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Vanadium Pentoxide Inhalation Provokes Germinal CenterHyperplasia and Suppressed Humoral Immune Responses

G. Pinon-Zarate, V. Rodriguez-Lara, M. Rojas-Lemus, M. Martinez-Pedraza,A. Gonzalez-Villalva, P. Mussali-Galante, and T.I. Fortoul A. Barquet F. Masso L.F. Montano

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May 1, 2008 9:44 801 UIMT˙A˙308740

Journal of Immunotoxicology, 5:1–7, 2008Copyright c© Informa Healthcare USA, Inc.ISSN: 1547-691X print / 1547-6901 onlineDOI: 10.1080/15476910802085749

Vanadium Pentoxide Inhalation Provokes Germinal CenterHyperplasia and Suppressed Humoral Immune Responses

G. Pinon-Zarate, V. Rodriguez-Lara, M. Rojas-Lemus, M. Martinez-Pedraza,A. Gonzalez-Villalva, P. Mussali-Galante, and T.I. FortoulDepartamento de Biologıa Celulary Tisular Facultad de Medicina, UNAM, Mexico5

A. BarquetLaboratorio de Genoma de Patogenos, InDRE, SS, Mexico

F. MassoDepartamento de Biologıa Celular, Instituto Nacional de Cardiologıa “Ignacio Chavez”, Mexico

L. F. Montano10

Laboratorio de Inmunologıa, Departamento de Bioquımica, Facultad de Medicina, UNAM, Mexico

Vanadium, an important air pollutant derived from fuel prod-uct combustion, aggravates respiratory diseases and impairs car-diovascular function. In contrast, its effects on immune response15are conflicting. The aim of our work was to determine if spleens ofvanadium-exposed CD1 mice showed histological lesions that mightresult in immune response malfunction. One hundred and twelveCD-1 male mice were placed in an acrylic box and inhaled 0.02M vanadium pentoxide (V2O5); actual concentration in chamber20≈1.4 mg V2O5/m3) for 1 hr/d, twice a week, for 12 wk. Control miceinhaled only vehicle. Eight mice were sacrificed prior to the expo-sures. Eight control and eight V2O5-exposed mice were sacrificed24 hr after the second exposure of each week until the 12-wk studywas over. Another 8 mice that completed the 12-wk regimen were25immunized with recombinant Hepatitis B surface antigen (HBsAg;three times over an 8-wk period) before sacrifice and analyses oftheir levels of anti-HBsAg antibody (HBSAb) using ELISA. In allstudies, at sacrifice, blood samples were obtained by direct heartpuncture and the spleen was removed, weighed and processed for30H-E staining and quantitation of CD19 cells. The results indicatedthat the spleen weight of V2O5-exposed animals peaked at 9 wk (546± 45 vs. 274 ± 27 mg, p<0.0001) and thereafter progressively de-creased (321 ± 39 mg at 12 wk, p < 0.001; control spleen = 298± 35 mg). Spleens of V2O5-exposed animals showed an increased35number of very large and non-clearly delimited germinal centers(that contained more lymphocytes and megakaryocytes) comparedto those of control mice. In addition, their red pulp was poorlydelimited and had an increase in CD19+ cells within hyperplasicgerminal nodes. The mean HBsAb levels in immunized control mice40were greater than that in the exposed hosts (i.e., OD = 0.39 ± 0.03vs. 0.11 ± 0.05, p < 0.01). HBsAb avidity dropped to a value of 40

Received 15 October 2007; accepted 15 January 2008.Address correspondance to: Teresa I. Fortoul, Departamento de Bi-

ologıa Celular y Tisular, Edificio A, Third Floor, Facultad de Medicina,UNAM, Mexico 04510; e-mail: fortoul@servidor.unam.mx

in V2O5-exposed animals vs. 86 in controls (p < 0.0001). We con-clude that the chronic inhalation of V2O5, a frequent particle PM2.5

component, induces histological changes and functional damage to 45the spleen, each of which appear to result in severe effects on thehumoral immune response.

