Preparation method of lamprey antisera and activity assay

Cellular Immunology 280 (2012) 85–91

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Preparation method of lamprey antisera and activity assay Peng Su a,b,1, Liyong Chen a,b,1, Xu Qiao a,b,1, Fenfang Wu a,b, Bo Feng a,b, Yinglun Han a,b, Ge Liu a,b, Qingwei Li a,b,⇑ a b

College of Life Science, Liaoning Normal University, Dalian 116029, China Institute of Marine Genomics & Proteomics, Liaoning Normal University, Dalian 116029, China

a r t i c l e

i n f o

Article history: Received 12 July 2012 Accepted 28 November 2012 Available online 10 December 2012 Keywords: Lamprey Immune system Complement Immunization schedule

a b s t r a c t Lampreys, the surviving representative of jawless vertebrates, have been a focal point in the search for the evolutionary origin of adaptive immunity. They have independently evolved the variable lymphocyte receptor (VLR)-based adaptive immune system that protects themselves from infection by a variable of microorganisms. The standard immunization schedule for Japanese lamprey (Lampetra japonica) was established to prepare antisera by injection of Escherichia coli, Bacillus proteus, Staphylococcus aureus, Mycobacterium smegmatis, RRBCs, SRBCs, NB4 cells and Hela cells. In this study, we demonstrated the activities of lamprey antisera, which might be helpful to research the collaboration between VLR-based adaptive immune system and complement system in jawless vertebrates. Ó 2012 Published by Elsevier Inc.

1. Introduction Jawless vertebrates (with Lamprey for example) have been a focal point in the search for the evolutionary origin of adaptive immunity because of their unique position in chordate phylogeny [1]. Prior to the jawless vertebrates, the invertebrates relied on germline-encoded molecules and phagocytes for immune recognition and microbial defense, which are known as the innate immunity [2]. Then the jawed vertebrates have evolved adaptive immune system which is based on highly diverse antigen receptors. A characteristic of the system is its capacity to maintain a memory of previous pathogenic encounters. Moreover, this memory provides an accelerated response for the organism to repel a second invasion. In 2004, Pancer et al. [3] confirmed the adaptive immune system of jawless vertebrates is based on variable lymphocyte receptors (VLRs) which are generated by tandem array of highly diverse leucine-rich-repeat (LRR) motifs as basic structural units. Therefore, lamprey could be the best model organism to investigate the adaptive immune system that is not based on immunoglobulin. Three types of VLR genes (VLRA, VLRB and VLRC) have been identified [4,5]. Only VLRB+ lymphocytes could undergo

Abbreviations: VLR, variable lymphocyte receptor; IgSF, immunoglobulin superfamily; LRR, diverse leucine-rich-repeat; RRBCs, rabbit red blood cells; SRBCs, sheep red blood cells; i.p., intraperitoneal injection; i.m., intramuscular injection. ⇑ Corresponding author at: College of Life Science, Liaoning Normal University, Dalian 116029, China. Fax: +86 0411 85827799. E-mail address: [email protected] (Q. Li). 1 These authors contributed equally to this work. 0008-8749/$ - see front matter Ó 2012 Published by Elsevier Inc. http://dx.doi.org/10.1016/j.cellimm.2012.11.015

lymphoblastoid transformation, proliferation, and differentiation into plasmacytes and finally secrete multivalent VLRB antibodies [6–8]. In this study, we established the immunization schedule for Japanese lamprey (Lampetra japonica) to prepare antisera by injection with various kinds of antigens: Escherichia coli (E. coli), Bacillus proteus (B. proteus), Staphylococcus aureus (S. aureus), Mycobacterium smegmatis (M. smegmatis), rabbit red blood cells (RRBCs), sheep red blood cells (SRBCs), NB4 cells and Hela cells. Moreover, the remarkable activities of lamprey antisera against corresponding antigens have also been testified. 2. Material and methods 2.1. Animals, bacterial strains and cells Adult male and female Japanese lampreys were obtained from the Songhua River region of Heilongjiang province, China. These lampreys were kept in aquariums before being immunized according to the schedule. The bacteria were kindly supplied by Mingjie Xie, Department of Microbiology of Liaoning Normal University, RRBCs and SRBCs were obtained from rabbit and sheep, respectively. Hela and NB4 cells were presented by Prof. Jianing Zhang of the Institute of Dalian Medical University. 2.2. Immunization schedule with various doses of Escherichia coli 96 healthy lampreys (without trauma) were divided equally into four groups. They were separately immunized with 100 ll

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ranging from 0.5% to 40% (v/v). The optimum time research of antisera cytotoxicity was carried out through incubating respective antigens at 4 °C for different period (5 min–120 min). To determine the effects of Ca2+ and Mg2+ on cytotoxic activity, the antisera were pre-incubated with serial dilutions of EDTA, and then mixed with RRBCs and incubated at 4 °C for 30 min before evaluation of the cytotoxic activity.

