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Passage of immunoglobulins from plasma to the oral cavity in rhesus monkeys

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Passage of immunoglobulins from plasma to the oral cavity in rhesus monkeys
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  Immunology 197835 923 Passage of immunoglobulins from plasma to the oral cavity in rhesus monkeys S. J. CHALLACOMBE , M.W. RUSSELL JANE E. HAWKES, LESLEY A. BERGMEIER  T. LEHNER Departmentof Oral Immunology and Microbiology, Guy s HospitalMedical and Dental Schools, London Received 9 February 1978; acceptedfor publication16 March 1978 Summary. The passage of immunoglobulin fromplasma to the oral cavity was studied in rhesus monkeys.ImmunoglobulinsG, A and M were puri- fied from pooled rhesus monkey serum, radio- labelled with 125I and injected intravenously into twelve monkeys. Sequential samplesof oral fluids weretaken overa 24 h period and were assayed forradioactivity. Radioactivity could be detected in crevicular fluid washings after 0-5 h in monkeys injected with IgG and IgA, and after 2 h in monkeys given IgM. Maximal levels were found after 4 h with eachimmunoglobulin. Radioactivity in parotid and mixed saliva couldbe detected in all animals after 30min,reaching a maximum levelafter 4 h. Ultra- centrifugation on sucrose densitygradients revealed that most of the radioactivity in crevicular fluid washingswas in the 7S zone in the animals given IgG and IgA, and in a 19S zone in animals given IgM. The radioactivity in partoid saliva did not represent intact immunoglobulin molecules, since all the activity was present in zones of low molecular weight in animals given IgG, IgA or IgM. In mixed saliva a small amount of radioactivity was found in the immunoglobulin zones. The results suggest that *Present address: Department of Immunology, The Mayo Graduate School ofMedicine, Rochester, Minnesota,U.S.A. Correspondence: Dr M. W. Russell, Department of Oral Immunology and Microbiology, Guy s Hospital, London SEI 9RT. 0019-2805/78/1200-0923 02.00   1978Blackwell Scientific Publications intact molecules of IgG, IgA and IgM canpass fromplasma to the oral cavity via crevicular fluid, and could contribute to oral defence mechanisms particularly in the crevicular domain. The volume of crevicular fluid in the approximal space of deciduousmolars of rhesus monkeys was estimated to be approximately 0 3 1I. INTRODUCTION IgA is the predominant immunoglobulin class pre- sent in human secretions and at mucosal surfaces  Tomasi, Tan, Solomon   Prendergast, 1965; Tomasi   Bienenstock, 1968). Almost all of the IgA in human saliva is of the secretory type  Brandtzaeg, Fjellanger   Gjeruldsen, 1970) and is secreted locally by the major salivary glands  Hurlimann   Zuber, 1968) and by minor salivary glands  Craw- ford, Taubman   Smith, 1975). Approximately 10 of the IgA in mixed saliva is in the monomer form  Brandtzaeg et al. 1970). Not all of this is accounted for by the amount present in parotid and submandi- bular saliva and some may be derived from extra- glandular sources. IgG and IgM also contribute to the total globulin content of saliva. The concentra- tion of IgG in particular is considerably higher in mixed saliva than in parotid or submandibular secretions  Brandtzaeg et al. 1970) and is probably  riv from extraglandular transfer either entering the oral cavity directly through thesurface mucosal 923  S. J. Clhallacombe, M. W. Russell   Jane E. Hawkes epithelium, or viathe gingival crevice surrounding the teeth  Brandtzaeg et al. 1970 .   