Xenopus laevis (G-line) mounts a primary plaque forming cell (PFC) response either in vivo or in vitro following challenge with foreign erythrocytes. Methods are described for generating and assaying the response, which specify criteria such as antigen dose, antigen choice, response kinetics, and complement source. The results suggest that at the peak of the primary response (approximately day 6), animals of different ages produce predomi-nantly different ‘classes’ of antibody which display markedly different complement-fixing characteristics. Antibodies produced by larvae and 4-month-old postmetamorphic animals appear here to be unable to fix either guinea pig complement (GPC′) or adult Xenopus complement, but can readily fix complement from 6-month-old Xenopus. The proportion of spleen PFC’s producing antibody capable of fixing GPC′ progressively increases from about six months to 18 months of age. Possible explanations for such ontogenetic changes are discussed.

Adult frog and toad immunoglobulins are able to fix guinea pig complement (Ohnishi, 1980; Romano, Geczy & Steiner, 1973) and this combination is commonly used in direct plaque-forming-cell (PFC) assays to study haemolytic antibody responses to foreign erythrocytes. Antibodies of Xenopus tadpoles (and those of young postmetamorphic Xenopus) appear less able (Du Pasquier & Cunningham, unpublished data) or altogether unable (Williams, 1981) to fix guinea pig complement, and rosette-forming-cell assays often must be used as an alternative to PFC assays (Kidder, Ruben & Stevens, 1973). In this report primary in vivo and in vitro responses of Xenopus have been examined in animals ranging in age from 2 months (tadpole) to several years (full-grown adults). The results describe a modified Mishell-Dutton (1966) culture system for generating primary PFC responses in Xenopus and outline possible onto-genetic changes in the complement-fixing ability of Xenopus antibodies. Three sources of complement have been compared for activity in the PFC assay, including 6-month-old and adult Xenopus serum and commercial guinea pig serum.

Animals

Inbred Xenopus laevis have been used throughout these experiments. Inbred Xenopus (G-line) (Katagiri, 1978), which appear to be MHC homozy-gous (JJ) (Di Marzo & Cohen, 1982), were donated by C. Katagiri (Hokkaido University, Sapporo, Japan) or purchased commercially (Nippon Life Sciences, Sapporo, Japan). Animals used for breeding or experimentation were healthy, feeding, and free from obvious infection. Offspring were ob-tained through artifical breedings induced by repeated injection of human chorionic gonadotrophin (Griffin and George, Sussex, England). Animals were reared at constant temperature (23 °C) and were immunized at elevated temperature (26 °C). Prior to metamorphosis animals were fed nettle powder, for 3–4 months following metamorphosis, they were fed live Tubifex worms, and thereafter minced ox liver.

Antigens

Sheep red blood cells (SRBC) from a single sheep were purchased commer-cially (Tissue Culture Services, Slough, England) and rabbit red blood cells (RRBC) from a single rabbit were collected via an ear vein. Prior to storage, erythrocytes were centrifuged (350 g) and the buffy coats were aspirated and discarded. Erythrocytes were stored in Alsever’s solution (Flow Labs, Irvine, Scotland) for at least one week and up to four weeks prior to use.

In vivo immunization

Immunization of Xenopus was performed by injection of washed and intact sheep or rabbit red blood cells suspended in amphibian strength saline. Postmetamorphic Xenopus received standard 0·05ml per gram body weight injections of a 0·0025% suspension or 10% suspension, via the dorsal lymph sac. Xenopus tadpoles received 0·005ml injections of a 50% suspension, intraperitoneally.

