The release of soluble H-2 alloantigens during disaggregation of mouse embryo tissue by a chelating agent

Antigens of the H-2 system function as transplantation antigens determining the fate of allogeneic homografts, as immunogens stimulating the production of circulating antibody, and as antigens of erythrocytes and leucocytes reacting with antibody (Stimpfling & Snell, 1961; Snell, Hoecker, Amos & Stimpfling, 1964). The antigens extracted by Versene are most conveniently detected by their ability to inhibit the cytotoxic action of antibody produced by tissue allografts made between mouse strains differing at the H-2 locus. The potency of the antigens extracted is thus expressed in terms of their inhibition of a standard cytotoxic alloantiserum. This technique allows determination of very small amounts of material and it avoids the problems of immunization and quantitative scoring of skin graft survival, which are encountered when H-2 antigens are tested in terms of their ability to sensitize animals to subsequent skin grafts. All the results presented here concern H-2 antigens rather than H-2 immunogens. These antigens, which might be considered H-2 haptens, are of interest both because of the manner of their extraction, and because they are the smallest forms of H-2 antigens yet reported and characterized (Herzenberg & Herzenberg, 1962; Lejeune-Ledant, 1962; Brent, Medawar & Ruskiewicz, 1962; Davies, 1962; Kandutsch & Stimpfling, 1963; Manson, Foschi, Dougherty & Palm, 1964; Haughton, 1965; Heberman & Stetson, 1965; Kahan, 1965; Nathenson & Davies, 1966).

Mice. Animals of inbred strains A (H-2^a) C57B1/6 (H-2^b), Balb/c (H-2^d), C3H (H-2^a) and (DBA/2 × Balb/Fj hybrids (H-2^d/H-2^d), referred to as D₂C, were used in the experiments reported. These animals came either from the colony maintained by the Weizmann Institute of Science, Rehovoth, Israel, or from the Roscoe B. Jackson Memorial Laboratory, Bar Harbor, Maine. No difference between embryos from the two colonies, or in the materials extracted from them, was noted; hence, no distinction of mouse origin will be made in reporting the results. The principal H-2 antigens of the strains used are listed in Table 1.

Matings for embryos were made between D₂C males and C57B1/6 various females. This combination gave the greatest frequency of successful matings of those tested. Females were brought to oestrus by injection of hormones, after McLaren & Michie (1959), and pregnancies were timed from the day of observation of a copulation plug.

Alloimmune sera were prepared by injecting appropriate mice with spleen and lymph node cell suspensions at 1- to 2-week intervals. Approximately 25 × 10⁶ nucleated cells were given per injection for a total of four to six injections. Animals were bled on days 4-6 after the last injection; the sera were pooled and stored at - 50° C.

The cytotoxic test employed measures cell death in terms of permeability to dye; it has been previously described in detail (Gorer & O’Gorman, 1956; Boyse, Old & Thomas, 1961; Edidin, 1964). In the present series of tests the diluent for cells and sera was 0·1 % crystalline bovine serum albumin (Armour, Fraction V), in tris-buffered Hanks’s solution, rather than protein-free Hanks’s solution.

Target mesenteric lymph node cells were suspended at a concentration of 1 × 10⁶ cells per ml. Ten microliters of this suspension was mixed with 10 μ1 of antiserum on a slide, and incubated in a moist-box at 37 °C. After 20 min, complement (normal guinea-pig serum) was added and the slides were incubated for a further 40 min. Nigrosin, 0·6 % in heated normal mouse or calf serum, was then added and after a few minutes the cells were examined at × 200-250 magnification. One hundred to two hundred cells per drop were counted and classified as stained or unstained. Five replicate tests were placed on a single slide.

Soluble H-2 antigens, SAg, were prepared from batches of 9 to 10-day-old embryos. After removal from the uterus and washing in Hanks’s solution, the embryos were staged according to number of somites and transferred to 0-02 % Versene in phosphate-buffered NaCl (PBS). The embryos were minced with fine forceps and incubated for 2-3 h at 37 °C with constant swirling or stirring. After incubation the suspension was made 5 % in trichloroacetic acid, held at 4 °C for 10 min and centrifuged at 10,000 g for 30 min, removing a variety of precipitated proteins and nucleic acids, as well as the embryo tissue fragments, but leaving antigenic activity in solution. The TCA supernatant was dialysed overnight in the cold against one change of distilled water. This brief dialysis removed most of the TCA without appreciable loss of antigen activity. The dialysed material was lyophilized and either stored at — 20 °C, or immediately made up in PBS, at 2-5 × concentration, for testing with antiserum.