proportion of IgM is secretedlocally and has secretory com- ponent attached  Brandtzaeg, 1972 , but since the amount of IgM in mixed saliva is significantly correlated with the degree of gingival inflammation  Orstavik   Brandtzaeg, 1975 , much of the IgM in mixed saliva may be derived from the gingival crevice. The source of immunoglobulins and antibodies in the oral cavity is currently of considerable interest. Protection against dental caries in animal models has been achieved by invoking salivary antibodies against Streptococcus mutans in rats  Taubman   Smith, 1974; Michalek, McGhee, Mestecky, Arnold   Bozzo, 1976 and serum antibodies in monkeys Bowen, Cohen,Cole   Colman, 1975; Lehner, Challacombe   Caldwell, 1975,1976 . Recently protection against dentalcaries has been found in rhesus monkeys passivelyinfused intravenously with antibodies of the IgG class, but not withim- mune serum or antibodies of the IgM or IgA classes  Lehner, Russell, Challacombe, Wilton, Scully   Hawkes, 1978 . Antibodies givenintravenously must therefore be able to reach the tooth surface, and it has been shown that IgG can pass in an intact formfromplasma to the oral cavity via the gingival crevice  Challacombe, Russell   Hawkes, 1978 . The objectives of this investigation were to deter- mine whether IgA and IgM could pass from plasma to the oral cavity viacrevicular fluid parotid saliva or directly across the mucosal epithelium and to compare the results with those for IgG. M TERI LS  ND METHODS Monkey immunoglobulins Rhesus monkey immunoglobulins were isolated frompooled monkey serum. IgG was purified by a combination of DE E cellulose chromatography and gelfiltration, IgM by gel filtration and zone electrophoresis and IgA by gel filtration DE E cellulose chromatography, hydrophobic chromato- graphy and zone electrophoresis  M. W. Russell   L. A. Bergmeier, unpublished results . Each immuno- globulin gave a single precipitation line against rabbit anti-monkey serum. The purified immunoglobulins were labelled with 1251  IMS-3,Radiochemical Centre, Amersham by a modification of the Chloramine T method, as described elsewhere  Challacombe et al. 1978 . Injection of labelled immunoglobulins Twelve rhesus monkeys,which weighedbetween 2X3 kg and 4-7 kg were injected with radiolabelled immunoglobulins intothe small saphenous vein on the hind lim four each for IgG, IgA andIgM. The animals were given approximately 50  ug of the purified immunoglobulin containing between 5 x 107 and 108 counts per minute  c.p.m. . Collection of samples Samples ofserum, crevicular fluid washings  CFW , mucosalwashings and parotid andmixed saliva were taken at 0, 0 5, 1 2, 4 and 24 h as described previously  Challacombe et al. 1978 . CFW were taken from the gingivalcrevices and approximal spaces of the left deciduousmolars and first per- manent molar teeth. The animals were fed on a high sucrose diet and all had mild gingival inflammation with a mean gingival indexof 11  Loe   Silness, 1967 . Immunoglobulin concentrations The concentrations of immunoglobulin in samples were assayed using a modification of the single radial immunodiffusion method  Challacombe, 1976 . For this purposemonospecific anti-rhesus monkey immunoglobulin antisera were raised in rabbits and calibrated against astandard monkey serum using purified monkey IgG, IgA or IgM as standards  Russell   Bergmeier, 1978 . Sucrose density gradient rate-zonal ultracentrifugation Sucrose density gradients wereprepared as described previously  Challacombe et al. 1978 . For ultra- centrifugation of IgG samples, linear 10 to 25 w/w sucrose densitygradients were used, for IgA samples 10 to 30 and for IgM samples 10 to 40 gradients were used. For ultracentrifugation of radioactive serum, samples of 20,ul were made up to 100MAl with saline. For ultracentrifugation of CFW, 20  ul of sample was added to 20  ul of unlabelled carrier serum, and diluted to 100 ul with saline; for parotid and mixed saliva, 50,ud were added to 20 ul of unlabelled carrier serum and diluted similarly. After centrifugation at 65,000 rev/min for 16 h at 4° about twenty-four fractions were collected and assayed for radioactivity and protein concentration as pre- viously described  Challacombe et al. 1978 . The positions of IgG, IgA and IgM in the carrier serumwere determined by single radial immunodiffusion. 