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Alterations in the pulmonary and systemic immune response in rats exposed to coal fly ash
ELSEVIER
Immunopharmacology 29 (1995) 103-109
Alterations in the pulmonary and systemic immune response in rats exposed to coal fly ash* Shashi Dogra*, Ashok Kumar Khanna, Jawahir Lal Kaw lndustricd Toxico1og.v Research Centve, P.O. Box No. 80, Mahatma Gandhi Mug. Lucknow-226001, India
(Received 27 June 1994; revision received 30 August 1994; accepted 5 September 1994)
Abstract The effect of intratracheally injected fly ash on the development of pulmonary and systemic immunity was studied in rats. Following intratracheal and intraperitoneal immunisation with sheep red blood cells (SRBC) there was an appearance of antibody forming cells (AFC) in lung associated lymph nodes (LALN) of animals exposed to either fly ash or physiological saline. Enumeration of AFC in LALN after immunisation by either of the routes, revealed a reduction in the number of AFC in LALN of fly ash exposed rats in comparison to saline exposed animals. The reduction in the number of AFC was more pronounced after exposure of Cd-coated fly ash. The AFC appeared in the spleen only after immunisation through intraperitoneal route and the number of AFC in spleen of the fly ash and saline exposed group of animals did not show any significant difference. These results demonstrate that fly ash burden of lungs results in an impairment of the local immune response of the lungs without an associated effect on the systemic immunity. Key\+jor&s: Fly ash; Lung; Immunity; Antibody-forming
cell; Lymphocyte
1. Introduction Fly ash, a common air pollutant produced during coal combustion for energy generation in thermal power plants, has been reported to produce pneumoconiosis (Golden et al., 1983). It contributes significantly to air pollution due to the presence of a larger number of potentially toxic metals and carcinogenic compounds (Aranyi et al., 1979; Fisher and Wilson,
1980; Weisman
et al., 1983). By virtue
of
offering a large surface area, fly ash can adsorb heavy
metals released into the environment from the toxic fumes of diesel exhaust, motor vehicles and numerous industrial activities. Inhalation of Cd, one of the toxic metals reported to be contained in fly ash (Hayes et al., 1980), has been reported to produce pulmonary disorders and an alteration in immunologically specific mechanisms (Morgan et al., 1982; Mellman and Ukkonen, 1985; Vredevae et al., 1985). Earlier studies on the immune status of lungs in animals
exposed
to fly ash after lung immunisation
have demonstrated immunosuppressive potential of fly ash (Burns and Zarkower,
* Corresponding author. Abbreviations: AFC. antibody forming cells; FCS, foetal calf
serum; HA, haemagglutination; LALN. lung-associated lymph nodes; SRBC, sheep red blood cells * ITRC Communication No. 188I. 0162-3109:95:SY.S00 1995 Elsevier Science B.V. All rights reserved SSDI 0162-3109(94)00049-2
1982; Bite et al., 1987;
Dogra and Kaw, 1993). In the present investigation we report studies on systemic immunity in rats exposed to fly ash and extend our earlier studies to include the effect of Cd-coated fly ash on local lung immunity.
104
2. Material
S. Dogru et al. : Immunopharmacology
and methods
2.1. Dust Fly ash was obtained from a thermal power station in India and an approx. 5-pm particle was prepared. The details of the method of preparation of the fly ash samples and elemental composition was reported earlier (Kaw et al., 1990). The fly ash particles used contained more than 90% of the particles less than 5 pm in diameter. The fly ash particulates were suspended in autoclaved saline, sonicated at 100 amp for 2 min and well agitated before intratracheal instillation into rats.
29 fl99j)
IO3- IO9
oculated I/T with 11.6 mg of native fly ash SUSpended in 0.5 ml saline. Animals of group 2 were inoculated with 11.6 mg of Cd-coated fly ash, while group-3 animals served as controls and were inoculated I/T with 0.5 ml saline alone. This set of rats was subsequently employed for I/T immunisation to evaluate local immunity. The dose of fly ash was selected on the basis of pilot experiments designed to select amount of fly ash that could adsorb an amount of Cd equal to its l/5 LD,O value. 2.4. Immunisation of animals for studying systemic immune response
2.2. Coating of$y ash with Cd Cadmium chloride (CdCl,, BDH) was used to coat native fly ash by the method described by Aranyi et al. (1979). Briefly, a 0.5 ‘& solution of CdCl, was prepared in distilled water and 100 mg of fly ash was added to 5 ml of CdCl, solution. Precipitation of the metal hydroxide was carried out with 1 N NaOH and the precipitate was dried at 100°C. The dried precipitate was heated at 675 oC in a platinum crucible for 1 h in a muffle furnace. The amount of Cd adsorbed on fly ash was determined in an atomic absorption spectrophotometer after acid digestion. Dried Cd-coated fly ash, thus prepared, was stored for further use. 2.3. Animals and experimental protocol Female albino rats from the Industrial Toxicology Research Centre (Lucknow, India), animal colony, weighing 200-250 g, fed freely on pellet diet (Hindustan Lever, Bombay, India) and having free access to drinking water were used throughout the experiment. Broadly two sets of experiments were performed. In set I the animals were divided into 2 groups. The rats of group 1 were inoculated intratracheally (I/T) 11.6 mg of native fly ash suspended in 0.5 ml saline under light ether anaesthesia. The rats of group 2 served as controls and were similarly inoculated I/T with 0.5 ml saline alone. The animals were subsequently employed for I/P immunisation to evaluate systemic immunity. In a second set of experiments the animals were divided into 3 groups. The rats of group 1 were in-
