United States
                                   Environmental Protection
                                   Agency
                                   Health Effects Research
                                   Laboratory
                                   Research Triangle Park NC 27711
xvEPA
                                   Research and Development
                                   EPA-600/S1-81-020  May 1981
Project  Summary
                                  Determining  Effect  of
                                  Pollutants on  the
                                  Immune  System

                                  A. Zarkower, J. Davis, F. Ferguson, and D  Stnckler
                                    The purpose of this project was to
                                  determine the effects of fly-ash inhala-
                                  tion on the ability of animals to resist
                                  infections, neoplastic growth, and the
                                  development of hypersensitive re-
                                  sponses. Mice were exposed to fly ash
                                  from two sources, carbon black, and
                                  filtered air only. Following various
                                  exposure  periods  (days  to months),
                                  the immunologic competence of lymph-
                                  cytes, neutrophiles, and macrophages
                                  was assessed.
                                    Fly-ash inhalation resulted in a
                                  decreased response in the spleens and
                                  mediastinal lymph nodes to Escherichia
                                  coli antigen given by aerosol.  This
                                  suppression  was  much  less severe
                                  than that following inhalation of carbon
                                  black and silica quartz.  Fly ash had
                                  little  effect on the ability of T and B
                                  lymphocytes to respond  to mitogens
                                  PHA  (phytohemagglutin (PHA) and
                                  lipopolysaccharide) and to  be stimu-
                                  lated for cytolytic response against
                                  tumor cells. Both the in vitro response
                                  of the (BCG)-BACILLE CALETTE GUER-
                                  IN-sensitized micre to purified protein
                                  derivative of tuberculin and  in  vivo
                                  delayed-type hypersensitive reactions
                                  were  increased.
                                    Exposure to fly ash had the following
                                  more pronounced  effects on  macro-
                                  phages: a decrease in the number of
                                  pulmonary macrophages capable  of
                                  phagocytosis; a decrease in  antibody-
                                  dependent cell-mediated cytolysis
                                  (ADCC),  in contrast to enhancement
                                  of ADCC after intratracheal injection
                                  of fly ash and silica and inhalation  of
                                   silica; an increase in cytotoxie activity
                                   against HBO tumor cells after intratra-
                                   cheal injection of fly ash and a decrease
                                   after inhalation of fly ash; and decreas-
                                   ed ability to activate T cell mitogenesis
                                   after fly-ash inhalation.
                                    This Project Summary was develop-
                                   ed by EPA's Health Effects Research
                                   Laboratory, Research Triangle Park,
                                   NC. to announce key findings of the
                                   research project that is fully document
                                   edd in a separate report of the same
                                   title (see Project Report ordering in-
                                   formation at back).


                                   Introduction
                                    The lung is the primary exposure site
                                  for gases and for particles smaller than
                                  2 /urn in diameter After inhalation, toxic
                                  substances may interact  with surface
                                  proteins and cells or may enter the body
                                  and be carried to such organs as the
                                  lymphoid tissues, liver, and kidneys,
                                  which are the principal organs of clear-
                                  ance, detoxification, and excretion.
                                  Although exposure  to airborne sub-
                                  stances can also result in absorption by
                                  the gastrointestinal tract and the skin,
                                  the lung accepts the greatest burden.
                                    Inhalation of toxic substances can
                                  cause disease either by direct damage
                                  to lung  tissues or by affecting other
                                  organ systems. Direct lung damage
                                  often follows exposure to high concen-
                                  trations of toxic minerals (silica, asbes-
                                  tos) or gases (ozone, nitrogen dioxide).
                                  Substances  that can have  indirect
                                  effects include lead, which can affect a

