Download Directional Selection for Specific Sheep Cell Antibody Responses

IMMUNOLOGY, HEALTH, AND DISEASE
Directional Selection for Specific Sheep Cell Antibody Responses
Affects Natural Rabbit Agglutinins of Chickens
P. F. Cotter,1 J. Ayoub, and H. K. Parmentier
Framingham State College, Framingham, Massachusetts 01701-9101; and Wageningen Institute of Animal Sciences,
Wageningen University, 6700 AH Wageningen, The Netherlands
intermediate, and line L was lowest. The correlation between SRBC and RRBC titers was 0.43 (P = 0.0). Females
had higher titers than males, but the difference was only
significant for the SRBC antibody (P = 0.028). Qualitative
changes in anti-Gal accompanied SRBC selection. Rabbit
agglutinins of 4 types were recognizable: classic, granular,
annular, and one negative or very weak reaction. The
score type means in line L were highest, in the control line
were intermediate, and in line H were lowest, suggesting
avidity differences now exist among these lines. The results show integration of natural and acquired immune
systems because selection for one temporarily affected
the other. Given the importance of anti-Gal in primates,
our results should stimulate further study of this antibody
in poultry species.
ABSTRACT Agglutination data from generations 8
through 19 indicate that bidirectional selection for specific
SRBC antibody responses was successful in a line cross of
ISA × Warren medium heavy layers. After 11 generations
titers of the high SRBC selected line (H line) were nearly
1:32,000; those of the low SRBC selected line (L line) were
less than 1:2, but titers of the randombred control line
remained stable at 1:32. Directional SRBC selection also
affected levels of a naturally occurring rabbit cell agglutinating antibody (RRBC), presumably the avian form of
α-galactose antibody (anti-Gal). This indirect response
was biphasic and opposite in direction to the SRBC responses through generation 14 after which anti-Gal titers
of all 3 lines increased. At generation 19, line H had the
highest agglutinin titers; of both types, control line was
(Key words: natural rabbit antibody, anti-α-galactose antibody, sheep red blood cells, directional selection)
2005 Poultry Science 84:220–225
diseases (Biozzi et al., 1979). Those observations
prompted several groups to undertake similar experiments with chickens. The collective results have shown
that acquired sheep agglutinin levels respond to selection
in either direction (reviewed in Pinard-van der Laan et
al., 1998).
Selection for one antibody can also affect the response
to other antigens. As a recent example, Yunis et al. (2002)
found that chickens selected for Escherichia coli vaccine
titers had correlated responses to Newcastle disease virus
(NDV) and infectious bursal disease virus (IBDV). It
would be of interest to determine whether selection for an
acquired response also affects natural antibodies. Because
rabbit agglutinins exist at high levels in several avian
species (Cotter, 1998) they may provide a convenient test
system to study interactions between acquired and natural antibody. Previously, higher levels of natural antibodies directed to exogenous and endogenous proteins were
detected in the present H line (Parmentier et al., 2004).
Furthermore, adoptively transferred natural antibodies
enhanced subsequent specific antibody responses (Lam-
INTRODUCTION
The presence of natural antibodies to xenogenic erythrocytes in chickens was described by Bordet (1898) more
than a century ago. Quantitative data obtained by Bailey
(1923) established that in chickens, antibodies capable of
agglutinating rabbit cells have the highest titers among
14 species tested. She also found that natural sheep cell
agglutinins were typically at low levels in unsensitized
chickens and only slightly above those for turtle, horse,
and goat. The same author found that absorbing pooled
chicken serum with guinea pig cells reduced the rabbit
titers by 75%. The remaining 25% of the rabbit cell antibodies were refractory to further absorptions demonstrating their heterogeneity. Thus the rabbit agglutinin
system of chickens is complex and composed of multiple types.
Genetic selection for specific sheep cell antibody in mice
is accompanied by greater resistance to some infectious
2005 Poultry Science Association, Inc.
Received for publication April 10, 2004.