Sheep erythrocytes (SRBC) used to immunise rats were washed thrice with sterile saline. Based on earlier preliminary studies SRBC were given I/P to rats at a dose of 12 x 10’ cells 4 days before killing, to elicit a systemic immune response. The animals were sacrificed at an interval of 7, 15 and 30 days of fly-ash exposure. 2.5. Immunisation of animalsforstudying local immune response Preliminary experiments were carried out to fix the dose of SRBC which could induce maximum antibody formation in lung-associated lymph nodes. Based on this an immunisation dose of 1 x 1O’SRBC was I/T administered to animals 7 days before killing to elicit local immune response in lungs. The animals were killed at 15 and 30 days after fly-ash exposure. 2.6. Haemagglutination assay Blood was drawn from control and experimental rats and serum separated. The antibody titer of serum was assessed by the method of Hudson and Hay (1989) using round-bottomed microtiter plates. 2.7. Adsorption of guinea-pig serum (complement) and foetal cay serum (FCS) with SRBC Guinea-pig serum, as a source of complement, and heat-inactivated foetal calf serum (56°C of 1 h) were adsorbed with SRBC (10: 1) in the cold for
1 h so as to remove antibody against SRBC, if present. Aliquots of adsorbed sera were stored at -20°C until use. 2.8. Antibod,v forming
cell (AFC) ussay
Lung-associated lymph nodes (LALN) and spleen of control and treated rats were disrupted gently in cold RPMI-1640 medium. The lymphocytes from LALN were passed through a sieve filter, repeatedly pipetted to obtain a single-cell suspension and centrifuged at 1200 rpm for 10 min at 4°C. The cell button was washed twice with saline, resuspended in cold RPMI-1640, total leukocytic count determined and the cell concentration was adjusted at 10 x 106/ ml. The cell viability was assessed by Trypan blue exclusion (Hanks and Wallace, 1958). The spleen cell suspension was allowed to stand for 2 min and the supernatant cell suspension centrifuged at 1200 rpm for 10 min at 4°C. The cells were given a brief hypotonic shock treatment with distilled water to lyse RBC, washed immediately with cold RPMI1640 twice and total number of nucleated cells counted. A cell suspension (10 x lob/ml) was prepared in RPMI-1640 after assessing the cell viability. Both LALN and spleen cell suspensions were supplemented with 10“/, heat-inactivated, SRBCadsorbed foetal calf serum. The number of AFC in LALN and spleen was estimated in vitro by the modified Jerne Plaque Techniques (Cunningham et al., 1968). Slide chambers were prepared to accomodate single cell layer of lymphocyte-SRBC suspension. The assay mixture consisted of 200 ~1 each of lymphocytes (10 x 106/ml), SRBC (1000 x lO’iml), 50 ~1 of adsorbed complement and 50 ~1 RPMI-1640. Aliquots of 30-40 ~1 of assay mixture were charged into each chamber and incubated at 37 ‘C for 60 min in moist trays. The number of IgM AFC were counted microscopically by counting the areas of lysis, surrounding lymphocytes. The number of AFC, in 2 to 3 chambers was counted for each sample and data were expressed as mean number of AFC/106 lymphoid cells. 2.9. Estimution of Cd Cadmium was estimated in lungs and LALN of native and cadmium-coated fly-ash-exposed animals
at day 15 and 30. A weighed amount of each sample was acid digested. After complete digestion and appropriate dilution, the amount of Cd was determined in an atomic absorption spectrophotometer (Belcher and Brooks, 1963; Schroeder and Nason, 1971).
3. Results 3.1. Effect ofj$ nsh on systemic immune response The number of AFC in the spleen of fly ash treated animals at various time intervals after exposure was not significantly different from controls (Fig. 1). The alterations in the serum haemagglutination titer of fly ash exposed animals were not statistically significant, when compared to respective controls at different time periods (Fig. 2). Fig. 3 shows the number of nucleated cells in LALN at different periods after fly ash exposure. The total number of nucleated cells was increased in LALN 7 days after fly-ash treatment (PcO.05). At day 15 and 13 of exposure the total number of cells in LALN of fly ash exposed rats was not significantly different from that of controls. It is apparent from Fig. 4 that the number of AFC in LALN was comparable to controls 7 days after exposure. At day 15 there was a 16.167; reduction and at day 30 a reduction of 26.157; in the number of AFC. These changes were not significant.
5001
0
Saline
a
Fly Ash
Fig. I. Effect of native fly ash on the AFC number in spleen. Route of exposure I/T; route of immunisation I/P. The data represent mean k S.D. of triplicate readings from 4 separate experiments.
S. Dogra et al. : lmmunopharmacology 29 (1595) 103-109
106 0
Wine
f-J
saline
m
Fly Ash
a
Fly Ash
m
Cd-Coated
Fly Ash
0 T
Fig. 2. Effect of native fly ash on H.4 titer of blood serum. Route of exposure I/T; route of immunisation I/P. The data represent the mean + S.D. of log, values in triplicate of 4 separate experimenls.
DOVS
Fig. 5. Effect of native and Cd-coated fly ash on total leucocytic count in LALN. Route of fly ash exposure I/T; route of immunisation I/T. The data represent the mean? S.D. of triplicate readings from 4 separate experiments. aP
3.2. Effect of Cd-coatedjy sponse in lungs
Fig. 3. Effect of native fly ash on total leucocytic count in LALN. Route of exposure I/T; route of immunisation I/P. The data rcpresent the mean + S.D. for triplicate readings from 4 separate experiments. C = Pt0.05.
u
Saline
a
Fly Ash
ash on local immune re-
Fig. 5 shows the alteration in the number of nucleated cells in LALN following intratracheal exposure to fly ash and Cd-coated Ay as at days 15 and 30. At both the time intervals the number of cells in fly ash and Cd-coated fly-ash-exposed animals was increased (PtO.O1 and t0.05). At day 15 the increase was 103.4% and 258.5% and at day 30 it was 80.13 7; and 186.14 y/,, respectively. Exposure to fly ash or Cd-coated fly ash at day 15 showed
a
Saline
a
Fly Ash
a
Cd-Coated
Fly Ash
300 Y u' f,
a 200
a
0
Q -I
0 100
': :: "04
0 0I
0
!ifil 7
15
30
30
Days Fig. 4. Effect of native fly ash on the number of AFC in LALN. Route of exposure I/T; rout of immunisation l/P. The data represent the mean & S.D. of triplicate readings from 4 separate experiments.
