AJKBAK 53 (I't. :{) 205-214 (1975)
THE ORIGIN OF THE IMMUNOCLOBULINS
IN INTESTINAL LYMPH OF SHEEP
by J. W. gUIN», A. J. HUSHANDf AND A. K. LASGELLES*
(From the Dair\- Research Unit, Svdnex- Universit\- Farms, Camden, N.S.W.
and the Department of Experimental Pathology,
John Gnrtin School of Medical Research, Au.stralian National University.)
{Accepted for publication April 21, 1975.)
Summary. N'arioiis iinmunoglobulins were labelled with radioaetive iodine and
their distribution between intestinal lymph and plasma followed in order to
determine the origin of the immunoglobulins found in intestinal lymph. By
comparing specific activities in plasma and lymph, it was computed tbat 25'i nf
the IgG, and IgGs and 90*^ of the IgA in intestinal lymph were locally
synthesised. The results sugge.st that \irtuall\ all of the IgA and a proportion
i»f the IgGj computed to be synthesised locally were derived from plasma cells
of corre.sponding specificity in the lamina propria of the intt.stine.
INTRODUGTION.
Previous studies have shown that most of the IgA and at least a proportion
of the IgM in mammary secretion of ruminants is synthesised localK b\' plasma
cells located in close association with the glandular epithelium (Lee and
Lascelles, 1969, 1970; Wat.son and Lascelles. 1973a). In contrast, the majority
of the IgG] and IgG2 in mammar\ secretion is derived from the blood (Mackenzie and Lascelles, 1968; Hrandon, Watson and La.scelle.s, 1971). Recentlv,
attention has been directed towards obtaining a similar understanding of immnne
mechanisms in the intestine (Lascelles and McDowell, 1974).
Studies on the transfer of radio-labelled immunoglobulins into intestinal
secretion of sheep demonstrated that, as in the mammary gland, the IgA in
intestinal secretion was almost exclnsively of local origin, whereas the IgM,
IgG. and most of the IgG, were derived from the blood (Gripps, Hnsband and
Lascelles, 1974). The absence of signifieant amounts of locally produced IgG,
• Present address: Department of Experimental Pathology, John Curtin School of Medical
Re.search. Australian National University, Ganberra, Australian Capital Territor\'. Australia 2fi00.
f Dairy Research l^nit. Sydney Uni\ersit\ Farms. Guniden, New South Wales Australia
2570.
t C.S.I.R.O. Divi.sion of Animal Health, Private Bag x\o. 1, P.O., Parkville. Victoria,
Australia 3052.
206
|. W g U I \ . A. I H I S H A M ) \M> A. K. LASCKLI.ES
in iiite.stiiKil sccrt'tiuii was surprising in vit'w of the high proportion oi IgC,•specific colls found in the laminii propria (Lee und Lascelles, 1970; Curtain and
Anderson, 1971),
Beh, Watson and Liiscelles (1974) concluded that most ol tlic IgA and
significant (jnantitics of IgG, and IgCj in intestinal lymph eonld not bo
accounted for hy eapillan filtraticni alone. This conclusion wa.s l>ast'd on comparisons l>ctwecii the concentration ratios (concentration in intestinal lympli
divided by the concentration in plasma^ of the immimoglohnlins with that of
albumin. In the present experiments a more detailed study of the origin of
immunoglolmlins iii intestinal lymph has lieen nndertaken nsing labelled IgGj,
Igt'2, IgNt, IgA, mid ulbnmin and the extent of plasma contribution determined
by eomparing specific activities in plasma luid lymph. These resnlts are discnssed
in relation to the origin of immimoglolmlins in intestinal secretion (Cripps
etaL 1974.)
MATEHIAUS
Animals.
Crosshn-d ucthtTN IH'IWI'CII I and 2 ><';irs of ;iyc were hiiiisrd ind(>i>rs In small pens
and pro\'jdf(l with Ititi'inc chafl' and water ad lihiltitn.
Surgical techniques ami .sample collection.
The inleslirml lyinphiitiV duct was taiiinilutt'd a.s descril)ed h> Liisti'lk's ami Morris
(1961). Lxiiiph was ttillrcti-d c-ontinuomly into stcrilt' blood infusion haRs which ctmtained
antifoagulant (Tuta LalMuatories (Australia) Ity. Ltd., Lane Cove). The Ivniph W;LS infused
l»ack into the animals even- .'^6 h throiiiih un indw-flling t-athftcr in tin- jiimilar vein.