Accepted for publication September 2, 2004.
1
To whom correspondence should be addressed: 39 Hathaway Cir.
Arlington, MA 02476; e-mail: [email protected]; [email protected].
Abbreviation Key: anti-Gal = α-Gal antibody; H = high SRBC selected; L = low SRBC selected; RRBC = rabbit red blood cell.
220
ANTIBODY SELECTION AFFECTS NATURAL RABBIT AGGLUTININS
mers et al., 2004), suggesting genetic and or functional
linkage between natural and specific antibodies. Moreover the pathophysiology of protozoan and clostridia disease in primates is related to α-galactose antibody (antiGal; Pothoulakis, 1999). These observations provide a further strengthening of the case for the study of chicken
anti-Gal because both are important avian pathogens.
The objectives of the current research were to demonstrate that directional selection for sheep agglutinins also
affected titers of natural rabbit agglutinins and that this
was accompanied by changes in the rabbit antibody quality as well.
MATERIALS AND METHODS
Sera
Blood samples were obtained from wing veins during
the course of a divergent selection experiment begun in
1986 (Van der Zijpp and Niewland, 1986). Twenty samples from both selected lines were available at generations
8, 11, 14, 17 and 19; n = 200. Twenty additional samples
were available at generations 8 and 19 from the randombred control; n = 40. These samples were kept in
frozen storage (−20°C) in the Netherlands and shipped
by airfreight to the United States, where they were analyzed further.
Experimental Lines
In brief, the lines described here originated from an
ISA × Warren cross of medium heavy layers. Lines high
(H) and low (L) SRBC selected were subjected to upward
and downward selection for SRBC agglutinins, respectively. In addition an unselected control line was also
maintained. Selection was based on individual merit using plasma microtiter levels determined at d 37 of age
(Pinard, et al. 1992). This date represented the fifth day
postimmunization i.m. with 25% SRBC.
Rabbit Cells
Whole blood of domestic rabbits was collected from
the ear vein into chilled Alsever’s anticoagulant solution
and stored at 4°C for no more than 1 wk. Erythrocytes
were separated by low speed centrifugation, and residual
serum was removed by 3 washes with PBS. Packed cells
were adjusted to 2% vol/vol prior to use.
Agglutinin Testing
Microtiters were performed in 96-well U-bottom
plates.2 Sera were diluted with PBS so that column one
2
Catalog number UN 2-9064, Marsh Bio-Products, Rochester, NY.
Sea-Kem type ME, catalog number 50015, FMC Inc., Rockland, ME.
4
Goat-anti chicken IgM, catalog number 643951, MP Biomedicals
(formerly ICN) Costa Mesa, CA.
5
Release 14, Minitab Inc., Malvern, PA.
3
221
was at 1:10 followed by doubling dilutions thereafter. Ten
microliters of (2%) rabbit cells was added to each well and
mixed with the serum dilutions. Plates were incubated at
room temperature for 2 to 4 h, after which the cells settled.
They were resuspended by vigorous tapping of the plates
and allowed to resettle. Agglutination was determined by
examination of each well containing serum by mirrored
fluorescent light. Wells not containing serum served as
negative (cell) controls. The column number of last well
showing clear-cut evidence of agglutination was recorded
as the titer.
Qualitative differences in rabbit red blood cell (RRBC)
agglutinins were recognized and scored by type as follows: 1 classic (blanket), 2 granular (gritty), and 3 annular
(ring). Type 4 reactions (buttons) were considered as nonagglutinated and scored as 0 for the purpose of calculating
the line means.
Modified Ouchterlony Double Diffusion
Glass plates were flooded with 0.75% agarose3 to a
depth of 0.75 mm. Serum or IgM specific antibody4 was
added to well rows punched at a distance of 10 mm. Based
on an earlier method of diffusion distance measurements
(Cotter, 2000) the locations of the precipitates formed
between the sample and antibody wells were used as a
gauge of molecular weights. This is possible because sample well-precipitin distances are determined by diffusion
coefficient (molecular weight) differences. Precipitate
lengths were taken to represent quantity as these are
determined by the amount of IgM in the sample that has
entered the zone of equivalence (Crowle, 1973).