Keywords Vanadium, pentoxide, humoral immune response

INTRODUCTION 50

Vanadium (V) has recently become recognized as an impor-tant air pollutant (Fortoul et al., 1996, 2002) in the atmosphereof Mexican cities. There, as elsewhere, it is a component ofresidual oil fly ash (ROFA; Samet et al., 1999) that enters theorganism, mainly by inhalation (Brook et al., 2004; Nemmar 55et al., 2004). Another important source of vanadium is as anatmospheric contaminant generated from combusted fuel prod-ucts. Occupational exposure to one major chemical form thatis also found in urban air, vanadium pentoxide (V2O5), occursduring the cleaning of oil-fired boilers and furnaces, during han- 60dling of catalysts in chemical plants, and during the refining,processing, and burning of vanadium-rich fossil fuels, espe-cially Venezuelan or Mexican oils (Nriagu, 1998; Ivancsits et al.,2000).

There is ample epidemiological evidence showing that ex- 65posure to particulate matter with diameters less than 2.5 µm(PM2.5)-bearing air pollution aggravate respiratory diseasesand impairs the cardiovascular function (Dockery et al., 1993;Ackermann-Liebrich et al., 1997; Pope et al., 1999; Gold et al.,2000; Laden et al., 2000), especially if the individuals are ex- 70posed to peaks of air pollution (Peters et al., 1999). At thisparticle size, metals are adsorbed by inhalation and enter the

1

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2 PINON-ZARATE ET AL.

systemic circulation, thus exerting its toxic effect in other or-gans and tissues (Dockery and Pope, 1994).

There are conflicting results in relation to Vanadium exposure75and the immune response. Inhalation of vanadium decreases thephagocytic index and the inducible production of interleukin(IL)-6 and interferon (IFN)-γ by rats pulmonary macrophages(Cohen et al., 1997). Similarly, the exposure of peripheral bloodmononuclear cells to vanadium reduces the release of IFNγ80and the proliferation of PHA-stimulated cultures, although IL-5 release shows a vanadium concentration-dependent bi-modalbehavior (Di Gioacchino et al., 2002). Although there are slightdifferences between those studies probably related to species dif-ferences or method of administering the vanadium, the tendency85is to suppress the immune response. We have previously shownthat the chronic inhalation of V2O5 in CD1 mice induces anincrease in the number and size of platelets (Gonzalez-Villalvaet al., 2006). We have also seen that these vanadium-treated miceshow macroscopic splenic lesions that might lead to histologic90and functional alterations. Therefore, the aim of the work re-ported here was to determine if the spleens of vanadium-exposedCD1 mice showed histological lesions that might induce immuneresponse malfunctions.

MATERIALS AND METHODS95

MiceEight-week-old CD-1 male mice (bred in the Instituto de

Investigaciones Biomedicas, UNAM) weighing 33 ± 2 g werehoused in hanging plastic cages kept in an animal facility (withaverage 21◦C temperature, 57% humidity, controlled lighting100[12:12 hr light/dark regime]) and fed Purina rat chow and waterad libitum. The experimental protocol was in accordance to theAnimal Act of 1986 for Scientific Procedures.

Exposure RegimensInhalation exposures were performed as described by (Avila-105

Costa et al., 2004). Briefly, exposures were performed with a0.02 M V2O5 (99.99% purity, Sigma-Aldrich, St Louis, MO)suspension in deionized water containing 100 µl Tween-20/100ml of suspension. The aerosol inhalation chamber was an acrylicbox measuring 45 cm × 21 cm × 35 cm that could house11025 mice/session and had a total volume of 3.3 L. The ultra-nebulization to generate the atmospheres was carried out usinga DeVilbiss Ultraneb 99 (Somerset, PA) system that maintained aconstant flow of 10 L/min; according to the manufacturer, about80% of the aerosolized particles reaching the mice would be ex-115pected to have a mass median aerodynamic diameter (MMAD)of 0.5–5.0 µm.