Dctt-nninatioas of total protein concentration ((Hiriiall. Hardawill and Da\i(l. 1949) and cell
founts (ohtained tLsiii),; a '(Toulter' rniKli'l FN clcc-tronic cell counter) in hliKKl and lymph
revealed that ihe altovc procedure prevented the loss of protein and cells that (iccurs when
lymph is ullowcd tn drain Iri'fly from the iiite.stinai duel.
Lymph sample.s wvw collected into ((mduatvd gia.ss ccntrifnKf tnln-s coiitaiuiiiH stnall
amounts of heparin. and plasma samples were prepared from blood withdrawn from the
jugular catheter into ht'parinised containers.
Preparation of immuno(ilobulin.i and albumin.
Pure ovine IftC:,. IftCij and albumin were prcparwl by the melhtx) of \VatM>n. Brandon
and Lascelles (19721. O\inf IgM was prepari'd from Rivanol (2-ethox>-0.9-diaininoacridine
lactate. KiKh-Li«ht LaimratoHes. Ltd.. I'.K.) precipitated senini ( K. P. AdaniN and M. R. P.
Miller, paper in prfjiaration).
Ovine IfiA was prepared by pooling frattioiLs finm tlie trough rt'Kitm fiiMowiny the largest
peak of a DKAK Sepha<lex Kradient eluticin (OOIM to ()-40M pliosplmie Inilit-r. pH 8-0)
of sheep intestinal Uiiiph. 10 ml of .sheep intestiniil I> iiipli were ap]>lied lo u .') cm .\ 20 cm
glass column and the (wiolcd (raction> regularly caiiu' from clution volumes .'150-450 nil. The
chyloinicrons were removed from the intcNtinal Kniph In ultraL-eiitrtfugation ( 19.2(X) (,' for
1 .5 h) before the lymph was appliefl to the DE.M'" colunni. After concentration the pooled
material was fractionated on a C200 Sephadex i-ohimii using Tn's IICL buffer (pH 8-0).
Three peaks were obtained, with IgA lieing found in the ascending side of the eschision peak.
The purity of the proteins was tested on inimunoe!ectrophoresi.s and double diffusion
against monospeclRc antiscra. Antisera uere prepared as dt'StrilH-d b> Watson, Brandon and
Lascelles (1972). The iimnuiiiielectropluirctic put tern of the immunoglobulins lalH'iled in
experimcnl I nre sliDwti in Tigs 1. 2, '^ am! I.
IMMUNOGLOBULINS IN INTESTINAL LYMPH
207
Fig. 1. An imniunoelectrophoretic slide with pure IgG^ in the top well and normal .sheep
serum in the bottom well. Rabbit anti-sheep serum was used to fill the trough.
Fig. 2. An immunoelectrophoretic slide with pure IgG^, in the top well and normal sheep
serum in the bottom well. Rabbit anti-sheep serum was used to fill the trough.
Fig. 3. An immunoelectrophoretic slide with pure IgA in the top well and sheep intestinal
lymph in the bottom well. Ralibit anti-sheep .serum was used to fill the trough,
Fig. 4.
An immunoelectrophoretic slide with pure IgM in the top well and liormal sheep
senini in the bottom well. Rabbit anti-sheep serum was used to fill tht; trough.
]. W. QUIN, A. ]. HUSBAND AND A. K. LASGELLES
Quantitation of immimoglohutins and albumin.
A modificati[)n of the single railial iinnumodiflusi(ui method of Maneini. Carbonara and
llercmaiis (1965) ua.s u.sed for tbe quautitation of the tour iimuuiioglohuHns aud albnmin
(Brandon. Watson and Lascelles, 1971; Watson, Brandon and La.scellcs. 1972: Wat.son and
Lascelles, L973b).
Radioactive laheUint; and assay.