In vitro immunization

Immunization of Xenopus spleen cells was performed by culturing 3 × 106 spleen leukocytes (at 10 × 106 per ml) in flat-bottom 24-well plates (Linbro No. 76–033–05, lot No. 76091102, Flow Labs, Irvine, Scotland) with varying num-bers or equal numbers of intact sheep or rabbit red blood cells. The complete culture medium consisted of 60% Leibovitz-15 (L-15) supplemented with 10% heat-inactivated foetal calf serum (Lot number 29072126), 10mM-HEPES buffer, 20mM-sodium bicarbonate, 2mM-l-glutamine, 50 units/ml penicillin, 50μg/ml streptomycin, 2· 5μg/ml fungizone (all from Flow Labs, Irvine, Scot-land) and 0· 05mM-2-mercaptoethanol (British Drug Houses, Poole, England). Cultures were fed on days 1, 3 and 5 with one drop (30μl) of a nutritive mixture consisting of 60% L-15 supplemented with 20% FCS, 20mM-HEPES buffer, 40mM-sodium bicarbonate, 10 mM-l-glutamine, 5 × Eagles non-essential amino acids, 5 × Eagles essential amino acids (all from Flow Labs, Irvine, Scotland) and 1% (w/v) D-glucose (Sigma Chemical Co., Poole, England). Cultures were incubated (at 26– 27 °C), in a water-saturated atmosphere of 5% CO2 in air prior to harvest. Cultures were harvested by pipetting and gentle scraping and the contents were transferred to 12 × 75mm Falcon tubes (A. J. Beveridge Co., Newcastle, England).

PFC assay

In vivo and in vitro PFC responses were measured by the thin-layer direct-slide technique (Cunningham & Szenberg, 1968). Cells to be assayed for PFC activity were suspended at varying dilutions in 60% L-15 supplemented only with 10% FCS. A 150μl sample of each spleen cell suspension was combined with 15JLH of a 25% suspension of indicator erythrocytes and 40μl of antigen-absorbed, 1/10 diluted guinea pig or 1/10 diluted Xenopus serum.

Guinea pig serum used as a source of complement was purchased commerci-ally in lyophilized form (Wellcome Reagents Ltd., Beckenham, England) and reconstituted immediately prior to use. Xenopus serum used as a source of complement was collected from non-immune animals by cardiac puncture, maintained on ice, and used fresh (not frozen).

The mixtures were pipetted into double microscope slide chambers which were sealed with a 2:1 mixture of paraffin wax and petroleum jelly. After 1– 2 h at 30 °C, PFC were counted on two separate slides at dilutions which gave up to 100 plaques per slide, under low power of a dissecting microscope and only plaques with a clear central lymphocyte were scored as PFC. Data is expressed as in vivo PFC per 106 originally recovered spleen leukocytes, or as in vitro PFC per 106 originally cultured leukocytes.

In vivo and in vitro PFC responses of 6–9-month-old Xenopus

In vivo challenge with sheep or rabbit erythrocytes results in a specific primary anti-SRBC or anti-RRBC PFC response which peaks on approxi-mately day 7 (Tables 1 and 2). Responses are elicited by low doses and by high doses of RBC. Using Xenopus (homologous) serum rather than guinea pig (heterologous) serum as a source of complement increases the sensitivity of the PFC assay (no further increase occurs when both Xenopus and guinea pig sera are used together). Both types of complement must be absorbed and Xenopus serum must be used fresh (not frozen).

Table 1.

Antigen and complement dependence of the in vivo Xenopus anti-SRBC and anti-RRBC PFC response

Antigen and complement dependence of the in vivo Xenopus anti-SRBC and anti-RRBC PFC response
Antigen and complement dependence of the in vivo Xenopus anti-SRBC and anti-RRBC PFC response
Table 2.

Time courses of the in vivo Xenopus anti-SRBC and anti-RRBC PFC response

Time courses of the in vivo Xenopus anti-SRBC and anti-RRBC PFC response
Time courses of the in vivo Xenopus anti-SRBC and anti-RRBC PFC response

In vitro challenge with sheep or rabbit erythrocytes also results in a specific primary anti-RRBC PFC response which peaks on approximately day 7, but not an anti-SRBC PFC response (Tables 3 and 4). In absence of RRBC or SRBC low levels of PFC arises spontaneously against both types of red cell. The presence of spontaneous PFC’s following in vitro culture contrasts with our failure to record background anti-SRBC or anti-RRBC PFC’s in spléno-cytes assayed directly after removal from the animal. It is possible that in vitro conditions (where FCS is present) achieve polyclonal differentiation of B cells, which include anti-red-cell reactive clones. Thus FCS added to L15 medium enhances tritiated thymidine uptake of spleen lymphocytes (Williams, 1981) and anti-RRBC PFC numbers increase during culture of splenocytes (in the absence of RRBC’s) taken from early-thymectomized Xenopus (Lallone, 1984). In the presence of SRBC, and at all RBC-to-leukocyte ratios tested, the formation of anti-SRBC PFC is specifically decreased, while RRBC specifically increase the formation of anti-RRBC PFC. The reasons for this differential effect of SRBC and RRBC remain unclear, but may be a unique feature of the in vitro PFC response of inbred (G-line) animals reared in our laboratory. Detecting an in vitro PFC response is at least as dependent on the use of Xenopus serum rather than guinea pig serum as a source of complement as is the in vivo response.