Fig. 6. Effect of native and Cd-coated fly ash on the number of AFC in LALN. Route of exposure I/T; route of immunisation l/T. The data represent the mean + S.D. of triplicate readings from 4 separate experiments. “PiO.01.
S. Dogra et al. / Immunopharmacology 29 0
Fly Ash
m
Cd-Coaled
Fly Ash
"1 -6
103-109
107
treated animals. The Cd contents in the lungs burdened with Cd-coated fly ash were increased at both day 15 and 30 post-exposure when compared to the values obtained in fly-ash-exposed and control animals.
Saline
a
119951
-I 4. Discussion
Fig. 7. Effect of native and Cd-coated fly ash on HA titer in blood serum. Route of exposure I/T; route of immunisation I/T. The data represent mean k S.D. of log, values in triplicate from 4 separate experiments. “P
27 “/, and 5 1.23 “/ decrease in the number of AFC in LALN (P < 0.0 1). On day 30 the results were broadly similar to those observed at the earlier time interval (Fig. 6). The results of the effect of exposure to native or Cd-coated fly ash on serum hemagglutination titer (HA) are shown in Fig. 7. The increase in the HA titer following fly ash treatment at 15 days was nonsignificant. With Cd-coated fly ash treatment the titer was increased by 72.48 “/; (PC 0.05). At 30 days there was a decrease of 22.8% (PC 0.01) and 19.4% (P= not significant) in the HA titer of fly-ashexposed and Cd-coated fly-ash-treated rats, respectively, when compared to controls. Table 1 shows the amount of Cd detectable in the lungs of control, fly ash and Cd-coated fly-ash-
The results of this investigation demonstrate a suppression in local immunity of lungs following pulmonary exposure to native and Cd-coated fly ash. These results confirm our earlier investigations and those of others which revealed that inhalation or intratracheal instillation of native fly-ash particles suppresses immune response in lungs (Burns and Zarkower, 1982; Bite et al., 1987; Dogra and Kaw, 1993). The observed effects appeared to be localised and not systemic since a similar depression in the number of AFC in the spleen of the fly-ash-exposed animals was not observed. An increased number of total nucleated cells in LALN following fly-ash exposure and immunisation through intratracheal route has been attributed to an enhanced cell division and recruitment of new lymphocytes in response to flyash exposure (Shami et al., 1984; Bite et al., 1987). In animals exposed to fly ash intratracheally and immunised through intraperitoneal route the increase in the number of cells of LALN was significant only at 7 days but not at days 15 and 30, as observed in intratracheally immunised rats. This discrepancy could possibly be due to difference in the route of immunisation in the two cases. To further validate these findings and to exclude the possibility of the observed effects being due to the filtration of the antigen in LALN the number of
Table I Change in Cd content (~9) and whole tissue weight in lungs (g) after exposure to native and Cd-coated fly ash Treatment
Saline Fly ash Cd-coated d P
15 days
30 days
Cd in lung
Lung weight
Cd in lung
Lung weight
0.130 * 0.066 0.107~0.064 5.111 + 1.176”
1.26 2 0.37 l.OSkO.16 1.47kO.016
0.121 kO.023 0.102 + 0.030 0.256 + 0.093
1.15~0.11 1.05 * 0.20 1.52 + 0.42
AFC were determined in the LALN and spleen of rats exposed intratracheally to fly ash and immunised intraperitoneally with SRBC. Such experimental manipulations resulted in the appearance of AFC in both spleen and LALN of control and fly-ashexposed animals. In spleen the number of AFC was similar in both the groups, though in LALN of fly-ash-exposed rats, the number of AFC was reduced. The results substantiate the hypothesis that pulmonary exposure to fly ash impairs the local lung immunity (Bite and Schnizlein, 1980; Bite et al., 1987). The mechanism by which the local immunity of the lung is effected is not clear, though the role of macrophages in alteration of immune response cannot be ruled out. Our earlier observations and those of others have demonstrated that coal fly ash particulates are readily phagocytosed by macrophages. Even though these cells retain their morphology, their functional abilities appear to be markedly reduced (Hill et al., 1982; Zarkower et al., 1982; Dogra et al., 1987). Alveolar macrophages play a key role in processing and presenting the antigen to the immunocompetent cells, and any derangement in this normal physiological mechanism would jeopardise the regulation of immune mechanisms in the lungs (Cory et al., 1984; Harmsen el al., 1985; Kaltreider et al., 1986; Lipscomb et al., 1986; Bite and Shopp, 1988). The immunosuppression in local immunity of lungs could be due to metallic constituents of Ay ash, since fly-ash particulates are known to contain a number of heavy metals many of which possess immunotoxic potential (Wei et al., 1982; Hanson et al., 1983; Weissman et al., 1983). The macrophages could play a significant role in dissolution of the metal from fly ash particulate (Lundborg et al., 1985) and translocate it to the LALN to elicit an effect on the appearance of AFC. Estimation of Cd in the lungs of rats exposed to Cd-coated fly ash revealed a significant decrease in Cd concentration in lungs with lapse in time. Since the fly ash exposure took place through lungs in the present experiment the metal dissolved in the lungs may not have reached spleen in optimal concentrations to elicit a response. To what extent Cd-coated fly ash brings modulation of systemic immunity is a subject matter of our current research.
Acknowledgement The authors wish to thank Mr. R.P. Singh for technical help and Mrs. A.P. John for secretarial assistance.