Pure preparations of the various umuunoglobulins were labelled with radioisotope according
to the method of Hehnkamp et ai (J960). IgC, and IgCg were labelled «itb '-'U and IgC^,
IgM and IgA witb ' -''I. Measureuicnts of radioactivity were carried out on 2 • 5 ml of
plasma and 5-0 ml of lympb using a well type detector with a 5 em tballiuui-acti\atecl sodium
iodide crystal eonnected to a Packard Model 2oH (^imputing Sealer/Timer antl Model 271
AnaK'ser.
The radioactivity of at ]ea.st one sample of plasma aud iute.stinai lyniph fit)ui each sheep
was characterised on C200 Sephadex. Iu this way, radioactivity was shown to be entirely
associated with the appropriate optical density peak.
Labctiing of plasma albumin.
Ry injecting sheep with T-1824 (Evans Blue) dyt* it was possible to follow the transfer
of albumiu between plasma and K'lupli. The amount {)f dye reco\'ered in samples of plasma
and lymph was deteniiinctl colorimctrically after its extraction from the samples according
to the method of Allen (1951).
Experimental proeedure.
TliL" intestinal lymphatic ducts of a total of 10 sheep were eannulated and 2-3 da\s
allowed for sbeep to recover from the effects of the operation prior to intra\enous injection
of the labelled protein. Four .sheep were injected with i^U-IgC, and 3 of these were also
injected with '--'I-IgM. Another .3 sheep were injected with '-'U-IgC^ and ^-''t-IgA. At
regular intervals over a 44 li period lynipli was collected for 10 min and a simultaneous blood
sample wa.s taken in the middle of each lymph collection. At the completion of sampling,
3 of these 7 .sheep were given jntra\enous injections of T-Ifj24 and sauiplcs of lymph and
plasma were obtained as abo\c over a further 22 h. In a second experiment 3 sheep were
injected with '-"I-IgC, and i-''I-lgC., and were sampled as described above.
The specific radioactivities and the radioactive counts injected into each sheep are
given below. Tbe specific activitj- varied with tbe concentratitui of protein used in the
labelling method.
Immunofilohnlin
Iodine
Speeifie iietiviit/
(cpm/mg)
5-0
1-5
9-3
4-1
IgM
IgA
X
X
X
X
10"'
10"
10''
10"
Do.sc injected
(cpm X 10")
15-0
15-0
14-S
13-4
RESULTS.
A plot of specific activity iu plasma and lymph agaiust time after injection
of the label is presented for each immunoglobulin (Fig. 5) and for albumin
(Fig. 6). In each case there was a characteristic sharp decline in the specific
activit} in plasma and, with the exception of IgA, thi-s was accompanied by a
rapid increase in specific activity in lymph. In the case of IgA in all sheep
the maximiun .specific activitv iu lyuiph was only a small fractiou of the .specific
activitv in plasma al coiiiparablt- tiiiics. Equilibrutioii was judged lo ha\e
IMMUNOGLOBULINS IN INTESTINAL LYMPH
209
480
360
120
7
1000
750
500
2S0
3600
125
I-IgM
2700
1800
900
2500Q
15000
sooc
"P ~ 9 ~ 9
01
-9- •
3
TIME
AFTER
9-
O
-9
24
30
34
INJECTION
(hours)
Fig. 5. Changes in mean specific activities (cpm/nig) of IgC^ IgG.,, Ij;M and IyA in plasma
and lymph with time after injection of labelled iminnnoglolnilins. The means were calculated
from obser\atioiis from 3 sheep for IgG^. IgM and IgA and frnni 1 ^heep for IgCp
• - • Plasma. O--O Lynipli,
210
. W. QUIN. A. |. HUSBAND AND A. K. LASCELLES
occurred when .specific activity curves for plasma aud lymph had merged or
become parallel. On this basis, equilibration of the inimunoglohulins between
blood and lymph occurred b\ 24 h whereas the .smaller molecular weight
albumin had eciuilibrated by 6-8 h. The computations appearing in Tables 1 and
2 are based on 4 post-ecjuilibration samples from each sheep for the immunoglobnliiis {24, 30, 34 and 44 h after injection) and 3 post-equilibration samples
from each sheep for albumin (8, 12 and 22 h after injection). In each sheep
the specific activities for IgGi and IgCj in lymph were always less than eomparablc specific activities in plasma (Fig. 5). In contrast, specific activities for
IgM and albumin in plasma and lymph of all sheep were similar after
equilibration had occurred.