The dialysed, concentrated, TCA supernatant was an active inhibitor of alloimmune sera tested as cytotoxins against mouse lymphoid cells. A typical assay with the serum tested at two dilutions is shown in Fig. 1. This figure also indicates that the extracted activity is strain-specific, since Balb/c anti-A anti-serum, as expected, did not react with the (D₂C × C57B1/6)F₁ embryo extract. The specifically inhibiting extracts appear to contain a full range of H-2 anti-gens. This is shown in Table 2, in which antigens peculiar to H-2^b and H-2^d, numbers 2, 33 and 31 (Snell et al. 1964), are detected by appropriate antisera and, in addition, antigens common to a number of H-2 loci are also detected by means of the appropriate cross-reactive serum.

It appears that Versene is necessary for the extraction of SAg, and that its action may be blocked by the presence of excess calcium and magnesium ions. Table 3 presents experiments in which embryos were extracted by a variety of salt solutions either containing an excess of calcium ion, or lacking Versene, or containing another disaggregating agent, tetraphenylboron (Rappaport & Howze, 1964), which binds potassium. Of all these solutions, only that containing Versene in the absence of excess calcium was effective in extracting the antigens.

The experiments also suggest that Versene does not act by changing the permeability of cell membranes, allowing contents to leak out, but rather that it removes SAg from cell surfaces; ineffective agents gave at least the same percentage of damaged cells as shown by staining with nigrosin (Kaltenbach, Kaltenbach & Lyons, 1958) as did Versene. Further evidence for the surface action of Versene is derived from the data on re-extraction of once extracted broken cells; this treatment did not yield more SAg activity.

Another parameter in the extraction of soluble H-2 antigens was embryo age. SAg could not be obtained from batches of embryos with an average age greater than 40-44 somites. While it is difficult to establish the exact cut-off point for extraction it appears to be no later than the 11th day of gestation. Certainly much older embryos, as well as new born and adult tissues, do not yield active SAg.

The molecular size of the soluble materials extracted from 10- to 11-day embryos was determined by chromatography on Sephadex. Whole extracts, shown to be active inhibitors, separated into two peaks on Sephadex G-25 (Fig. 2). A slow-eluting peak, estimated to be material of approximately 2000 M.W. was variably present, depending on the degree of dialysis of the preparation. A faster-eluting peak, running in the excluded volume of the column, was rerun on Sephadex G-50. Here the activity peak eluted just after the excluded volume of the column (determined by simultaneously running blue dextran, with a molecular weight of approximately 2000000); this leads to an estimate of about 8000 for the molecular weight of the peak material (Fig. 3).

Table 4 gives data on the chemical composition of whole extracts, before column fractionation. Fig. 4 shows various determinations, including amino sugars, on a series of G-25 fractions. In the latter case, no correlation is apparent between any of the chemical entities measured and serum-inhibiting activity.

The effect of proteolytic enzymes on SAg was next examined. SAg was treated with a crude pancreatic protease mixture, Difco Trypsin 1:300, acidified with TCA to remove enzyme protein and then dialysed to remove TCA appeared to lose all activity. The same result was also obtained when highly purified trypsin (Worthington, 5 × crystalline) was substituted for the crude enzyme mixture.

However, when the trypsin-treated materials were run through Sephadex to remove the enzyme, and were not dialysed it was seen that trypsin acts to reduce the molecular size of the extracts, without destroying their antigenic activity. Fig. 5 shows the effect of crystalline trypsin on the G-25 void-volume peak (molecular weight about 8000) of SAg. The material was treated with 1 mg % trypsin for 1 h at 37 °C and then rechromatographed on the G-25 column. It will be seen that after treatment antigen activity is considerably retarded in its elution from the column, and appears to be associated with molecules of molecular weight less than 2000. Similar results were obtained when SAg was treated with crystalline pepsin.

The results of tryptic digestion of SAg suggested that it might be possible to remove antigenic fragments from tissues of older embryos, or even from adult mouse tissues by vigorous treatment with trypsin. Such treatment is known to remove a variety of cell surface components (Cook, Heard & Seaman, 1962; Eylar & Madoff, 1962; Langley & Ambrose, 1964), and it appeared that the H-2 determinants themselves are resistant to proteolytic enzymes. Table 5 indicates the activity extracted from newborn liver after digestion for various times with crude trypsin, followed by extraction with Versene, acidification with TCA and overnight dialysis against distilled water; the treatment also yielded H-2 activity from older embryos and from adult spleens. All of these extracts appear to be far more heterogeneous, in both chemical composition and fragment size, than are extracts of 9- to 10-day embryos. Work is currently in progress on the purification of SAg extracted from adult tissues.