References Aranyi C, Miller FJ, Andres S, Ehrlish R, Fencers J, Gardner D and Waters MD. Cytotoxicity to alveolar macrophages of tract metals absorbed on fly ash. Environ. Res. 1979; 20: 14-23. B&her CB and Brooks KA. Determination of strontium in coal fly ash by atomic absorption spcctrophotometry. Anal. Chim. Acta 1963; 29: 202-205. Bite DE, Hahn FF. Benson J, Carpenter RL and Hobbs CH. Comparative lung immunotoxicity of inhaled quartz and coal combustion fly ash. Environ. Res. 1987; 43: 374-389. Bite DE and Schnirlein CT. Cellular immunity induced by lung immunisation of Fischer 344 rats. Int. Arch. Allergy Appl. Immunol. 1980; 63: 438-445. Bite DE and Shopp MG. Antibody response after lung immunisalion. Exp. Lung Res. 1988; 14: 133-155. Burns CA and Zarkower A. The effects of silica and fly ash dust inhalation on alveolar macrophage effector cell function. J. Reticulocndothel. Sot. 1982; 32: 449-459. Cory D. Kulkarni P and Lipscomb MF. The migration of broncho-alveolar macrophages into hilar lymph nodes. Am. J. Pathol. 1984; 115: 321-328. Cunningham 45 and Srenberg A. Further improvement in the plaque technique for detecting single antibody forming cells. Immunology 196X; 14: 599-600. Dogra S and Kaw JL. Pulmonary immunotoxic potential of coal fly ash. Int. J. Toxicol. Occup. Environ. Health 1993; 2: 6367. Dogra S, Khanna AK and Kaw JL. In viva effect of fly ash on surface receptors of mice peritoneal macrophages. Environ. Sci. Health 1987: A 22: 189-203. Fischer GL and Wilson FD. The effect of coal fly ash and silica inhalation on macrophage filnction and progenitors. J. Reticuloendothel. Sot. 1980; 27: 513-523. Golden EB, Warnock ML, Hulett LD and Chung AM. Fly ash lung: A new pneumoconiosis. Am. Rev. Rcspir. Dis. 1983; 125: 108-111. Hanks JH and Wallace JH. Determination of ccl1 viability. Proc. Sot. Exp. Biol. Med. 1958; 98: 188-192. Hanson RL, Henderson TR, Hobbs CH, Clark CR, Carpenter RL, Dutcher JS. Harvey TM and Hunt DF. Detection of nitroaromatic compounds on coal combustion particles. J. Toxico]. Environ. Health 1983; 11: 971-980. Harmsen AC. Muggenburg BA, Snipes MB and Bicc DE. The role of macrophages in particles translocation from lung to lymph nodes. Science 1985; 230: 1277-1280. Hayes TL, Pawely JB, Fischer CL and Goldman M. A model for the cxposurc of individual lung cell to the foreign elements contained in fly ash. Environ. Res. 1980; 22: 499-509.
Hill JO, Gray RH, DeNee PB and Newton GH. Comparative damage to alveolar macrophages after phagocytosis of respirable particles. Environ. Res. 1982; 27: 95-109. Hudson L and Hay FC. Antibody interaction with antigen. In: Practical Immunology, 3rd ed. Edinburgh: Blackwell, 1989; 207-263. Kaltreider HB, Caldwell JL and Byrd PK. The capacity of normal murine macrophages to function as antigen presenting cells for the initiation of primary antibody forming cell response to sheep erythrocytes in vitro. Am. Rev. Respir. Dis. 1986; 133: 1097-l 104. Kaw JL, Khanna AK and Waseem M. Modification of pulmonary silicotic reaction in rats exposed to coal lly ash. Exp. Pathol. 1990; 39: 49-57. Lipscomb MF, Lyons CR, Nunez G, Ball EJ, Stastny P, Vial W, Lern V, Weissler J, Miller LM and Towcur GB. Human alveolar macrophages: HLA-DR positive macrophages that are poor stimulators of primary mixed lcucocyte reaction. J. Immunol. 1986; 136: 497-504. Lundborg M, Eklund A, Lind B, Comner P. Dissolution of metals by human and rabbit alveolar macrophagcs. Br. J. Int. Med. 1985; 42: 642-645. Mellman IS and Ukkonen P. Interlisation and fate of macrophage FC receptors during receptor mediated endocytosis. In:
VF Ralph (ed.), Mononuclear Phagocytes. Oxford/Edinburgh: Blackwell, 1985; 75-85. Morgan RM, Kundomal YR and Hupp EW. SAS prabit analysis for cadmium mortality. Environ. Res. 1982; 29: 233-237. Shami SG, Salbaugb SA, Hahn FF, Griffith WC and Hobbs CH. Cytokinetic and morphological changes in the lungs and lung associated lymph nodes of rats after inhalation of fly ash. Environ. Res. 1984; 35: 373-393. Schroeder HA and Nason AP. Trace element analysis in clinical chemistry. Clin. Chem. 1971; 17: 461-474. Vredevac D, Levy EL, Knutson D, Cook G and Cohen P. Recovery of murine mononuclear phagocytic system following chronic exposure to cadmium. Environ. Res. 1985; 37: 373382. Wei CL. Raabe OG and Rosenblalt LS. Microbial detection of mutagenic nitroorganic compounds in filtrates of coal fly ash. Envsiron. Mutagen. 1982; 4: 249-258. Weisman SH, Carpenter RL and Newton CF. Respirable aerosols from fluidized bed coal combustion 3. Elemental composition of fly ash. Environ. Sci. Technol. 1983; 17: 65-71. Zarkower A, Eskcw ML, Scheuchenzuber WJ and Graham JA. Effects of fly ash inhalation on murine immune function. Changes in macrophage mediated activities. Environ. Res. 1982; 29: 83-89.