Table 1 Immunization schedule of Escherichia coli. Time of Dose of E. coli immunization 1: 2: 3: 4:

Way of immunization/test

1  102 1  104 1  106 1  108

each Intraperitoneal injection each each each

1 (Day 1)

Group Group Group Group

Day 5

Antiserum preparation Test of bacteriolytic activity of antisera

2 (Day 10)

Group Group Group Group

1: 2: 3: 4:

1  102 1  104 1  106 1  108

Day 15

Antiserum preparation Test of bacteriolytic activity of antisera

3 (Day 20)

Group Group Group Group

Day 25

Antiserum preparation Test of bacteriolytic activity of antisera

4 (Day 30)

Group Group Group Group

Day 35

1: 2: 3: 4:

1: 2: 3: 4:

1  102 1  104 1  106 1  108

1  102 1  104 1  106 1  108

2.7. Analysis of cytolytic effects of lamprey antisera

each Intraperitoneal injection each each each

Tumor cells (Hela cells and NB4 cells), and RBCs (RRBCs and SRBCs) were suspended to 5  107 cells/ml in normal saline. Lamprey antisera were added to corresponding antigens and incubated at 4 °C for 20 min. The treated cells were washed and re-suspended in normal saline. The numbers of survival cells were directly counted with a hemocytometer. Normal saline was used as blank control; the sera stimulated by normal saline and naive sera were used as negative controls.

each Intraperitoneal injection each each each

each Intraperitoneal injection each each each

Percentage of cell lysis % ¼

4

6

5  107

 100%

Antiserum preparation Test of bacteriolytic activity of antisera

2

5  107  The numbe of survival cells treated by lamprey antiserum

8

0.01 M PBS containing 10 E. coli, 10 E. coli, 10 E. coli and 10 E. coli at 10 day (named Day1, Day10, Day20 and Day30) intervals by four intraperitoneal injections. The control animals were injected with normal saline. Then the bacteriolytic effect of lamprey antiserum was testified 5 days after the immunization each time. The specific details of immunization schedule with E. coli are in Table 1. 2.3. Immunization schedule with various kinds of antigens Lampreys were immunized with eight kinds of antigens at 10day intervals by four intraperitoneal injections. The antigens were 108 E. coli, 108 B. proteus, 108 S. Aureus, 108 M. smegmatis, 107 RRBCs, 107 SRBCs, 106 NB4 cells and 106 Hela cells.

ð1Þ

Bacteria (E. coli, B. proteus, S. Aureus and M. smegmatis) were suspended to 5  107 cells/ml in normal saline. Lamprey antisera were added to corresponding antigens and incubated at 4 °C for 30 min. The treated bacteria were washed and re-suspended in normal saline. The bacteria were inoculated onto LB agar plates and incubated for 14 h at 37 °C. Colony-forming units were counted for viability and the average number of colonies was estimated from three plates. At time zero (before incubation with antiserum), the number of colony-forming units (CFU) was taken as 100%. Normal saline was used as blank control; the sera stimulated by normal saline and naive sera were used as negative controls. Percentage of cell lysis % The number of CFUðtime zeroÞ  The number of CFUð14hours laterÞ The number of CFUðtime zeroÞ  100% ð2Þ

¼

2.4. Collection of lampreys antisera stimulated by various kinds of antigens

2.8. Analysis of antigen specificity

Each lamprey was sterilized with 75% alcohol first. Total blood was collected and incubated at 4 °C for 1 h before centrifugation at 7000 rpm for 15 min. The upper antiserum were extracted and preserved at 20 °C.

In order to research the antigen specificity of lamprey antisera, E. coli, B. proteus, S. Aureus, RRBCs and Hela cells were incubated with the antiserum stimulated with E. coli. The cytolytic and agglutination effects of lamprey antisera were analyzed as mentioned above.

2.5. Agglutination assay The antisera were heated at 56 °C for 30 min, and then serially diluted in 2-fold increments from 1/2 to 1/256 with normal saline. 50 ll of each antisera dilution was added to 50 ll antigen in 96well flat-bottom plates and incubated at 37 °C for 1 h and 4 °C overnight before visual inspection for antigen agglutination by light microscopy. 2.6. Determination of cytolytic effects of lamprey antisera To determine the temperature dependence of the stimulated sera, the lamprey antisera were pre-treated at various temperatures (4 °C–65 °C) for 20 min. Then the treated sera were incubated with RRBCs. Determination of the optimum sera concentration for cytotoxicity was analyzed according to the method of Liang et al. [9]. In brief, antisera were mixed with RRBCs at sera concentrations

Fig. 1. Bacteriolytic activity of lamprey antisera immunized by E. coli. Data represent mean percentages ± SE, Error bars indicated standard error of mean (s.e.m), n = 3.