Statistical Analysis
Data were analyzed using the Minitab5 statistical package. Titer data were analyzed using the GLM and interaction-main effects plot procedures. Correlations were
determined by matching individual SRBC titers (obtained
in the Netherlands), and rabbit cell agglutinin data were
determined in the United States. One-way analysis of
variance was used for IgM diffusion distances and RRBC
agglutination scores.
RESULTS
The results of divergent SRBC antibody selection and
its effect on RRBC agglutinin levels are shown graphically
in Figures 1 and 2. These results represent the mean titers
for the respective erythrocyte over the course of the experimental run. A direct comparison of SRBC and RRBC
titers requires alignment because of the initial 1:10 dilution used with the latter. This is accomplished by addition
of 3.3 (log2 of 10) to the RRBC values.
The response to SRBC selection in these lines has been
presented in more detail elsewhere (Bovenhuis, et al.,
2002). It is necessary to include some SRBC information
here as well because those results are interconnected with
the current data, and together they indicate a certain degree of integration of acquired and natural immunity.
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COTTER ET AL.
FIGURE 1. Titers of SRBC agglutinins, determined by doubling dilutions (log2) are shown for control (C) and lines selected for high (H)
and low (L) SRBC over the course of divergent selection. Data for line
C were available at generations 8 and 19 only.
Generation means shown by Figure 1 indicate that at
generation 8 the SRBC titers were already clearly separated among lines. The log2 titer of the control line was
4.95, near to the exact average of lines H (8.6) and L (2.8).
The response to downward selection was immediate in
line L, and titer means continued to decline through generation 19. The response to upward selection in line H
appeared to exhibit a slight lag because the mean for
generation 11 was slightly less than at generation 8 (8.3
vs. 8.6). This delay was only temporary as H line titers
rose and reached 10.1 by generation 14. The upward response of line H continued through generation 17 when
the mean titer reached 12.9. Titers of line H increased
FIGURE 2. Titers of rabbit red blood cells (RRBC) agglutinins are
shown for control (C), high SRBC selected (H), and low SRBC selected
(L) lines over the course of SRBC selection. Sera were diluted to1:10
initially, followed by doubling dilutions (log2). Titers of RRBC are lower
than SRBC titers by a factor of 3.3 (log2 10) due to the initial dilution.
Means of lines H and L within a generation not sharing a common
superscript are significantly different (P < 0.05). Data for line C were
available at generations 8 and 19 only.
further to nearly 15 by generation 19. Thus by the end of
the experiment SRBC titers of the H line would be greater
than 1:32,000, and L line titers would be less than 1:2
when expressed using conventional terminology.
There appeared to be some directional asymmetry in
the responses of lines H and L. This difference was reflected by differences in the values of coefficients of quadratic fits to the data, where H = 9.9 − 1.45x + 0.07x2, and
L = 0.45 − 0.08x + 0.01x2. This result is not uncommon in
bidirectional selection experiments. Such asymmetry is
sometimes considered to arise as a consequence of the
scale of measurement (Falconer, 1981). Here it was probably due to the initial SRBC titer of line L, which was 2.8
at generation 8, already being close to its ultimate lower
bound of zero, preventing further downward selection.
Conversely titers of the H line were not subject to a similar
constraint, but ultimately they might be expected to reach
a plateau.
In contrast to the directional changes that occurred in
selected lines over the course of the experiment, SRBC
titers of the control line remained virtually unchanged.
Their final level of 5.05 at generation 19 was scarcely
higher than the initial level of 4.95 at generation 8. Thus
when expressed conventionally control line titers remained at a constant 1:32 over the course of the experiment.