Immunopharmacology 29 (1995) 103-109
Alterations in the pulmonary and systemic immune response in rats exposed to coal fly ash* Shashi Dogra*, Ashok Kumar Khanna, Jawahir Lal Kaw lndustricd Toxico1og.v Research Centve, P.O. Box No. 80, Mahatma Gandhi Mug. Lucknow-226001, India
(Received 27 June 1994; revision received 30 August 1994; accepted 5 September 1994)
Abstract The effect of intratracheally injected fly ash on the development of pulmonary and systemic immunity was studied in rats. Following intratracheal and intraperitoneal immunisation with sheep red blood cells (SRBC) there was an appearance of antibody forming cells (AFC) in lung associated lymph nodes (LALN) of animals exposed to either fly ash or physiological saline. Enumeration of AFC in LALN after immunisation by either of the routes, revealed a reduction in the number of AFC in LALN of fly ash exposed rats in comparison to saline exposed animals. The reduction in the number of AFC was more pronounced after exposure of Cd-coated fly ash. The AFC appeared in the spleen only after immunisation through intraperitoneal route and the number of AFC in spleen of the fly ash and saline exposed group of animals did not show any significant difference. These results demonstrate that fly ash burden of lungs results in an impairment of the local immune response of the lungs without an associated effect on the systemic immunity. Key\+jor&s: Fly ash; Lung; Immunity; Antibody-forming
cell; Lymphocyte
1. Introduction Fly ash, a common air pollutant produced during coal combustion for energy generation in thermal power plants, has been reported to produce pneumoconiosis (Golden et al., 1983). It contributes significantly to air pollution due to the presence of a larger number of potentially toxic metals and carcinogenic compounds (Aranyi et al., 1979; Fisher and Wilson,
1980; Weisman
et al., 1983). By virtue
of
offering a large surface area, fly ash can adsorb heavy
metals released into the environment from the toxic fumes of diesel exhaust, motor vehicles and numerous industrial activities. Inhalation of Cd, one of the toxic metals reported to be contained in fly ash (Hayes et al., 1980), has been reported to produce pulmonary disorders and an alteration in immunologically specific mechanisms (Morgan et al., 1982; Mellman and Ukkonen, 1985; Vredevae et al., 1985). Earlier studies on the immune status of lungs in animals
exposed
to fly ash after lung immunisation
have demonstrated immunosuppressive potential of fly ash (Burns and Zarkower,
* Corresponding author. Abbreviations: AFC. antibody forming cells; FCS, foetal calf
serum; HA, haemagglutination; LALN. lung-associated lymph nodes; SRBC, sheep red blood cells * ITRC Communication No. 188I. 0162-3109:95:SY.S00 1995 Elsevier Science B.V. All rights reserved SSDI 0162-3109(94)00049-2
1982; Bite et al., 1987;
Dogra and Kaw, 1993). In the present investigation we report studies on systemic immunity in rats exposed to fly ash and extend our earlier studies to include the effect of Cd-coated fly ash on local lung immunity.
104
2. Material
S. Dogru et al. : Immunopharmacology
and methods
2.1. Dust Fly ash was obtained from a thermal power station in India and an approx. 5-pm particle was prepared. The details of the method of preparation of the fly ash samples and elemental composition was reported earlier (Kaw et al., 1990). The fly ash particles used contained more than 90% of the particles less than 5 pm in diameter. The fly ash particulates were suspended in autoclaved saline, sonicated at 100 amp for 2 min and well agitated before intratracheal instillation into rats.
29 fl99j)
IO3- IO9
oculated I/T with 11.6 mg of native fly ash SUSpended in 0.5 ml saline. Animals of group 2 were inoculated with 11.6 mg of Cd-coated fly ash, while group-3 animals served as controls and were inoculated I/T with 0.5 ml saline alone. This set of rats was subsequently employed for I/T immunisation to evaluate local immunity. The dose of fly ash was selected on the basis of pilot experiments designed to select amount of fly ash that could adsorb an amount of Cd equal to its l/5 LD,O value. 2.4. Immunisation of animals for studying systemic immune response
2.2. Coating of$y ash with Cd Cadmium chloride (CdCl,, BDH) was used to coat native fly ash by the method described by Aranyi et al. (1979). Briefly, a 0.5 ‘& solution of CdCl, was prepared in distilled water and 100 mg of fly ash was added to 5 ml of CdCl, solution. Precipitation of the metal hydroxide was carried out with 1 N NaOH and the precipitate was dried at 100°C. The dried precipitate was heated at 675 oC in a platinum crucible for 1 h in a muffle furnace. The amount of Cd adsorbed on fly ash was determined in an atomic absorption spectrophotometer after acid digestion. Dried Cd-coated fly ash, thus prepared, was stored for further use. 2.3. Animals and experimental protocol Female albino rats from the Industrial Toxicology Research Centre (Lucknow, India), animal colony, weighing 200-250 g, fed freely on pellet diet (Hindustan Lever, Bombay, India) and having free access to drinking water were used throughout the experiment. Broadly two sets of experiments were performed. In set I the animals were divided into 2 groups. The rats of group 1 were inoculated intratracheally (I/T) 11.6 mg of native fly ash suspended in 0.5 ml saline under light ether anaesthesia. The rats of group 2 served as controls and were similarly inoculated I/T with 0.5 ml saline alone. The animals were subsequently employed for I/P immunisation to evaluate systemic immunity. In a second set of experiments the animals were divided into 3 groups. The rats of group 1 were in-