The concentration of V2O5 in the inhalation chamber wasquantified as previously described (Fortoul et al., 2002). A 0.22µm filter was positioned at the external outlet of the ultranebu-120lizer during each exposure period. At the end of each exposure,the filter was removed and weighed. Vanadium concentration

was analyzed in each filter in a graphite furnace atomic absorp-tion spectrometer (Perkin Elmer Mod. 2380, Shelton, CT), usinga 318.4 nm wavelength and a slit of 0.7 nm. The assay detection 125limit was 0.37 ppm. Accuracy was assessed by three randomdeterminations of seven different standard solutions, preparedwith the same chemical reagents used during the metal analysis.Each sample was analyzed in triplicate.

In these studies, sets of 25 mice were placed in the acrylic 130box to inhale V2O5 for 1 hr/d, twice a week, for 12 wks. Previousexperiments in our laboratories have shown that the V2O5 con-centration and the particular regimen used here truly induced amodel of chronic vanadium exposure (Avila-Costa et al., 2005).There were a total of 112 mice in the vanadium exposure cohort 135at start of the experiment; initially, 5 separate runs per day toaccommodate this number, decreasing thereafter as the numbersof mice in the cohort were reduced due to need for analyses ofendpoints. Control mice (112 also) inhaled only the vehicle —deionized water/Tween—during each exposure. 140

Tissue Sampling and PreparationMice from each group (n = 8/cohort) were sacrificed 24 hr

before the first V2O5 or vehicle exposure and, thereafter, 8 con-trols and 8 exposed mice were sacrificed 24 hr after the secondV2O5 exposure of each week until the 12-wk period was over. 145Each time, the mice were anesthetized with sodium pentobar-bital. Blood samples from direct heart puncture were obtained.The spleen was then removed, weighed, and processed for his-tological examina-tions (i.e., paraffin embedding and stainingwith Hematoxylin-Eosin for subsequent light micro-scopy eval- 150uation). Images from the spleen of control and exposed animalswere captured at 10X and 20X in a BX51 Olympus microscope.All samples were evaluated by 2 independent observers.

To identify B-lymphocytes in the spleens, immunohisto-chemical staining for CD-19 (Cell Signaling, Technology Inc., 155Danvers, MA) was performed on spleen frozen sections (5µm thickness) using previously-published protocols (Mussali-Galante et al., 2005). Immuno-reactivity of the anti-CD19 an-tibody was visualized by incubation in a solution contain-ing 0.05% 3,3′-diaminobenzidinetetrahydrochloride substrate 160(Zymed Labs Inc., San Francisco, CA)

Determination of Antibodies to Hepatitis B SurfaceAntigen (HBsAg)

To evaluate antibody production in the vanadium-exposedanimals, the animals were inoculated with a well-defined 165T-dependent antigen (Woo et al., 2006), since these requirean adequate B-lymphocyte-T-lymphocyte collaboration to ini-tiate an immune response. Sixteen animals that had completedthe 12-wk regimen (8 control and 8 V2O5-exposed mice) wereimmunized intraperitoneally with 4 µg recombinant HBsAg 170(EngerixB, GlaxoSmithKline, Mexico) in FCA (Freund’s com-plete adjuvant). The first two doses were applied at 3-wk in-tervals and a final dose of the HBsAg without adjuvant was

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VANADIUM AND HUMORAL IMMUNE RESPONSE 3

applied intravenously via the tail 5 wk after the second dose.All mice were then sacrificed three days later. Blood from eight175non-immunized CD-1 mice (maintained under same conditionsas the experimental animals) served as negative-HBsAg con-trols. Blood and spleen samples were isolated as previouslydescribed.