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variety of tissues (gastrointestinal tract,
nervous system, and blood); cadmium,
which can cause kidney damage and
osteomalacia; and mercury, which can
affect the central  nervous system.
Gases and organic and inorganic parti-
culates have a variety  of effects on
immunological responses in the body,
including suppression or enhancement
of antibody formation, cellular immuno-
responses, and  phagocytic activities of
macrophages. Decreased antibody re-
sponses may increase susceptibility to
infectious diseases; increased stimula-
tion of specific types of antibodies may
increase resistance to infectious dis-
eases, but may also lead to  immuno-
pathological conditions such as imme-
late hypersensitivity and  immune com-
plex diseases. Decreased cellular im-
mune responses may decrease resis-
tance  to certain infectious agents and
certain neoplastic processes, while
increased  cellular immunocompetence
may  manifest  itself as delayed-type
hypersensitivity. Changes in neutrophile
and macrophage functions  also can
affect  resistance to both infectious and
neoplastic diseases
  In the near future, expanded use of
coal for power generation and as stock
for liquid and gaseous fuels is expected
to yield a number of products (e g., SO?,
NOa, hydrocarbons, and particulates)
that could react with biological systems.
Fly ash will be a large component of the
se coal-derived  air pollutants, and it is
composed of many elements that can
cause cell damage. Since inhaled parti-
culates will come  into intimate contact
with cells of the immune system (macro-
phages and lymphocytes), changes in
immunological  responses may occur,
                                        leading ultimately to decreased ability to
                                        cope with infectious agents and neopla-
                                        stic cells. This project was designed to
                                        test the effects of fly-ash inhalation on
                                        cells involved in the immune response
                                        and their functions.

                                        Exposure Facilities
                                          Female BALB/c mice were exposed
                                        to fly ash (and in some cases, carbon) in
                                        specially designed  chambers. Com-
                                        pressed  air was pulsed into a dustbed,
                                        and the particles entered an air stream
                                        passing through the dust-generator
                                        cavity. This air stream was mixed with a
                                        stream of filtered air before  it entered
                                        the exposure chamber. Control cham-
                                        bers received airstreams from the same
                                        sources, though without the  particles.
                                        After varying lengths of time, the animals
                                        were removed from the chambers, and a
                                        variety of tests  was done to determine
                                        the immunologic competence of lym-
                                        phocytes, neutrophiles, and macro-
                                        phages.


                                        Results
                                          In thef irst series of experiments, mice
                                        were  exposed  to carbon and fly ash
                                        (from the Pennsylvania State  University
                                        (PSD) power plant) for 7, 21, 35, and 56
                                        days. The total  concentrations of the
                                        generated particulates and the propor-
                                        tions in the respirable size range «2.1
                                        (im) are  given in Table 1. Inhalation of
                                        both fly ash and carbon decreased the
                                        numbers of antibody-forming cells in
                                        the spleen following stimulation of the
                                        mice with Escherichia  coli antigen
                                        aerosols. The effect of carbon was much
                                        greater than that of  fly ash.  Antibody-
                                        forming activity in the mediastinal
lymph nodes was enhanced after 7 days
of exposure, followed by suppression
after 21 days and recovery after 35 and
56 days. Carbon and fly ash both caused
hypertrophy of the lymph nodes, with an
increase in the number of lymphocytes
at all times after exposure. Serum
agglutinating activity also decreased.
  Little change occurred in the lympho-
cyte responses to T and B cell mitogens.
The recognition  ac.tivity of the cells was
decreased after a 21-day exposure, as
was lymphocyte response to Con A. No
significant change was found in the
cytolytic capacity of T  cells  after  any
period of exposure.
  A nine-month exposure to fly ash at
somewhat higher concentrations  re-
sulted in a highly significant decrease in
splenic antibody-forming cell response
in mice exposed to E. coli lipopolysac-
charide antigen by either aerosol or
intratracheal injection. Plaque-forming
cell responses in the mediastinal lymph
nodes were lower in both cases, though
not significantly.
  In a second series of experiments,
mice were exposed to carbon and fly ash
provided by the U.S  Environmental
Protection Agency (EPA), as well as that
from the PSU power plant.  The total
concentrations of the particulates  and
the proportions in the respirable size
range are given  in Table 2.
  The number of antibody-forming cells
in the spleens decreased after 7 and 24
days of exposure to fly ash, and de-
creased serum  antibody activity was
found after 7, 21, and  56 days of ex-
posure. Carbon dust caused a very
pronounced and progressive  reduction
in the number of antibody-forming cells
in the spleens  after all times of ex-
Table 1.     Paniculate Exposure Concentrations Expressed as fjg/m3 of Air

                                                Mean Fly Ash ± Standard Error
                                                                                Mean Carbon Black ± Standard Error
Experiment
Responses to
antigenic
stimulations
in vivo
Responses to
mitogens in
vitro and
recognitive
activity of
T cells
Cytolytic
activity of
stimulated
T cells
Exposure Period
7 days (7/28-8/4/77)
21 days (7/28-8/1 8/77)
35 days (7/28-9/2/77)
56 days (7/28-9/22/77)
7 days (8/8-8/1 5/77)
21 days (8/8-8/30/77)
35 days (8/8-9/13/77)
56 days (8/14-10/11/77)