ALBUMIN
0
2
E
TIME
8
AFTER
12
INJECTION
(hours)
Fig. 6. Changes in mean specific acti\ity of albumin (njj T-1824/mg albumin) in plasma and
lymph with time after injection of T-1824. The means were calculated from observations
from .} sheep. • - • Plasma. O-"O Lymph.
The mean concentration of each immunoglobulin and of an)nmin in plasma
and lymph and their lymph:pla.sma label (radioactivity or T-1824) ratios are
presented in Table 1. The results show that the concentrations of IgGi, IgG^,
IgM and albinnin in intestinal lymph were only 35-5()^ of ttiose in plusma,
whereas the concentration of IgA in 1\ mph was about 3 times greater than that
in plasma.
The !\ inph:plasma label ratios were calculated b\' dividing the amonnt of
label in lymph b\' that in p]a.sma taken at the same time. The results show that
the IgG] ratio was greater than that observed for IgG^.. However, it was concluded that the lower IgGg ratio was due to between .sheep variation since in a
second experiment, in which ''"I-labelled IgG| and '-''I-labelled IgG^. were
injected simultaiieously into each of 3 sheep, the lymph :plasma radioactivity
ratios were similar (Table 2). In all sheep the ratio for albumin was significantly
higher than ratios for the immunoglobulins. It can be seen from Table 1 that
the concentration ratios for albumin were similar to the label ratio.s.
so " t r--1-% —
+1+1+1+1+1
Q^ \C v^ ^ m
r^ r'l ^c i^ r*j
T t 1-^ CNOC O^
p^ r-- ^> Ov
^
—. ^ ^
^
OC (N O — - ^
OOOOO
1 ! - 2 0 + 1 26
2 1 4 + 0 64
0 74 + 0 1 5
0 48 + 0 10
1 1 18 + 2-06
+1 +1+1+1+1
ri •c — 5 o
odddo
6 bboo
+1+1+1+1+1
OO OO o O c^
OOOOO
•n, O ^ O TjO — O O f-J
+1 +1 +1 +1 +1
iC t ^ fN *C O
— u-1 p^i O (N
fN
r-i
No. of 1
sheep
,
(A)
Plasma
concentraiion
(mg/ml)
(B)
Lymph: plasma
label ratio
(C)
Lymph
concentration
(mg/ml)
{ C - A x B)
Amount locally
synthesised
(mg;ml)
% Plasmaderived
Significance of*
local synthesis
lMMUN()(;iX)in'I,INS IN TNTF.STINAl, LYMPH
CJZ
c
E
Q Si
211
[. W. yUJN. A. 1. Hl'SHAND \MT) .\. K. I.ASCELT.ES
212
TABLE 2.
The lymplt:plasma lahel ratio observed when ' ^ ^l-lahellcd IgG, ami ' ^ ^l-lahelled l,i,'C • are injeeled
into the same sheep.
Sheep 1
Sheep 2
Sheep 3
0 45 + 0 0 8 *
0 33 + 0 09
0 38 + 0 07
0 43 + 0-05*
0 38 + 0 09
0 42 + 0 07
* The values presented are the mean ± standard error for 3 observations on each sheep.
An estimate of the amount of eaeh immunoglobuhn and albumin in intestinal hmph derive<l from blood plasma wa.s computed as the product of the
lymph:plasma label ratio and the plasma concentration. Thus, the proportion of
plasma-derived protein in lyniph was obtained by expre.ssinj; the amount eoinputed above as a percentage of the concentration in lymph. The amount
synthesised locally was computed as the difference between the amount derived
from plasma and the Kmph concentration. The significance of local synthesis
was determined b\ anal)sis of variance of the difference between the amount
of plasma-derived protein and the protein concentration in lymph (Table 1).
These calculations indicate that almost all of the IgM and albumin were derived
from blood plasma. However, for IgCi, IgGo and IgA there was a highly
significant difference (P < 0 001) between the amount derived from plasma
and the lymph eoneentration. The resnlts in Table 1 indicate that about 75% of
the IgGi iiiid IgG:; 'ind only about 10^' of the IgA were derived from plasma.