Figure 2 shows the corresponding generation means
for the rabbit agglutinin levels (RRBC) in each of the 3
lines. As was the case with SRBC titers, lines were already
separated by generation 8 with control line titers at intermediate levels.
The initial titer of line H was about 5.4 (corrected to
account for the initial 1:10 serum dilution) and actually
declined to about 4.6 by generation 14. Conversely rabbit
agglutinins of line L increased from 3.7 at generation 8
to 4.2 by generation 14. After generation 14, RRBC titers
of both selected lines rose, and this continued through
generation 19 in line H, but line L appeared to plateau.
Thus the RRBC antibody titers of the selected lines
were characterized by biphasic response kinetics. Initially
(through generation 14) the direction of the response was
opposite to what was occurring as the result of directional
(SRBC) selection. The second phase began after generation 14 during which time rabbit agglutinin levels of both
selected lines rose and reached their highest levels at
generation 19.
Figure 2 also includes the RRBC levels of the control
line. Data were available at generations 8 and 19 only,
which was also the case for the SRBC titers. Unlike the
unchanged SRBC titers, the rabbit titers for the control
line increased from 4.6 to about 7.5 by generation 19.
Expressed conventionally this would correspond to an
initial titer of less than 1:32 and a final titer greater than
1:128 or an increase of about 3 log2 dilutions.
Although the kinetics of indirect response to selection
were different from those of the direct response at generation 19, the rank of the 3 lines was the same for both
agglutinin types. Line H was highest, the control line was
223
ANTIBODY SELECTION AFFECTS NATURAL RABBIT AGGLUTININS
TABLE 2. Mean location of IgM precipitate diffusion distances
Location of IgM precipitate2
Line1
Gen 83
n
Gen 19
n
H
Control
L
6.1y
6.9x
6.0y
10
10
10
5.4y
5.9x
5.4y
174
20
174
x,y
Means in the same column sharing no common superscript differ
significantly (P < 0.05).
1
H = high SRBC selected; L = low SRBC selected.
2
Distance in mm between sample well center and precipitate meniscus.
3
Gen 8, Gen 19 = generations 8 and 19.
4
IgM was undetectable in 3 samples.
FIGURE 3. Individual SRBC agglutinin titers, sex means, and their
connection for control, high SRBC selected, and low SRBC selected lines.
Data for the control line were available at generations 8 and 19 only.
intermediate, and line L was lowest for each agglutinin
type.
Gender had an important effect on SRBC titer. Over all
generations, mean titers of hens were higher (6.6, n = 127)
than those of cocks (5.1, n =113; F = 4.87, P = 0.028).
Individual values are shown in Figure 3, which also includes the location of the gender means and a connecting
line. RRBC titers were higher in hens compared with
cocks (dilution corrected, 5.6 vs. 5.4), but this difference
was not significant, F = 2.4, P = 0.13.
Qualitative differences in the forms of the agglutination
of rabbit cells accompanied the observed quantitative
changes. These were easily recognized, and a scoring system was used to categorize settling patterns. The 3 distinctly positive agglutination types were found reinforce
the point that these antibodies represent a heterogeneous
population. Type 4 reactions resembled buttons (which
typically represent an absence of agglutination in the
SRBC system) and were very weak and suspended easily
after abrupt manual agitation of the microtiter plates. For
the purpose of calculating line means, these values were
regarded as negative and recorded as zero. Thus lower
scores (except zero) result from higher antibody avidity.
A significant difference was found with line H having a
lower score than the control line or line L (Table 1).
Qualitative differences in IgM were detected among
lines using Ouchterlony double diffusion data (Table 2).
TABLE 1. ANOVA of RRBC agglutination type line mean scores
Line1
H
Control
L
n
100
40
100
Mean
agglutination
score2
x
1.90
2.25x
2.41y
SD
0.72
0.78
0.85
Means sharing no common superscript differ significantly (P < 0.05).