ELISA plates (Maxisorp, NUNC) were incubated with 20 ng180recombinant HBsAg/well in 0.05 M carbonate buffer (pH 9.2)for 2 hr at room temperature, followed by an extensive wash withPBS-0.3% Tween 20. The plates were then incubated another 90min with 1% bovine sera albumin in PBS before another exten-sive wash was performed. Aliquots (100 µl) of 1:100, 1:200,1851:400 and 1:800 dilutions of mice sera in PBS-0.1% Tween 20(PBS-T) were then added to triplicate designated wells and theplates incubated for 2 hr at room temperature. At the end ofthe incubation, the plates were extensively washed with PBS-Tbefore wells received 100 µl of a 1:2000 dilution of peroxidase-190labeled goat anti-mouse IgG (Sigma) in PBS-T for 1 hr at roomtemperature. The plates were then extensively washed with PBS-T and 0.05% o-phenylene-diamine in 0.1 M citric acid sub-strate was then added to each well. After 20 min, the reactionwas stopped with 3 N HCl and the plates read at 490 nm in195a BioTek ELx808 ELISA reader with Gen5 software (BioTekCorp, Winooski, VT).

Avidity of antibody to HBs (HBs-Ab) was determined by theurea method as described by Wilson et al. (2006). Briefly, a Max-isorp ELISA plate was incubated with 20 pg/well of recombinant200HBs-Ag (diluted 1:100) as previously noted. After washing theplate, a 1:200 dilution of mice sera in PBS-T were incubated for90 min before adding 150 µl of 8 M urea for 10 min; a dupli-cate plate was made where no urea was added. Both plates werethen extensively washed with PBS-T before wells received 100205µl of a 1:2000 dilution of peroxidase-labeled goat anti-mouseIgG in PBS-T for 1 hr at room temperature; the plates were thenprocessed as above. The avidity results were derived from theformula: 100% × (mean OD of urea-treated wells/mean OD ofnon-treated wells) and each value represents the percentage of210specific antibody bound to the plate after urea treatment. Eachserum sample was tested in triplicate.

Statistical AnalysisThe results were analyzed by means of a Mann–Whitney U-

test and an ANOVA test with a Tukey’s post-hoc test (SPSS215software, 10.0 release). The difference between control and ex-posed animals was considered significant at a p-value less thanor equal to 0.05.

RESULTSThe V2O5 concentration in the inhalation chamber, based on220

the average concentration calculated from examination of all thefilters from the 24 exposures was 1436 ± 273 µg/m3. Levels ofvanadium in the control systems were consistently below thelevels of detection of the system (i.e., < 0.37 ppm).

Animal Health 225

Eight of the V2O5-exposed and 5 control mice perished of anapparent ear infection during Wk 5 of the regimen. The infec-tions that led to these deaths in both control and V2O5-exposedmice appeared to be due to animal (male-male) aggression (i.e.,ear biting is one of the tactics observed in these actions) that 230led to subsequent infection and ultimately death. None of theremaining mice in either treatment group showed signs of de-veloping this or any other health condition such as asthma, upperrespiratory tract infection, diarrhea. Importantly, no mice diedas a direct result of the vanadium exposures. 235

Spleen WeightThere was no difference in the basal versus the final weight

of mice spleen in control animals (262 ± 19 mg vs. 298 ± 35mg at 12 wk; p = 0.28). There was a gradual increase in themice spleen weight as the number of exposures to vanadium 240was progressing (274 ± 27 mg to 546 ± 45 mg after 9 wk; p <

0.0001), afterwards the spleen weight started to diminish untilit reached a 321 ± 39 mg value at the end of the experimentalperiod (12 wk; p < .001) (Figure 1).

Spleen Histological Findings 245The spleens of control mice contained well-delimited areas

of white and the red pulp (Figure 2A). In the white pulp, lympho-cytes and well-structured germinal centers with their respectivecentral artery were clearly defined, as were some megakary-ocytes (Figure 2B). The spleens of animals exposed to vanadium 250showed an increased number of non-clearly delimited germinalcenters that were larger and contained more lymphocytes thanthose in control spleens (Figures 2C). These samples also con-tained an important increase in numbers of megakaryocytes andtheir red pulp was poorly delimited (Figures 2D). The germinal 255centers boundaries were well defined in controls as compared tothose in the exposed mice. Germinal centers from control spleens

FIG. 1. Spleen weight (mg) of mice exposed to vanadium for different timelengths, compared with controls. The results are expressed as the mean ±SD.