It was also of interest to measure the How of intestinal lymph in order to
estimate the dailx' output of IgA from the mesenteric region. The mean and
standard error of 7 measurements of flow for each of 6 sheep was 59-47 ±
4-06 ml/h and the mean IgA concentration in lymph was 0-48 mg/ml {Table 1).
Thus, intestinal lymph was contributing about 0 7 g of IgA to the blood each
day.
DISGUSSION.
The specific activity in lymph relative to that in plasma gives an indication
of the amount of labelled immunoglobulin deri\ed from plasma. Thus, the very
low specific activity of IgA in lympli relative to pla.snia (Fig. 5) indicates that
the great majority of the IgA in lymph is locally synthesised \vith only a small
proportion derived from plasma (Table 1) in accordance with an earlier report
(Beh et ai, 1974). These results are also in conformity with those of previous
workers (Lee and Lascelles, 1970; V^aermau, 1970) who reported the occurrence
of large numbers of IgA-.specific cells in the lamina propria and their virtual
absence from lymph nodes. Althongh most of the IgA produced lay these cells
probably enters intestinal .secretion, it is apparent that a significant amount
enters the lymph. It is considered that only tri\'ial amounts of IgA were contributed by mesenteric lymph nodes, since Beh and Lascelles (1974) failed to
find significant numbers of IgA-specific cells in this tissue.
IMMIIN(X;L()HUL!\S
I X T K S ' I I X A L LV\II>11
21.3
The present results also demonstrate that about 25'^ of IgG, and lg(ij is of
local origin. Thi.s confirms the suggestion of Heli et al. (1974) that a proportion
of IgC, and IgC^. in intestinal lymph was not accounted for by eapillaiy filtration
alone. It is probalile that a considerable amount of tlie newly svnthesised IgC,
originated from plasma cells in the lamina propria of the intestine since histological studies ha\e demonstrated the pre.sence of large numbers of IgC,-speeific
cells ill this region {Lee and Lascelles, 1970; Curtain and Anderson, 1971). In
addition, eells of this specificity are distributed in areas more rem<jtc from the
intestinal epithelium than eells of IgA specificity {Brandtzaeg, liaklieii. Fausa
and Hoel, 1974; A. J. Husband, unpublished data) which may account for the
escape of locally .s\'nthesi.sed IgC| into lymph rather than its secretion into the
intestinal lumen (Cripps el ai, 1974), Significant local eontril)ution of IgC,
could also come from the mesenterie Ivniph nodes, particularly in view of the
high proportion of IgCpContaining cells in cell suspensions from mesenteric
nodes (Beh and Lascelles, 1974). This is also c(JMsidered to be the most probable
source of all locally synthesised IgC:., since plasma cells of IgGa specificity are
rare in the lamina propria (Lee and Lascelles, 1970; Curtain and Anderson, 1971).
The computation of IgA output yielded a lower figure than the 2 0 g/day
calculated by Beh et al. (1974), This resulted from the lower concentration of
IgA in intestinal Ivniph of the sheep u.sed in the* present studies. The intestinal
lymphatic preparations used b)- Beh at al. (1974) were allowed to drain freeh
and this appeared to result in a progressive elevation of IgA concentration in
lymph as drainage continued (Beh, personal conimunication). This may in part
explain the discrepancy in the findings,
Neverthele.ss, it is apparent that there is a lai-ge amount of IgA produced
by sheep eaeh day. Oipps et al. (1974) reported the IgA concentration in
secretion from isolated intestinal loops to be 4 9 mg/ml, at least 98!? of whieh
was of local origin. Since about 100 ml of secretion were collected from a 30 cin
loop of intestine each da\, this represents the production of close to 0-5 g of
IgA per da\ from only about 10-15% of the small intestine. When this is added
to the 0-7 g per da\ calctilated for intestinal lymph outj^nt, tho \ast majorit)of whieh was produced !ocall\. the (juantitati\e importance of the Ig.A ,sv,steni in
tho intestinal region becomes apparent.
Acknowtedgfmeut.\. We wisli to thank Mrs. ,S. Sliarpe for competent technical assistance.
This work was supported in part liy u grant from ihe Australian Dairy Produce Board. One of
us (A,J,H.) was supported hy a C.S,I.R,O, Postgraduate Seholarship award.
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