Sheep erythrocytes (SRBC) used to immunise rats were washed thrice with sterile saline. Based on earlier preliminary studies SRBC were given I/P to rats at a dose of 12 x 10’ cells 4 days before killing, to elicit a systemic immune response. The animals were sacrificed at an interval of 7, 15 and 30 days of fly-ash exposure. 2.5. Immunisation of animalsforstudying local immune response Preliminary experiments were carried out to fix the dose of SRBC which could induce maximum antibody formation in lung-associated lymph nodes. Based on this an immunisation dose of 1 x 1O’SRBC was I/T administered to animals 7 days before killing to elicit local immune response in lungs. The animals were killed at 15 and 30 days after fly-ash exposure. 2.6. Haemagglutination assay Blood was drawn from control and experimental rats and serum separated. The antibody titer of serum was assessed by the method of Hudson and Hay (1989) using round-bottomed microtiter plates. 2.7. Adsorption of guinea-pig serum (complement) and foetal cay serum (FCS) with SRBC Guinea-pig serum, as a source of complement, and heat-inactivated foetal calf serum (56°C of 1 h) were adsorbed with SRBC (10: 1) in the cold for
1 h so as to remove antibody against SRBC, if present. Aliquots of adsorbed sera were stored at -20°C until use. 2.8. Antibod,v forming
cell (AFC) ussay
Lung-associated lymph nodes (LALN) and spleen of control and treated rats were disrupted gently in cold RPMI-1640 medium. The lymphocytes from LALN were passed through a sieve filter, repeatedly pipetted to obtain a single-cell suspension and centrifuged at 1200 rpm for 10 min at 4°C. The cell button was washed twice with saline, resuspended in cold RPMI-1640, total leukocytic count determined and the cell concentration was adjusted at 10 x 106/ ml. The cell viability was assessed by Trypan blue exclusion (Hanks and Wallace, 1958). The spleen cell suspension was allowed to stand for 2 min and the supernatant cell suspension centrifuged at 1200 rpm for 10 min at 4°C. The cells were given a brief hypotonic shock treatment with distilled water to lyse RBC, washed immediately with cold RPMI1640 twice and total number of nucleated cells counted. A cell suspension (10 x lob/ml) was prepared in RPMI-1640 after assessing the cell viability. Both LALN and spleen cell suspensions were supplemented with 10“/, heat-inactivated, SRBCadsorbed foetal calf serum. The number of AFC in LALN and spleen was estimated in vitro by the modified Jerne Plaque Techniques (Cunningham et al., 1968). Slide chambers were prepared to accomodate single cell layer of lymphocyte-SRBC suspension. The assay mixture consisted of 200 ~1 each of lymphocytes (10 x 106/ml), SRBC (1000 x lO’iml), 50 ~1 of adsorbed complement and 50 ~1 RPMI-1640. Aliquots of 30-40 ~1 of assay mixture were charged into each chamber and incubated at 37 ‘C for 60 min in moist trays. The number of IgM AFC were counted microscopically by counting the areas of lysis, surrounding lymphocytes. The number of AFC, in 2 to 3 chambers was counted for each sample and data were expressed as mean number of AFC/106 lymphoid cells. 2.9. Estimution of Cd Cadmium was estimated in lungs and LALN of native and cadmium-coated fly-ash-exposed animals
at day 15 and 30. A weighed amount of each sample was acid digested. After complete digestion and appropriate dilution, the amount of Cd was determined in an atomic absorption spectrophotometer (Belcher and Brooks, 1963; Schroeder and Nason, 1971).
3. Results 3.1. Effect ofj$ nsh on systemic immune response The number of AFC in the spleen of fly ash treated animals at various time intervals after exposure was not significantly different from controls (Fig. 1). The alterations in the serum haemagglutination titer of fly ash exposed animals were not statistically significant, when compared to respective controls at different time periods (Fig. 2). Fig. 3 shows the number of nucleated cells in LALN at different periods after fly ash exposure. The total number of nucleated cells was increased in LALN 7 days after fly-ash treatment (PcO.05). At day 15 and 13 of exposure the total number of cells in LALN of fly ash exposed rats was not significantly different from that of controls. It is apparent from Fig. 4 that the number of AFC in LALN was comparable to controls 7 days after exposure. At day 15 there was a 16.167; reduction and at day 30 a reduction of 26.157; in the number of AFC. These changes were not significant.
5001
0
Saline
a
Fly Ash
Fig. I. Effect of native fly ash on the AFC number in spleen. Route of exposure I/T; route of immunisation I/P. The data represent mean k S.D. of triplicate readings from 4 separate experiments.
S. Dogra et al. : lmmunopharmacology 29 (1595) 103-109
106 0
Wine
f-J
saline
m
Fly Ash
a
Fly Ash
m
Cd-Coated
Fly Ash
0 T
Fig. 2. Effect of native fly ash on H.4 titer of blood serum. Route of exposure I/T; route of immunisation I/P. The data represent the mean + S.D. of log, values in triplicate of 4 separate experimenls.
DOVS
Fig. 5. Effect of native and Cd-coated fly ash on total leucocytic count in LALN. Route of fly ash exposure I/T; route of immunisation I/T. The data represent the mean? S.D. of triplicate readings from 4 separate experiments. aP
3.2. Effect of Cd-coatedjy sponse in lungs
Fig. 3. Effect of native fly ash on total leucocytic count in LALN. Route of exposure I/T; route of immunisation I/P. The data rcpresent the mean + S.D. for triplicate readings from 4 separate experiments. C = Pt0.05.
u
Saline
a
Fly Ash
ash on local immune re-
Fig. 5 shows the alteration in the number of nucleated cells in LALN following intratracheal exposure to fly ash and Cd-coated Ay as at days 15 and 30. At both the time intervals the number of cells in fly ash and Cd-coated fly-ash-exposed animals was increased (PtO.O1 and t0.05). At day 15 the increase was 103.4% and 258.5% and at day 30 it was 80.13 7; and 186.14 y/,, respectively. Exposure to fly ash or Cd-coated fly ash at day 15 showed
a
Saline
a
Fly Ash
a
Cd-Coated
Fly Ash
300 Y u' f,
a 200
a
0
Q -I
0 100
': :: "04
0 0I
0
!ifil 7
15
30
30
Days Fig. 4. Effect of native fly ash on the number of AFC in LALN. Route of exposure I/T; rout of immunisation l/P. The data represent the mean & S.D. of triplicate readings from 4 separate experiments.