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4 PINON-ZARATE ET AL.

FIG. 2. (a) Spleen from control mice in which red (RP) and white pulp were clearly delimited. Megakaryocytes are also identified (arrowhead). (b) The boundariesof the lymphatic nodules with their germinal centers (GC) are well delimited by red pulp (RP). In exposed mice (c) lymphocytes and germinal centers increase inquantity and in size (GC) and red pulp is almost inexistent. (d) Lymphatic nodules increase in size as well as its germinal centers. Megakaryo-cytes also increasein size and in quantity (arrowhead). Hematoxylin and eosin staining was used throughout.

contained scanty CD19 B-lymphocytes; interestingly, spleensfrom vanadium-exposed animals showed an increased numberof CD19 cells, with a well-defined location pattern within hyper-260plastic germinal nodules (Figure 3).

FIG. 3. Spleen frozen sections to identify B-lymphocytes by CD-19 stain-ing. Vanadium-exposed mice showed an increase in stained cells (arrows) (b)compared with controls, where a weak stain was observed (a). 20X.

HBs-Ab ResponseThe HBs-Ab IgG mean OD value in the sera of non-

immunized control animals was 0.070 ± 0.002, whereas themean value for control HBs-Ag-immunized mice was 0.390 ± 2650.030 (p < 0.001). Immunized mice exposed to vanadium hada mean OD value of 0.110 ± 0.050 (p < 0.01 vs. non-exposedanimals) (Table 1).

HBsAb AvidityThere was not only a difference in antibody concentra- 270

tion, but also in avidity. The mean OD value of antibodiesbound to the plate before and after urea treatment in the con-trol HBsAg-immunized group was 0.752 ± 0.249 and 0.682± 0.168, respectively; in contrast, these values were 0.608 ±0.259 and 0.259 ± 0.128 in the vanadium-exposed mice (latter 275value significantly different from control at p < 0.003). Themean of the individual avidity values in the control HBsAg-immunized group was calculated to be 86; this value droppedto 40 in mice that had previously been exposed for 12 wkto vanadium (p < 0.0001) (Table 1). These results demon- 280strate that there is quite likely a deficiency in the histologicallyabnormal spleens of vanadium-exposed mice to induce VDJrecombination(s).

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VANADIUM AND HUMORAL IMMUNE RESPONSE 5

TABLE 1Hepatitis B surface antibody concentration and avidity in

vanadium-exposed and control (non-vanadium-treated) CD1mice

Non-vanadiumtreated Vanadium-treated

Mean OD for HBsAb level 0.390 ± 0.030 0.110 ± 0.050∗

HBsAbs avidity endpoints:Mean OD without Urea 0.752 ± 0.249 0.608 ± 0.259Mean OD with Urea 0.682 ± 0.168 0.259 ± 0.128∗∗

Mean Avidity 86.00 ± 14.59 40.00 ± 8.22∗∗∗

HBs-Ab IgG mean OD value as determined by ELISA. ∗Statisticallysignificant vs. control at p < 0.01. Antibody avidity in the sera ofmice (n = 8 per group) that completed the HBsAg immunizationscheme was determined by urea-treatment assay. ∗∗Significant differ-ence between urea and non-urea assays (at p < 0.003). Avidity valuerepresents mean (± SD) of the individual avidity values in respec-tive control and 12-wk vanadium-exposed HBsAg-immunized mice.∗∗∗Significant difference in avidity between vanadium-exposed andcontrol mice at p < 0.0001.

DISCUSSION AND CONCLUSIONSIt is an established fact that certain environmental heavy met-285

als can alter the immune response of laboratory animals andprobably humans as well. Both stimulation and suppressionof immune responses have been demonstrated in contaminant-exposed animals (Exon, 1984). The source of heavy metals ex-posure is unlimited: paint, insecticides, fungicides, rat poisons,290household disinfectants, automobile catalytic converters, dieselexhaust, coal-burning power plants, steel and metal foundries,sewage sludge, industrial wastes, fish, mercury amalgam fill-ings, and many others. Certain people risk increased exposurebecause of where they live, their occupation or their lifestyle;295others may be genetically predisposed. Nevertheless, the ap-parent risk-free air pollutants have been linked to serious andlife threatening problems such as sudden infant death syndrome(Dales et al., 2004).

