice and treatments

Evaluation of signalling

Cells for assessment of signaling were obtained at selected times after LPS administration by peritoneal lavage using 7 ml of ice cold phosphate buffered saline with 1% bovine serum albumin. The cell pellet from each mouse (after centrifugation at 300 × g for 5 min) was then used for analysis of signaling. Evaluation of p-c-Jun and NF-B (p65) was done by preparing nuclear extracts as in our previous study [37] and using western blot analysis with antibodies to p-c-Jun and NF-B p65 from Santa Cruz Biotechnology (Santa Cruz, CA). Secondary antibodies conjugated with horseradish peroxidase were obtained from the same source. Bands were visualized using a chemiluminescence kit (Amerasham, Chalfont St. Giles, UK). Images were obtained using a VersaDoc 3000 (BioRad, Hercules, CA). Relative quantitation of band intensities was accomplished as described previously using NIH Image software [37]. Actin was used as a loading control in these analyses. The quantity of phospho-p38 and phospho-ERK (p-p38 and p-ERK) was determined using a cell based assay kit (FACE, Active Motif, Carlsbad, CA).

NF-B reporter mice

Transgenic mice with a reporter construct consisting of a luciferase gene with an NF-B dependent promoter were produced by Carlsen and colleagues [38] and obtained through Xenogen (Alameda, CA). Mice were treated with ethanol 30 min before stimulation of NF-B activation. Mice were treated with bacterial lipopolysaccharide (from E. coli 0128:B12, Sigma Chemical Co., catalog #L2755) by administering 60 g/mouse, intravenously. Two hr after LPS, mice were anesthetized with 150 mg/kg ketamine and 5 mg/kg xyalzine intraperitoneally. Mice were then treated with luciferin (15 mg/kg, intraperitoneally), and imaging was done 5-10 min later using an IVIS imager (Xenogen, Alameda, CA).

Evaluation of cytokine concentrations

Cytokines in serum and peritoneal lavage fluid were analyzed by multiplexed bead array analysis using a kit from BioRad and a Bioplex reader. Peritoneal lavage was done initially by removing the skin from the abdomen without puncturing the membrane of the peritoneal cavity then injecting 1 ml of ice cold phosphate buffered saline with 1% fetal bovine serum. The abdomen was massaged to dislodge cells and mix, and a 25 g needle and syringe was used remove the fluid (usually 0.7 ml of the original 1 ml was obtained). Cytokines were assessed at various times after challenge with LPS (indicated in figures).

Data analysis and statistics

Authors' contributions

SBP designed the study, identified appropriate methods, helped with harvesting blood and peritoneal fluid, and wrote the manuscript. RF performed most of the experimental procedures and edited the manuscript. Both authors reviewed and approved this manuscript.

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Figure 3
3Ethanol does not alter the translocation of the p65 component of NF-B to the nucleus. Nuclear extracts were prepared as described in materials and methods, and analyzed by western blot for p65. Values shown represent mean pixel density ± SEM (n = 5 mice per group). Values at each time point for LPS-treated and LPS plus ethanol-treated animals were compared by Student's t test and no significant differences were identified.

Area under the curve values for cytokine expression over time and activation of signaling molecules over time were calculated by entering the data in Prism 4.0 software (GraphPad, LaJolla, CA) and selecting area under the curve in the analysis menu. Statistical comparisons were done by Analysis of Variance (ANOVA) or Student's t test (to compare 2 groups) at each time point, using Prism 4.0 software (GraphPad Software, LaJolla, CA).

Table 1 :
1AUC Values for signaling and cytokine productionLPS
LPS + Ethanol
Ratio

p-p38
190694
106858
0.56
p-ERK
175677
129677
0.74
p-c-jun
33
5
0.15

IL-6
12885
4829
0.37
IL-12
1601
669
0.42
TNF-
65
12
0.19
IL-1
77
40
0.51

Animal care and use was conducted in accord with the NIH Guide and the regulations of Mississippi State University. The animal care and use program at MSU is accredited by the American Association for Accreditation of Laboratory Animal Care. Ethanol was administered by gavage using a 32% solution (v/v) in tissue culture grade sterile water. This has been shown to yield blood ethanol concentrations relevant to binge drinking and not to cause necrosis or other gross histopathological changes in the gastrointestinal tract[14]. This model has been cited and used by a number of other investigators. Peritoneal macrophages were activated in vivo by intravenous administration of lipopolysaccharide (LPS, Sigma Chemical Co., St. Louis, MO, from E. coli 0111:B4) at 60 g/mouse.Female C57Bl/6 × C3H F1 (B6C3F1) mice were obtained
from Charles River Labs through the National Cancer
Institute's animal program. Mice were allowed to recover
from shipping stress for at least two weeks before use in
experiments at 8-16 weeks of age. They were housed 5 per
cage in a temperature and humidity controlled facility.
Sentinel mice housed in the same room were negative for
common mouse pathogens during the period of this
study.

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AcknowledgementsThis work was supported by a grant from the National Institute on Alcohol Abuse and Alcoholism (#R01AA009505).
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