7 days (9/2-9/9/77)
21 days (9/2-9/23/77)
35 days (9/2-10/7/77)
56 days (8/19-10/14/77)
Total
2667 ± 1417
3357 ± 798
3220 ± 512
2658 ± 463
4976
3686 ± 647
3281 ±440
2277 ± 350


2417
1535 ± 563
1 732 ± 444
21 37 ±380
< 2. 1 fjm
655 ± 348
959 ± 236
1009 ± 153
860 ± 140
1433
1214 ± 110
11 30 ±85
840 ± 135


909
562 ± 230
643 ± 182
798 ± 151
Total
4736 ± 1683
4987 ± 1334
4805 ± 855
4615 ± 700
2461
4879 ± 1644
4301 ± 1049
4956 ± 762


2148
4237 ± 1458
4448 ± 1053
4445 ± 670
< 2.1 (urn
947 ± 555
1387 ± 522
1561 ±349
1472 ± 269
859
1815 ± 560
1679 ±363
1642 ± 296


924
1291 ±485
1220 ± 350
1449 ± 267

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fable 2.    Concentration of Particulates Expressed as /jg/m3 of Air

                                                 Mean Fly Ash ± Standard Error
  Experiment
                                                                                  Mean Carbon Black ± Standard Error
                         Exposure Period
           Total
      Total
< 2.1 urn
In vivo
responses to
antigenic
stimulations





In vitro
responses to
mitogens.
recognitive
activity of
T cells and
phagocytosis
by macrophages
Cyto/yt/c
activity of
stimulated
T cells




7 days
21 days
21 days
35 days

56 days

148 days

7 days

21 days
35 days
56 days

148 days

7 days

35 days

56 days

154 days

(11/16-11/23/77)
(11/18-12/9/77)
(11/23-12/14/77J
(12/8/77-1/12/78)

(12/15/77-2/9/78)

(12/22/77-5/19/78)

(12/8-12/15/77)

(11/15-12/6/77)
(11/16-12/21/77)
(1/10-3/7/78)

(12/13/77-5/9/78)

(1/13/77-1/20/78)

(11/29/77-1/3/78)

(1/11-3/8/78)

(12/20-5/23/78)

2232
2039
2143
2059
*1628
1502
*2634
1459
*7S37
2262
*1159
2042
2115
1334
*2674
1297
1879
1905
*3129
2113
*2819
1134
*2674
1459
*1831
±256
±343
±286
±289
±375
±285
±430
± 140
±221
±298
±228
±343
±235
±237
±97
± 131
±240
±387
+ 1281
±384
±839
±237
±429
± 140
±221
933
785
764
741
433
543
615
535
461
794
366
809
804
502
623
509
465
661
691
703
676
502
623
535
461
±77
± 139
± 130
±86
±77
± 100
±98
±50
±50
± 104
±72
± 136
±90
±86
±97
±48
±55
± 134
±283
+ 777
± 794
±86
±96
±50
±50
4492
4707
3608
2511