The effects of inhaled pollutants in the bronchoalveolar tract300have been widely analyzed. It has recently been shown that ul-tra fine particles are able to translocate from the lung into thesystemic circulation in hamsters and humans (Nemmar et al.,2004). These observations have proven to be of such interestthat the effect of inhaled pollutants on immune-related organs is305emerging as a new area of research.

Vanadium (V) has become one of the foremost studied ele-ments in the last few years, as its importance as an air pollutanthas become increasingly apparent (i.e., see Fortoul et al., 2002;Cohen, 2004). It enters the organism primarily by inhalation310(Brook et al., 2004); once entrained it cancause airway diseasesin rats that are similar to the pathologies of asthma and bronchi-tis in humans (Bonner et al., 2000). For these reasons, in part,our laboratories have undertaken studies to better understand the

toxicologic consequences from repeated exposures to airborne 315vanadium, as occurs regularly in the citizens exposed to urbanatmospheres in many cities, including Mexico City.

Because the route of administration/total numbers of expo-sures to vanadium can modify experimental outcomes (Avila-Acosta et al., 2005), we feel it is critical to emphasize that these 320experiments were done with mice chronically exposed to air-borne vanadium. The 0.02 M V2O5 atmospheric concentrationwas used because previous experiments with lower levels (e.g.,0.005 and 0.01 M) failed to induce any visible histologic dam-age. Similarly, these earlier studies indicated that clear differ- 325ences in the extent of tissue damage seemed to be proportional toboth the V2O5 concentration used and the number of inhalationexposures to any fixed dose of the agent.

The anatomical changes observed in vanadium-exposed an-imals were unanticipated. In contrast to the spleen, it was ex- 330pected that the thymus would be a more preferential target forthe toxicity of the inhaled vanadium. Unlike the spleen, the thy-mus has seemed to be a rather fragile organ; we have observedalterations in the percentage of T-lymphocyte subpopulationsafter just one V2O5 exposure (data not shown). Other Investiga- 335tors have seen similar effects on the thymus/ related functionsafter host exposure to vanadium. For example, the introduc-tion of vanadium into a phosphorus-deficient diet increased thenumber of CD4+ T-lymphocytes and antibody titer to SRBC(albeit afterwards there was a diminution of the effect) in chicks 340(Qureshi et al., 1999).

Our results also differ from those of other previous stud-ies. For example, al-Bayati et al. (1989) showed that while thekidneys in vanadate-injected animals suffered severe dose- andtime-dependent histological degenerative and necrotic changes 345that were accompanied by edema, vascular congestion, andglomerular membrane thickening, spleens of these hosts werenormal. Similarly, a recent report by the U.S. National Toxicol-ogy Program (NTP, 2002) showed that mice and rats exposedto doubling doses of V2O5 (up to 32 mg V2O5/m3) for 6 hr/d, 5 350d/wk, either for 16 d or for 3 mo, had an increase in lung weightas well as lung inflammation and hyperplasia. Interestingly, onlyrats exposed to the highest levels (i.e., 8 mg/m3) displayed signif-icant effects on their spleens’ weights and lymphocytic contents.These same doses had no comparable effects in the co-exposed 355mice.

Last, it is interesting to note that the mortality rates for theexposed rodents in the NTP study were very high relative to thatin our studies (i.e., none died here). It is likely that the variablesgoverning the NTP animals’ exposures (i.e., combining the fac- 360tors of exposure time, frequency, and vanadium concentrations,those rodents seeing ≈ 8 mg V2O5/m3 were ultimately exposedweekly to levels of V2O5 ≈ 85-fold greater than the mice in ourexperiments, i.e., 620 µg [5 regimens] vs. 8.4 µg [2 regimens])were a major reason for these differences in mortality. However, 365this still leaves open the question as to why even with this muchgreater amount of exposure, these NTP mice failed to displayany of the splenic changes that we note here.