Table 3.

Antigen and complement dependence of the in vitro Xenopus anti-SRBC and anti-RRBC PFC response

Antigen and complement dependence of the in vitro Xenopus anti-SRBC and anti-RRBC PFC response
Antigen and complement dependence of the in vitro Xenopus anti-SRBC and anti-RRBC PFC response
Table 4.

Time course of the in vitro Xenopus anti-SRBC and anti-RRBC PFC response

Time course of the in vitro Xenopus anti-SRBC and anti-RRBC PFC response
Time course of the in vitro Xenopus anti-SRBC and anti-RRBC PFC response

Choice of complement source in the 6-day PFC response of different-aged animals

Parallel changes occur in antibody and complement systems during development and aging in Xenopus which can be detected in either an in vivo or an in vitro PFC response. Tadpoles and 4-month-old postmetamorphic animals generate PFC in their spleens 6 days following in vivo SRBC chal-lenge and their antibodies appear able to fix 6-month-old Xenopus comple-ment (XLC′) but neither adult XLC′ nor guinea pig complement (GPC′) (Table 5). Adult animals also generate PFC in their spleens following SRBC challenge and their antibodies appear able to fix complement from any of these three sources, since responses can be detected equally well using complement from guinea pig, young Xenopus, or adult Xenopus donors. The development of in.vivo generated PFC able to fix GPC′ occurs gradually and the ratio of PFC qetected using young (6-month-old) XLC′ to PFC detected using GPC′ appeárs to be inversely proportional to the age of the spleen cell donor. PFC from the spleens of adult mice can be easily detected using guinea pig complement and yet their antibodies appear unable to fix complement from Xenopus of any age.

Table 5.

Parallel changes in the antibody and complement systems during aging in Xenopus, demonstrated in a primary in vivo anti-SRBC PFC response

Parallel changes in the antibody and complement systems during aging in Xenopus, demonstrated in a primary in vivo anti-SRBC PFC response
Parallel changes in the antibody and complement systems during aging in Xenopus, demonstrated in a primary in vivo anti-SRBC PFC response

Spleen cells from animals 4 months old generate PFC in culture 6 days following RRBC challenge and their antibodies appear able to fix 6-month-old XLC′ but neither adult XLC′ nor GPC′ (Table 6). As in the in vivo response, 12-month-old Xenopus possess in vz/ro-generated antibodies that can fix GPC′. However, the antigen-induced in vitro response recorded with GPC′ becomes comparable with assays using young XLC′ only when splenocytes are taken from 18-month-old animals.

Table 6.

Parallel changes in the antibody and complement systems during aging in Xenopus, demonstrated in a primary in vitro anti-RRBC PFC response.

Parallel changes in the antibody and complement systems during aging in Xenopus, demonstrated in a primary in vitro anti-RRBC PFC response.
Parallel changes in the antibody and complement systems during aging in Xenopus, demonstrated in a primary in vitro anti-RRBC PFC response.

These experiments provide two useful pieces of technical information con-cerning the generation and assay of primary spleen PFC responses in Xenopus. First, they suggest that Xenopus of any age may be able to mount a primary PFC response to foreign erythrocytes (including an in vitro response, which can be abolished by removal of nylon wool or glass bead adherent cells, by 3000 R irradiation, or by early larval thymectomy (Lallone, 1984)). Second, they suggest that in animals less than 6 months old, such a PFC response is detectable only using homologous serum (i.e. fresh serum from non-immune 6-month-old Xenopus) rather than heterologous serum (i.e. lyophilized guinea pig serum) as a source of haemolytic complement. The findings serve to illustrate the point made by the late W. Hildemann (1978) that complement from different vertebrates is not always naturally interchangeable and that the most efficient haemolytic activity is often obtained only with homologous serum. The sources of antibody, complement and target cell need careful choice in order to maximise lysis.