2934

3509

3180

5399
4027
2984

3520

3758

3172

2984

3509

±
±
±
+

±

±

±

±
+
+

+

±

±

±

±

1917
1192
355
346

459

312

550

1372
910
512

342

1933

526

512

312

1538 ±
1472 ±
1104±
857 ±

841 ±

1178±

932 ±

1407 ±
1248 +
885 ±

1162 ±

996 ±

909 ±

885 ±

1178 ±

1029
386
125
157

125

119

161

429
292
139

130

512

142

139

119

 * Exposure data for fly ash supplied by EPA.
posure, with almost complete suppres-
sion after  148 days of  exposure. The
effect of fly ash on the  mediastinal
lymph nodes varied from an increase in
the number of antibody-forming cells
after  7 and 21 days of exposure to no
effect or a  small decrease of response
after 56 days. The cellular immune
reactions were not affected, except for
suppression of cytolytic activity by PSD
fly ash after 35 days. Both fly ash and
carbon caused a progressive decrease in
phagocytic activity by lung-derived macro-
phages from 21 to 146 days of exposure.
  A third series of experiments exam-
ined the effects of fly ash on various
functions of the alveolar macrophage.
Exposure of mice to PSU fly ash at
concentrations of 742 /ug/m3 of air of
particles <2.1  /urn for up to four weeks
decreased  the proportions  of macro-
phages capable of phagocytosis and the
proportion of very active macrophages
(seven or more bacteria phagocytized).
  For practical reasons, golden hamsters
were  used instead of mice  in studies of
antibody-dependent cell-mediated cyto-
toxicity by alveolar macrophages. Intra-
tracheal injection of 2  mg silica  into
golden hamsters significantly enhanced
the ADCC 1, 7,14,42, and 70 days after
 injection; intratracheal injection of 2 mg
 fly ash resulted in enhancement at 14,
 42, and 70 days. Inhalation exposure of
 golden hamsters to respirable-sized
 silica at an average concentration of
 3102 /ug/m3 of air enhanced ADCC
 function slightly after 7 days and signi-
 ficantly after  14, 42,  and 70 days.
 Inhalation of fly ash (5886 A
-------
   particles < 2.1 /nm in diameter and
   68.36 ±5.56% < 4.7//m. Differences in
   viability of  macrophages from the fly-
   ash-exposed and control mice, as deter-
   mined with trypan blue absorption,
   were not statistically significant. Inhala-
   tion exposure of mice to fly ash for two,
   three, and four weeks significantly
   reduced the number of alveolar macro-
   phages that phagocytized Staphylococ-
   cus aureus in in vitro culture.
     The  elements present in respirable-
   sized fly ash. particles used  in this
   project were determined by energy
   dispersive X-ray analysis (EDXA). This
   type of analysis in both the scanning
   and transmission electron microscopy
   modes was used to determine the
   elements  present  in alveolar  macro-
   phages from mice exposed and not
   exposed to fly ash.
     Examination of control  alveolar
   macrophages in the transmission mode
   revealed so much variation in ultrastruc-
   ture that characteristic alveolar macro-
   phages could not be ascertained. Varia-
   tions were observed in the amounts of
   mitochondria, endoplasmic reticulum,
   phagosomes, lysosomes, secondary
   lysosomes, and myelin figures and in
   the shape of nuclei. Scanning electron
   microscopy also revealed much variation
   among control cells, including differ-
   ences in cell shape and size, membrane
   ruffling, and cytoplasmic projections.
   All  of the ultrastructural and morpho-
   logical differences  among control cells
   were also seen among cells exposed to
   fly ash, for all exposure periods.
     The tissues of mice exposed to fly ash
   for 6 weeks (short-term) and 31 weeks
   (long-term) were histologically exam-
   ined. Pigmented alveolar macrophages
        were observed in the lungs after both
        short- and long-term exposures. The
        black non-birefringent pigment resem-
        bled anthracotic pigment; particles that
        were  birefrmgent resembled silica.
        Pigment was more randomly dispersed
        in the lung after short-term than after
        long-term exposure. In  the long-term-
        exposed lungs, pigment was  more
        prominent and tended to localize in
        alveoli immediately adjacent to terminal
        bronchioles. Uptake of pigment by
        mediastinal lymph nodes was more
        intense in long-term-exposed  mice.
        Other reticuloendothelial tissues (i.e.,
        Peyer's patches, mesenteric lymph
        nodes, spleen, and liver) did not show
evidence of fly-ash-pigment uptake.
Lymphoid accumulation associated
with fly-ash-pigment  deposition was
more prominent in the long-term-exposed
mice. For both exposure times, lymphoid
accumulation is interpreted as a reaction
to the presence of fly ash pigment.


Conclusions
  Fly ash in relatively  high concentra-
tions had little effect on antimicrobial
and antitumor activities of lymphocytes
and macrophages. Inflammatory changes
suggestive of early pneumoconiosis
were seen in the lungs  after 217 days of
exposure to fly ash.
           A. Zarkower, J. Davis, F. Ferguson, andD. Stricklerare with the Pennsy/via State
             University, University Park, PA 16802.
           Judith A. Graham is the EPA Project Officer (see below).
           The complete report, entitled "Determining Effect of Pollutants on the Immune
             System," (Order No.  PB 81-171 829; Cost: $9.50, subject to change) will be
             available  only from:
                   National Technical Information Service
                   5285 Port Ftoyal Road
                   Springfield,  VA 22161
                   Telephone: 703-487-4650
           The EPA Project Officer can be contacted at:
                   Health Effects Research Laboratory
                   U. S. Environmental Protection Agency
                   Research Triangle Park,  NC 27711
United States
Environmental Protection
Agency
Center for Environmental Research
Information
Cincinnati OH 45268
               Postage and
               Fees Paid
               Environmental
               Protection
               Agency
               EPA 335
Official Business
Penalty for Private Use S300
                      t O

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