924  Passage of Ig from plasma to oral cavity Determination of volume of crevicular fluid CFW were collected from the approximal spaces of teeth in four monkeys, as described. Ten ul ofa solution of radiolabelled IgA of known c.p.m. was instilled into each space and as much as possible recovered. The volume recovered was measured  w and the c.p.m. determined. The volume  v of crevicular fluid is related to the dilution of c.p.m. according to the formula: v - x w)   10 where: s = c.p.m. at the start  total , r = c.p.m. recovered and w = volume  ,ui recovered. Immune precipitation ofradioactivity in serum samples Samples of serum were taken 1 h after intravenous injection of the radiolabelled immunoglobulins. Ten lI of serum were mixed with 50, 100 and 150 ,l of the appropriate antiserum to monkey immuno- globulin  M. W. Russell   L. A. Bergmeier, unpub- lished results and the volume made up to 200 p1 with saline. After incubation for   h at 370 and 18 h at 40, the samples were centrifuged for 10 min. at 2000 g. The precipitate was washed  x 3 in saline and the radioactivity in thepellet and the supernatant including thethree washes was counted. RESULTS Sequential samples The mean blood levels of radioactivity found 30 min after injection of radiolabelled immunoglobu- lins were 245,000 c.p.m./ml for IgG, 310,000c.p.m./ ml for IgA and 290,000 c.p.m./ml for IgM  Fig. 1 . This was followed by a fall in radioactivity over the first 4 h, particularly with IgAand IgM. After 24 h the IgG value had fallen by 55  , and the IgA and IgM by approximately 75   of the value found  t 30 min 30 8 o ,E 20   0 -o 0 0   2   Time  h Figure 2. Sequential levels of radioactivity in crevicular fluid washingsfrom rhesus monkeys given radiolabelled IgG  0 , IgA  V or IgM  -) intravenously  mean ± SE of four animals . Radioactivity was detectable in CFW within 30 min from all 4 animals which had been given IgA and the mean value increased to a maximum at 4 h  Fig.2 andhad fallen to a low level by 24 h. In the animals given IgM, significant radioactivity in CFW was not detectable until 2 h after injection. Maximal activity was found after 4 h, and this had decreased to low levels by 24 h  Fig. 2 . In animals given IgG, activity in CFW was detectable within 30 min and increased slightly to a maximal value at 24 h. The c.p.m./ml of CFW expressed as apercentage of the blood count at the same time increased over the periodofobservation for IgG and IgA  Table 1 and had reached 1 98   for IgA and 125   for IgG at 24 h. The value for IgM reached a maximal value ofonly 0-3   at 4 hand decreased to 015   after 24 h. In contrast to CFW radioactivity was found in samplesof parotid saliva from all animals by 30 min.  Fig. 3 . The mean values for IgG and IgM 00   a.   5   0-5 2 Time  h) Figure 1. Sequential blood levels of radioactivity in rhesus monkeys given radiolabelled IgG  0), IgA  V   or IgM  U) intravenously  mean ± SE of four animals . 7 4~~~~~~~~~~~~~~~~~~~~~~~~~~~  I I1>  I u u J 2 Time h) 4 Figure 3. Sequential levels of radioactivity in parotid saliva from rhesus monkeys given radiolabelled IgG  0 , IgA  v   or IgM  *) intravenously  mean iSE offour animals .  o  o o 0 925 24 4 24  S J. Challacombe, M. W. Russell   Jane E. Hawkes after 1, 2, 4 and 24 h was not significantly different from that found at 30 min. With IgA,however, the valuesincreased to a maximum at 2 to 4 h, and had fallen by 24 h  Fig. 3). The c.p.m./mlof parotid saliva expressed as a percentage of the blood value were found to increase throughout the experiment  Table 1 . WithIgA animals a mean value of 4-30 was found at 24 h compared with   mean valueof 2-1 for IgM, and a mean value of 1   for IgG animals. In mixed saliva, activity was found in all samples after 30min. The values followed a similar pattern to those found with parotid saliva and were maxi- mal at 2-4 h. Although the values were generally higher than those in parotid saliva, they were not significantlydifferent  Table 1 . Verylow levels of activity were found in mucosal washings. With IgM a positive value was found only at 24 h, whereas with IgG and IgA positive values were found with each sample examined. The radioactivity in mucosal washings was significantly less than in CFW at all sampling times  P < 0-05). Ultracentrifugalanalysis ofsamplesIgG. After injection of labelled IgG, greater than 99 of the 1251 count of serum was located in the 7S zone in sucrose density gradient analysis. Simi- larly, most of the 1251 counts in CFW were associa- ted with 7S IgG whereas in parotid or mixed saliva samples most of the counts were present in a broad band of salivary protein sedimenting at approxi- mately IS  Challacombe et al., 1978). IgA. Ultracentrifugal analysis ofsamples from monkeys injected with [125I]-IgA is shown inFig. 