Fig. 6. Effect of native and Cd-coated fly ash on the number of AFC in LALN. Route of exposure I/T; route of immunisation l/T. The data represent the mean + S.D. of triplicate readings from 4 separate experiments. “PiO.01.
S. Dogra et al. / Immunopharmacology 29 0
Fly Ash
m
Cd-Coaled
Fly Ash
"1 -6
103-109
107
treated animals. The Cd contents in the lungs burdened with Cd-coated fly ash were increased at both day 15 and 30 post-exposure when compared to the values obtained in fly-ash-exposed and control animals.
Saline
a
119951
-I 4. Discussion
Fig. 7. Effect of native and Cd-coated fly ash on HA titer in blood serum. Route of exposure I/T; route of immunisation I/T. The data represent mean k S.D. of log, values in triplicate from 4 separate experiments. “P
27 “/, and 5 1.23 “/ decrease in the number of AFC in LALN (P < 0.0 1). On day 30 the results were broadly similar to those observed at the earlier time interval (Fig. 6). The results of the effect of exposure to native or Cd-coated fly ash on serum hemagglutination titer (HA) are shown in Fig. 7. The increase in the HA titer following fly ash treatment at 15 days was nonsignificant. With Cd-coated fly ash treatment the titer was increased by 72.48 “/; (PC 0.05). At 30 days there was a decrease of 22.8% (PC 0.01) and 19.4% (P= not significant) in the HA titer of fly-ashexposed and Cd-coated fly-ash-treated rats, respectively, when compared to controls. Table 1 shows the amount of Cd detectable in the lungs of control, fly ash and Cd-coated fly-ash-
The results of this investigation demonstrate a suppression in local immunity of lungs following pulmonary exposure to native and Cd-coated fly ash. These results confirm our earlier investigations and those of others which revealed that inhalation or intratracheal instillation of native fly-ash particles suppresses immune response in lungs (Burns and Zarkower, 1982; Bite et al., 1987; Dogra and Kaw, 1993). The observed effects appeared to be localised and not systemic since a similar depression in the number of AFC in the spleen of the fly-ash-exposed animals was not observed. An increased number of total nucleated cells in LALN following fly-ash exposure and immunisation through intratracheal route has been attributed to an enhanced cell division and recruitment of new lymphocytes in response to flyash exposure (Shami et al., 1984; Bite et al., 1987). In animals exposed to fly ash intratracheally and immunised through intraperitoneal route the increase in the number of cells of LALN was significant only at 7 days but not at days 15 and 30, as observed in intratracheally immunised rats. This discrepancy could possibly be due to difference in the route of immunisation in the two cases. To further validate these findings and to exclude the possibility of the observed effects being due to the filtration of the antigen in LALN the number of
Table I Change in Cd content (~9) and whole tissue weight in lungs (g) after exposure to native and Cd-coated fly ash Treatment
Saline Fly ash Cd-coated d P
15 days
30 days
Cd in lung
Lung weight
Cd in lung
Lung weight
0.130 * 0.066 0.107~0.064 5.111 + 1.176”
1.26 2 0.37 l.OSkO.16 1.47kO.016
0.121 kO.023 0.102 + 0.030 0.256 + 0.093
1.15~0.11 1.05 * 0.20 1.52 + 0.42
AFC were determined in the LALN and spleen of rats exposed intratracheally to fly ash and immunised intraperitoneally with SRBC. Such experimental manipulations resulted in the appearance of AFC in both spleen and LALN of control and fly-ashexposed animals. In spleen the number of AFC was similar in both the groups, though in LALN of fly-ash-exposed rats, the number of AFC was reduced. The results substantiate the hypothesis that pulmonary exposure to fly ash impairs the local lung immunity (Bite and Schnizlein, 1980; Bite et al., 1987). The mechanism by which the local immunity of the lung is effected is not clear, though the role of macrophages in alteration of immune response cannot be ruled out. Our earlier observations and those of others have demonstrated that coal fly ash particulates are readily phagocytosed by macrophages. Even though these cells retain their morphology, their functional abilities appear to be markedly reduced (Hill et al., 1982; Zarkower et al., 1982; Dogra et al., 1987). Alveolar macrophages play a key role in processing and presenting the antigen to the immunocompetent cells, and any derangement in this normal physiological mechanism would jeopardise the regulation of immune mechanisms in the lungs (Cory et al., 1984; Harmsen el al., 1985; Kaltreider et al., 1986; Lipscomb et al., 1986; Bite and Shopp, 1988). The immunosuppression in local immunity of lungs could be due to metallic constituents of Ay ash, since fly-ash particulates are known to contain a number of heavy metals many of which possess immunotoxic potential (Wei et al., 1982; Hanson et al., 1983; Weissman et al., 1983). The macrophages could play a significant role in dissolution of the metal from fly ash particulate (Lundborg et al., 1985) and translocate it to the LALN to elicit an effect on the appearance of AFC. Estimation of Cd in the lungs of rats exposed to Cd-coated fly ash revealed a significant decrease in Cd concentration in lungs with lapse in time. Since the fly ash exposure took place through lungs in the present experiment the metal dissolved in the lungs may not have reached spleen in optimal concentrations to elicit a response. To what extent Cd-coated fly ash brings modulation of systemic immunity is a subject matter of our current research.
Acknowledgement The authors wish to thank Mr. R.P. Singh for technical help and Mrs. A.P. John for secretarial assistance.