Standard amphibian culture conditions (with minor modifications) have been used before to generate in vitro antibody responses in Xenopus (and antigen-binding responses in Bufo marinus; see Azzolina, 1975). A good PFC response to soluble protein antigens has been generated in cultures of dissoci-ated Xenopus spleen cells; however, no response could be detected without prior in vivo hapten and carrier priming (Blomberg, Bernard & Du Pasquier, 1980). A primary PFC response to foreign erythrocytes has been generated in cultures of Xenopus spleen fragments; however, this response required at least 14 days to become readily detectable by a direct PFC assay (Auerbach & Ruben, 1970).

Anuran amphibians (including Xenopus) are known to produce high and low relative molecular mass immunoglobulins (Hadji-Azimi, 1979) and a com-plement system which is superficially similar to that of mammals (Weinheimer, Evans & Acton, 1971; Legler et al., 1979; Ruben, Edwards & Rising, 1977; Moticka, Brown & Cooper, 1973; Donnelly & Cohen, 1977). Following metamorphosis there is an overall switch from larval to adult life and a significant increase in physical size. To accommodate these changes a variety of new adult proteins appear and replace larval proteins which subsequently disappear (Wald, 1958; Wise, 1970; Manwell, 1966). Not unexpectedly, changes occur in the nature and relative concentration of various serum proteins and this may include certain immunoglobulin classes and certain complement components (Richmond, 1968; Du Pasquier, Blomberg & Bernard, 1979; Geczy, Green & Steiner, 1973). A postmetamorphic shift in the predominant immunoglobulin class produced in a primary PFC response could stem directly from the differential activation of certain immunoglobulin genes (resulting either in the increased production of a low relative molecular mass Ig‘G’ class, see Du Pasquier & Haimovich, 1976, or the decreased production of a less-well-defined, possibly larval, Ig class; see Hadji-Azimi, 1979). That Xenopus may possess more than two Ig isotypes is suggested by very recent, preliminary, findings of Du Pasquier (personal communication). The shift could also arise indirectly from the progressive turnover and re-placement of certain B cell subsets (caused possibly by a decrease in the number of intrathymic- or thymus-derived B cells, see Du Pasquier, Weiss & Loor, 1972; Du Pasquier & Weiss, 1973; Hsu, Julius & Du Pasquier, 1984; Williams, Cribbin, Zettergren & Horton, 1983).

In most cases an obvious advantage can be associated with making a larval to adult protein switch. For example, tadpole and adult haemoglobins have markedly different oxygen-binding affinities (Hamada, Sakai, Tsushima & Shukuya, 1966). However, in the case of immunoglobulins such an advantage is not so obvious. Larval antibodies appear able to bind antigen specifically, to bind complement specificially, and appear perfectly adequate with respect to the size of their antigen repertoire, despite having far fewer lymphocytes (B cells included) than do their adult counterparts (Haimovich & Du Pasquier, 1973; Du Pasquier & Wabl, 1976). Perhaps young Xenopus (less than 6 months old) retain the ability to produce a predominantly or uniquely larval class of immunoglobulin (i.e. one which is capable of fixing only young Xenopus complement but not the (modified?) complement components from adult Xenopus) when antigen challenge places severe stress on their immune system. There are examples of other protein changes that can take place after meta-morphosis which set a precedent. For example, following metamorphosis, individual tadpole erythrocytes begin production of adult haemoglobin; eventually these cells are replaced by adult erythrocytes, and yet under severe anaemic stress (induced by phenyl-hydrazine) adult cells can be induced to reactivate production of tadpole haemoglobin (Moss & Ingram, 1965; Maniatis & Ingram, 1972; Maniatis, Steiner & Ingram, 1969; Hamada & Shukuya, 1966).

Supported by Medical Research Council Project Grant (G80/0720/2CB), a Research Grant from the North of England Cancer Research Campaign (to J.D.H.), and a Manpower Services Commission Grant (to M.R.C.). Thanks to Mrs. Jean Mather for typing the manuscript.

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