4. The two main protein peaks from serum samples correspond to sedimentation coefficients of7S and 4*5S, while saliva samples also contributed a broad band of approximately IS. Serum IgA was located in the 7S peak  Fig. 4a) which also contained the serum radioactivity  Fig. 4b). In samples of CFW taken 1 or 2 h after injection of the [I25I]-IgA some 70   of the counts were found in the 7S region, 15   in the 4S region and the remainder in the IS region apart from a little activity in a region of greater than 7S  Fig.4c). In contrast ultracentrifugation of parotid saliva revealed that no activity was present in the 7S region, and apart from a small amount of activity in the 4 5S region, all theradioactivity was found in the low molecular weight region  Fig. 4d) away from the srcin. Ultracentrifugation of mixed saliva samples revealed a spectrum midway between that of CFW and parotid saliva samples. Some activity was detectable in the 7S region, and in addition a little activity was found in thehigher molecularweight region. The bulk of activity was pre- Table 1. Radioactivity in oral fluids after intravenous injection of radiolabelled immunoglobulins expressed as percentageof serum levels Time  h) Immunoglobulin Fluid 0-5 1 2 424 mean ± SE IgG CFW 0400 50 0-490-77 1-25 0-68 ± 0-15Parotid saliva 0-560-71 1-23 1-42 1-08 1 00 ± 0-16 Mixedsaliva 047 1 00 0-89 1-45 2-27 1-22 ± 0-31 Mucosal washings n.d. 0-31 0-22 n.d. 0-91 0-48 ± 0-22* IgA CFW 0-21 047 0-961-27 1-98 1-07 ± 0-34 Parotid saliva 0-83 1-68 2-013-28 4-33 2-43 ± 0-62t Mixedsaliva 0 74 1-95 3-58 5 87 5-38   50 ± 0-98 Mucosalwashings 0-03 0-11 0-130-280-25 0-16 ± 0-05* IgM CFW 0 01 0 040 25 0 34 0-15 0-16 ± 0-06 Parotid saliva 0-770-890-95 1-52 2-11 1-25 ± 0-25t Mixed saliva 0-44 1-08   -36 1  46 2-42 1-35 ± 0-32 Mucosalwashings 000 0 00 000 0 00 0 03 0.01 + 0.01*  Significantly lowerthan CFW  P < 005 using t-test for dependent means). t Significantly greater than CFW  P < 0-05 using t-test for dependent means). CFW = crevicular fluid washings. 926  Passage of Ig from plasma to oral cavity i ~~~~~~~~   0   0 0-   E CFW  C c  3~~~~~~~~~~~   C   o 0-2 2g0 0 2 4 6 8 10 12 1416 8202224 Tubenumber Figure 4. Sucrose density ultracentrifugalanalysis influids from rhesus monkeys taken 1-2 h after intravenous injection of radio-labelled IgA:  a position of IgA assayed by singleradial immunodiffusion;  b radioactivity in serum  n   2 ;  c radioactivity in crevicular fluid washings  n = 4 ;  d radioactivity in parotid saliva  n = 4 ;  e radioactivity in mixed saliva  n = 4 ;  f radioactivity in urine  n = 1 Results expressed as mean of number of samples indicated. sent however in the region corresponding to I S  Fig. 4e . The profile with a urine sample  Fig. 4f was similar to that found with parotid saliva. No activity was found in t 7S IgA region, but was present in a broadband in the 1S region. IgM. Ultracentrifugation of samples in a 10 to 40 sucrose density gradient resulted in a protein peak of approximately 19S  Fig. 5 in addition to the 7S and 4S peaks. Serum IgM  Fig. 5a and most of the radioactivity in the serum samples was found in the 19S region Fig. 5b but in addition some activity was found in the 7S IgG region. In samples E of CFW approximately 55 of the total radio- activity recovered was present in the IgM region, and some 35   in the IgG region  Fig. Sc . In con- trast, activityin parotid saliva samples was largely confined to the low molecular weight  1S region, and only a trace of. activity wasfound in the IgM region  Fig. Sd . Precipitation of radioactivity in serum samples Radioactivity in samplesof serum taken 1 h after intravenous injection of radiolabelled immuno globulins could be precipitated with the correspond- ing antiserum to monkey immunoglobulin. Ineach 927
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