References Aranyi C, Miller FJ, Andres S, Ehrlish R, Fencers J, Gardner D and Waters MD. Cytotoxicity to alveolar macrophages of tract metals absorbed on fly ash. Environ. Res. 1979; 20: 14-23. B&her CB and Brooks KA. Determination of strontium in coal fly ash by atomic absorption spcctrophotometry. Anal. Chim. Acta 1963; 29: 202-205. Bite DE, Hahn FF. Benson J, Carpenter RL and Hobbs CH. Comparative lung immunotoxicity of inhaled quartz and coal combustion fly ash. Environ. Res. 1987; 43: 374-389. Bite DE and Schnirlein CT. Cellular immunity induced by lung immunisation of Fischer 344 rats. Int. Arch. Allergy Appl. Immunol. 1980; 63: 438-445. Bite DE and Shopp MG. Antibody response after lung immunisalion. Exp. Lung Res. 1988; 14: 133-155. Burns CA and Zarkower A. The effects of silica and fly ash dust inhalation on alveolar macrophage effector cell function. J. Reticulocndothel. Sot. 1982; 32: 449-459. Cory D. Kulkarni P and Lipscomb MF. The migration of broncho-alveolar macrophages into hilar lymph nodes. Am. J. Pathol. 1984; 115: 321-328. Cunningham 45 and Srenberg A. Further improvement in the plaque technique for detecting single antibody forming cells. Immunology 196X; 14: 599-600. Dogra S and Kaw JL. Pulmonary immunotoxic potential of coal fly ash. Int. J. Toxicol. Occup. Environ. Health 1993; 2: 6367. Dogra S, Khanna AK and Kaw JL. In viva effect of fly ash on surface receptors of mice peritoneal macrophages. Environ. Sci. Health 1987: A 22: 189-203. Fischer GL and Wilson FD. The effect of coal fly ash and silica inhalation on macrophage filnction and progenitors. J. Reticuloendothel. Sot. 1980; 27: 513-523. Golden EB, Warnock ML, Hulett LD and Chung AM. Fly ash lung: A new pneumoconiosis. Am. Rev. Rcspir. Dis. 1983; 125: 108-111. Hanks JH and Wallace JH. Determination of ccl1 viability. Proc. Sot. Exp. Biol. Med. 1958; 98: 188-192. Hanson RL, Henderson TR, Hobbs CH, Clark CR, Carpenter RL, Dutcher JS. Harvey TM and Hunt DF. Detection of nitroaromatic compounds on coal combustion particles. J. Toxico]. Environ. Health 1983; 11: 971-980. Harmsen AC. Muggenburg BA, Snipes MB and Bicc DE. The role of macrophages in particles translocation from lung to lymph nodes. Science 1985; 230: 1277-1280. Hayes TL, Pawely JB, Fischer CL and Goldman M. A model for the cxposurc of individual lung cell to the foreign elements contained in fly ash. Environ. Res. 1980; 22: 499-509.
Hill JO, Gray RH, DeNee PB and Newton GH. Comparative damage to alveolar macrophages after phagocytosis of respirable particles. Environ. Res. 1982; 27: 95-109. Hudson L and Hay FC. Antibody interaction with antigen. In: Practical Immunology, 3rd ed. Edinburgh: Blackwell, 1989; 207-263. Kaltreider HB, Caldwell JL and Byrd PK. The capacity of normal murine macrophages to function as antigen presenting cells for the initiation of primary antibody forming cell response to sheep erythrocytes in vitro. Am. Rev. Respir. Dis. 1986; 133: 1097-l 104. Kaw JL, Khanna AK and Waseem M. Modification of pulmonary silicotic reaction in rats exposed to coal lly ash. Exp. Pathol. 1990; 39: 49-57. Lipscomb MF, Lyons CR, Nunez G, Ball EJ, Stastny P, Vial W, Lern V, Weissler J, Miller LM and Towcur GB. Human alveolar macrophages: HLA-DR positive macrophages that are poor stimulators of primary mixed lcucocyte reaction. J. Immunol. 1986; 136: 497-504. Lundborg M, Eklund A, Lind B, Comner P. Dissolution of metals by human and rabbit alveolar macrophagcs. Br. J. Int. Med. 1985; 42: 642-645. Mellman IS and Ukkonen P. Interlisation and fate of macrophage FC receptors during receptor mediated endocytosis. In:
VF Ralph (ed.), Mononuclear Phagocytes. Oxford/Edinburgh: Blackwell, 1985; 75-85. Morgan RM, Kundomal YR and Hupp EW. SAS prabit analysis for cadmium mortality. Environ. Res. 1982; 29: 233-237. Shami SG, Salbaugb SA, Hahn FF, Griffith WC and Hobbs CH. Cytokinetic and morphological changes in the lungs and lung associated lymph nodes of rats after inhalation of fly ash. Environ. Res. 1984; 35: 373-393. Schroeder HA and Nason AP. Trace element analysis in clinical chemistry. Clin. Chem. 1971; 17: 461-474. Vredevac D, Levy EL, Knutson D, Cook G and Cohen P. Recovery of murine mononuclear phagocytic system following chronic exposure to cadmium. Environ. Res. 1985; 37: 373382. Wei CL. Raabe OG and Rosenblalt LS. Microbial detection of mutagenic nitroorganic compounds in filtrates of coal fly ash. Envsiron. Mutagen. 1982; 4: 249-258. Weisman SH, Carpenter RL and Newton CF. Respirable aerosols from fluidized bed coal combustion 3. Elemental composition of fly ash. Environ. Sci. Technol. 1983; 17: 65-71. Zarkower A, Eskcw ML, Scheuchenzuber WJ and Graham JA. Effects of fly ash inhalation on murine immune function. Changes in macrophage mediated activities. Environ. Res. 1982; 29: 83-89.