H = high SRBC selected; L = low SRBC selected.
2
Where 1 = classic, blanket pattern, 2 = granular or grit, 3 = annular
or ring, 4 = very weak reactions, cells easily suspended.
x,y
1
Measurements of the locations of the precipitates indicated that the molecular weight of IgM in lines H and L
was slightly higher than in the control line, which was
because their precipitates were located closer to the sample wells than those of the control line, indicating a slower
diffusion rate.
The average precipitate location of the control line at
generation 8 was 6.9 mm compared with 6.1 and 6.0 for
lines H and L, respectively. The corresponding measurements for generation 19 were 5.9 mm for the control and
5.4 mm for lines H and L. The apparent lower diffusion
distances found in generation19 could not be a consequence of SRBC selection because they were present in
the control line as well. It is more likely that the observed
generational difference was an artifact related to slight
variations associated with the assay method. However it
is important to note that the generational differences were
consistent in that the control line had higher values at
both times.
DISCUSSION
In humans, some of the mechanisms of innate immunity are based on cross-reacting natural antibodies. Examples include the hyperacute rejection of xenografts and
complement-dependent cell destruction. The best-characterized target antigen in xenograft rejection is α (1–3)
galactose, which is also widely distributed on the surfaces
of bacteria, protozoa, and viruses (Starzl and Zinkernagel,
1999). Apart from carbohydrates, natural antibodies directed to a variety of exogenous and endogenous antigens, including proteins, have been described
(Avrameas, 1991).
Natural antibodies that agglutinate rabbit cells are
likely to do so because these cells express high levels of
Galα1-3Galβ1-4GlcNAc-R, known as the α-Gal epitope
(Galili, et al., 1984). They are widely distributed among
animals and are found in several species of fowl including
chickens, ducks, emus, and turkeys (Cotter, 1998b). In
primates such antibodies are known as anti-Gal, and presumably those described here represent the chicken
equivalent. Their source is not known for certain, but
natural antibodies have long been believed to originate
from a stimulus provided by the surface of gut microor-
224
COTTER ET AL.
ganisms (Weiner, 1951). In human serum anti-Gal is
found between 30 and 100 µg/mL representing 1% of all
circulating antibody, and it is the largest single kind. It
is likely to be as important in chickens but is presently
incompletely characterized. However the following properties have been observed. The agglutination of rabbit
cells of inbred Leghorn chickens showed galactose concentration-dependent inhibition. Increasing galactose in
the diluent (from 0 to 1% in 0.25% intervals) was accompanied by a highly significant reduction in hemagglutination score. In a limited study of pooled serum from the
lines described here RRBC agglutination was reduced in
the presence of galactose, melibiose, and cellobiose. The
levels of RRBC agglutinins of turkey sera were compared
by using 2 diluents (PBS vs. 1% galactose in PBS). The
regression of hemagglutination score in the presence of
galactose vs. its absence was negative and highly significant (P. Cotter, unpublished data). Collectively these observations support the idea that these antibodies are
related to the anti-Gal of primates.
Anti-Gal isotypes of the IgG, IgM, and IgA classes are
found in primate serum, and IgG is the predominant type
(Galili, et al. 1995). Because chickens have isotype classes
corresponding to those found in mammalian species (Benedict, and Berestecky, 1987), the rabbit agglutinins described here are likely to have broad class membership
as well. Some rabbit agglutinins found in chickens were
inactivated by 2-mercaptoethanol (Cotter, 1998a), suggesting that that IgM isotypes are included, although
some chickens also have 2-mercaptoethanol–sensitive IgG
(Delhanty, and Solomon, 1966). Hemagglutination depends on many variables, including the amount and type
of antibody present; the size, number, and location of
available antigen sites; and the pH, temperature, and ionic
strength of the test medium (Quinley, 1993). Those antibodies described here represent a heterogeneous system
because some rabbit agglutinins are removed by guinea
pig cells, whereas others are not (Bailey, 1923). Because
a single diluent (PBS) and temperature were used
throughout, conditions governing agglutination were
probably optimal for only a portion of the antibodies
present in any one line at any generation, which would
account for some of the difference in the observed
patterns.