In the experiments presented here chelation, presumably of calcium and magnesium ions, effects the solution of rather small alloantigenic components of mouse embryo cells. Re-extraction of cells with fresh Versene, leading to further damage of their membranes, or damage to their membranes in the course of treatment with tetraphenylboron did not lead to release of soluble H-2 antigens, indicating that the mechanism of SAg release by Versene is not simply a change in membrane permeability, and suggesting that it releases the antigen directly from the cell surface.

It appears that Versene-extractable H-2 antigens are present only during early stages of mouse embryo development, at a time when the quantity of these antigens bound to cells is small (Edidin, 1964). Perhaps the soluble materials represent precursors of cell bound H-2 antigens requiring further covalent bond synthesis to be fully fixed to the surface, possibly the sort of in situ cross-linking proposed for the mucopeptide of gram-positive bacterial cell walls (Strominger & Tipper, 1965).

That the H-2 antigenic materials studied here contain polysaccharide determinants is suggested by the failure of proteolytic enzymes to inactivate them. It accords well with the finding by Nathenson & Davies (1966) that the H-2 activity in their preparations, made from adult mouse tissues, is associated with glycoprotein. On the other hand, the failure to abolish antibody-neutralizing activity by treatment with trypsin is in marked contrast to reports of its effects in at least partially inactivating a number of insoluble H-2 preparations (Billingham, Brent & Medawar, 1958; Basch & Stetson, 1962; Kandutsch & Stimpfling, 1962; Mann, Corson & Dammin, 1962). This difference suggests that the inactivation observed with trypsin in the experiments cited was due to loss of small, antigenic molecules after digestion, or to the failure of antigenic determinants on small carriers to elicit transplantation immunity.

Finally it should be noted that two treatments which result in disaggregation of tissue cells, Versene chelation and trypsinization, yield molecular fragments carrying H-2 specificities. Though there may well be no necessary connexion between the two, the association of tissue disaggregation and release of H-2 antigens suggests a functional site for H-2 at the cell surface. The suggestion is furthered by the correspondence in size of the SAg molecules, measured in calcium-free solution, and that of the subunits of sponge aggregation factor (Margoliash et al. 1965) which appear in the absence of calcium and magnesium ions. This interpretation is discordant with the observation that free lymphocytes and other leucocytes carry the highest per cell charge of H-2 (Hoecker & Pizarro, 1961; Basch & Stetson, 1962), though it does accord with the paucity of H-2 on erythrocytes. Though many other antigenic factors are associated with the phenomena of cell social behaviour, such as Forssman antigen (Fogel & Sachs, 1964), H-2 antigens are known to be involved in cell recognition (Moller, 1965) and may also be part of the material basis of cell adhesion.

1. Treatment of 10-day-old mouse embryos with the chelating agent Versene releases water-soluble materials carrying H-2 alloantigen activity, as measured by inhibition of cytotoxic sera.

2. The materials extracted (SAg) are specific for the strain of mouse embryo used.

3. SAg extracts may be resolved into two peaks of activity by chromatography on Sephadex G-25. The peaks correspond to molecular weights of approximately 8000 and 2000.

4. The molecular weight of SAg may be reduced to one-fourth its original value by treatment with trypsin.

1. Le traitement au Versène d’embryons de souris de 10 jours libère des substances hydrosolubles portant une activité alloantigénique H-2, telle qu’on la mesure par inhibition de sérums cytotoxiques.

2. Les substances extraites (SAg) sont spécifiques pour la souche de souris utilisée.

3. Les extraits SAg peuvent être séparés en deux pointes d’activité par chromatographie sur Sephadex G-25. Les pics correspondent à des poids moléculaires d’environ 8000 et 2000.

4. Le poid moléculaire de SAg peut être réduit au quart de sa valeur originelle par traitement des antigènes à la trypsine.

This research was performed during the tenures of a National Science Foundation Post-doctoral Fellowship and an American Heart Association Career Investigator Fellowship. I am grateful to Professor Michael Feldman and Professor Albert H. Coons for their hospitality and their interest in, and encouragement of, my work. I would like to thank Mrs Valerie A. Parsegian and Miss Ceyran Yilmaz for their able technical assistance.