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6 PINON-ZARATE ET AL.

We believe that the observed increases in organ weight andthe numbers of germinal centers (together with their abnormal370structures) are apparently a form of non-antigenic-based toxic-ity associated with chronic vanadium exposure. This is based,in part, on the fact that vanadium pentoxide has never been rec-ognized to act as an antigen and as such, is not acting to inducea “host humoral response” to the compound itself. Interestingly,375the increased amount of CD19+ cells found in the spleens of thechronically V2O5-exposed animals was also not related to anti-body production; the concentrations of anti-HBsAg antibody inthese mice was shown to be significantly lower in comparisonwith that in control HBsAg-treated hosts. Based on these two380sets of findings, it is becoming increasingly possible that, similarto what is seen with B-lymphocytes from chronic lymphocyticleukemia patients with spleen manifestations, the CD19+ cells ofV2O5-exposed mice have a higher expression of CD44 (Baireyet al., 2004) thus favoring their spleen “homing” through nu-385merous ligands (Naor et al., 2002). Further studies are neededto elucidate the mechanism(s) underlying our observations and,more importantly, the physiological relevance of these (immune)changes in the spleens of the exposed hosts.

Beyond these effects on the spleen, the results of these studies390also showed that chronic V2O5 inhalation affected the amountsof high avidity antibodies (i.e., these were greatly diminished)generated in the mice. These outcomes are somewhat akin tothose that have been observed in studies examining effects onantibody affinity from exogenous (like tetrachlorodi-benzo-p-395dioxin; Inouye et al., 2003) or intrinsic (i.e., like aging; Han et al.,2003) factors. Antibody avidity, also called functional affinity,depends on both antibody affinity and its ability to engage mul-tiple epitopes on the antigen. The former is the result of somaticmutation that is carried out in the germinal centers, mainly of the400spleen, although somatic mutations can occur in the absence ofgerminal centers (Kato et al., 1998; Kim et al., 2006). It is clearthat antibody avidity was severely affected by the histologicalchanges seen in the spleen.

Although the molecular basis of somatic mutation405remainselusive, recent discoveries suggest a role fortranscriptionor transcription-related factors (Diaz et al.,2001; Peters and Storb, 1996). Short-term exposure to vana-dium can stimulate activation of multiple transcription factors(Chen et al., 1999; Ding et al., 1999; Huang et al., 2000;410Wang et al., 2003), whereas the long-term exposure to V2O5

suppresses more than 1000 genes, including 298 genes relatedto regulation of transcription (Ingram et al., 2007), especiallyinterferon regulatory factors. It has been postulated that low-affinity B-lymphocyte clone recruitment and selection for both415long-lived plasma cells and memory B- lymphocyte pathwaysin the germinal center depends on the transcription factor IRF-4(interferon regulatory factor-4; Benson et al., 2007). IRF-4is necessary for class switch recombination and the plasmacell differentiation at exit from the germinal center (Cattoretti420et al., 2006); its absence would impact B-lymphocyte somaticmutation (as our results seem to suggest) and the long-term

humoral immune response. Preliminary results seem to supportthe latter.

In summary, the chronic inhalation of vanadium pentoxide, a 425frequent component of PM2.5 ambient pollution, induces his-tological changes and functional damage to the spleen, thusseverely affecting the humoral immune response of the exposedhost. This could be of particular interest to those who seek tounderstand the adverse health outcomes or the low response to 430vaccines (Idel et al., 1994) among children with strong environ-mental exposures to PM2.5.

ACKNOWLEDGMENTSThe authors are grateful to Ms. Veronica Rodriguez and

Ms. Judith Reyes Ruiz, for technical assistance. This work was 435partially supported by grants IN-224906, IN-200606 and IN-225106-2, from DGAPA-UNAM.

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