Some of the genes that control the SRBC response are
located on the sex chromosome (Boa-Amponsem, et al,
1997), which may account for the gender differences observed here. Although the gender difference in rabbit
agglutinin levels was not significant females had higher
titers than males. Thus the RRBC observations paralleled
the SRBC results. Perhaps there are general immunoregulatory genes located on the Z chromosome that influence
a broad range of antibody specificities. If such genes exist
they must act to down regulate antibody production because the hemizygous (Zw) females have higher titers.
Based on the observations of the control line it is likely
that any major gender difference in genes controlling
rabbit agglutinin levels were long ago removed by natural
selection. This observation may reflect the relative impor-
tance of the anti-Gal antibody in the overall immunity
scheme, which is why natural sheep agglutinins are at
low levels in unsensitized chickens, but rabbit agglutinins
are at moderate levels (Bailey, 1923). Recently, microsatellite regions for specific antibody responses to SRBC were
detected on chromosomes 3, 5, 16, and 23 by half sib
analysis, and 10, 16, and 27 by line cross analysis (Siwek
et al., 2003). Whether these regions are also involved in
anti-Gal or antibodies to lipopolysaccharides, and protein
antigens is being examined.
A comparison of the responses of the control line suggests that rabbit agglutinin levels are more sensitive to
environmental influences compared with sheep agglutinins. However, to our knowledge there was no dramatic
change with respect to husbandry or feed between these
generations. There was virtually no change in SRBC titers
of the control line between generations 8 and 19 but its
rabbit titer was near that found in line H. The possibility
that the present results can be accounted for because of
epitopes common to both erythrocytes is unlikely. RRBC
antibody titers declined while SRBC titers rose through
generation 14, and in the original survey data (Bailey,
1923) low levels of SRBC agglutinins were found in the
presence of much higher RRBC levels. Both observations
would be difficult to explain if the 2 erythrocyte species
shared epitopes.
It would be presumed that the heritability of the antiGal response would be lower than the SRBC response.
Thus artificial selection for anti-Gal titers would likely
be met by more resistance form natural selection and
progress in either direction would be slower than observed for sheep antibody.
Our data indicate that directional selection for SRBC
antibody was highly successful and it was accompanied
by an indirect effect on natural rabbit agglutinins. When
all data are included the correlation between SRBC antibody titer and RRBC titer was 0.43 (P = 0.0). This must
be considered a moderately high value given that the
indirect response was biphasic and partly opposite in
direction to that of the direct response. In addition the
SRBC levels of the control line remained virtually unchanged over the course of the experiment but RRBC
levels increased.
Natural antibodies may play an important regulatory
(often enhancing) role in specific immune responses (Ochsenbein et al., 1999). The high levels of natural antibodies
to proteins in the present lines (Parmentier et al., 2004)
and the functional relation between natural and specific
antibodies (Lammers et al., 2004) are in agreement with
this idea. It is difficult to speculate about the biological
significance of anti-Gal antibodies in poultry, although a
role in anti-retroviral resistance has been proposed in
primates. It is noteworthy that levels of natural antibodies
binding proteins are very low in young chickens, it remains to be established how age affects anti-Gal antibodies.
In summary the selection results show clear evidence
for the integration of genes that control the natural and
acquired immune systems. The exact nature of equiva-
ANTIBODY SELECTION AFFECTS NATURAL RABBIT AGGLUTININS
lence between the natural rabbit antibody described here
and the anti-Gal of primates awaits further experimentation. Given the established importance of anti-Gal in primates it is possible that the anti-rabbit agglutinins
described here represent a significant component of the
immune system of chickens. Further study of the properties of chicken anti-Gal is clearly indicated.
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