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Fig. 2. Cytolytic effects of lamprey antisera against RBCs, bacteria and tumor cells. Data represent mean percentages ± SE, Error bars indicated standard error of mean (s.e.m), n = 3. ⁄⁄P < 0.01.

Normal saline Antisera (i.m.)

2.9. Data analysis All statistical analyses were performed with the SAS proprietary software release 8.02 and student’s two-sample t-test.

3.1. Bacteriolytic activity of specific antisera for immune dose of E. coli As the result showed, the Bacteriolytic activity of specific antisera for E. coli was dose-dependent. And the percentage of cell lysis increased as the number of immunization rising. The percentage of cell lysis in the group immunized by 108 E. coli was above the mean value overall. Moreover, it reached the maximum (60% cells lysis) after the fourth time of immunization (Fig. 1). To investigate the highest dose of E. coli, the immunization of lampreys with 1010 E. coli was also carried out. Unfortunately, lampreys constantly lost in the immune process. The survivals of lampreys were so low that they were not enough to form a complete data.

80

Percentage of cell lysis (%)

3. Results

Naïve sera Antisera (i.p.)

60

40

20

0

3.2. Cytolytic effects of lamprey antisera for various kinds of antigens In order to research the cytolytic effects of lamprey antisera for various antigens, lampreys were immunized with eight kinds of antigens. Antisera from immunized lampreys possessed significant cytotoxicity against the corresponding antigens: Gram-negative bacteria (E. coli and B. Proteus), tumor cells (NB4 and Hela cells), and RBCs (RRBCs and SRBCs) (P < 0.01) (Fig. 2). It’s worth noting that, the antisera had no signally cytotoxicity against those Gram-positive bacteria (S. aureus and M. smegmatis).

SRBCs

NB4 cells

Fig. 3. Comparison of the immune effect between intraperitoneal injection and intramuscular injection. Data represent mean percentages ± SE, Error bars indicated standard error of mean (s.e.m), n = 3. ⁄⁄P < 0.01.

3.3. Comparison of the immune effect between intraperitoneal injection and intramuscular injection Lampreys were immunized with 107 SRBCs and 106 NB4 cells by either intraperitoneal injection (i.p.) or intramuscular injection

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B

A Naïve sera Antisera stumulated by RRBCs

90 80

80

Percent of of cell cell lysis lysis (%) Percent (%)

Percentage of cell lysis (%)

100

Naïve sera

Naïve sera Antisera stumulated by RRBCs Antisera stumulated by RRBCs

100

60

40

20

70 60 50 40 30 20 10 0

0 0.5

1

2 4 8 10 Sera concentration (% , v/v)

20

40

4

16 25 37 45 56 Incubation tempreture (℃) Incubation tempreture (℃)

65

D

C Naïve sera Naïve sera stumulated by RRBCs Antisera Antisera stumulated by RRBCs

100

90

EDTA Percentage of cell lysis (%)

Percentage of cell lysis (%)

80

80

60

40

20

70 60 50 40 30 20 10

0

0

5

10

20

60

120

Incubation time (min)

0

2

4

8

16

32

EDTA C once ntration EDTA C once ntration(mM) (mM)

Fig. 4. Bacteriolytic and hemolytic activity of lamprey antisera. Data represent mean percentages ± SE, Error bars indicated (s.e.m), n = 3. (A) The cytolytic effect of the antisera was dose-dependent. (B) The cytolytic effect of lamprey antisera was temperature sensitive. (C) The cytolytic effect of the antisera was time-dependent. (D) The cytolytic effect of the antisera was Ca2+ and Mg2+-dependent.

(i.m.). Antisera from i.p. group proved to be significant cytotoxic against the corresponding antigens (P < 0.01). However, there were no significant differences of cytotoxicity between i.m. group and naïve sera group (Fig. 3). It has been reported that, there are many principal lymphoid tissues [10], blood, kidneys and typhlosole in lampreys. The abundant blood vessels in lamprey’s celom could be the best media that allow antigens encounter with the immune molecules. In that case, the following immunizations were carried out through intraperitoneal injection only. 3.4. Cytolytic effects of Lamprey antisera were related to sera concentration, reaction time, temperature and divalent cation In order to characterize the cytolytic effects of lamprey antisera, the effective antisera concentration, the reaction time, the optimum reaction temperature and the correlation of antisera and divalent cation were observed. First, the cytotoxicity against RRBCs was dose-dependent. Approximately 80% RRBCs were lysed at 4% antisera concentration, and there were no additional effects at higher concentrations upto 40% (Fig. 4A). Second, the cytotoxicity

of the antisera was temperature sensitive. The optimum temperature for the cytotoxicity was between 4 °C and 37 °C which caused 75% cells lysis. As the temperature increased above 45 °C, the cytotoxic effects were diminished, and almost lost at 56 °C (Fig. 4B). Third, the cytotoxicity of the antisera was time-dependent. The optimal time was 20 min, and there were no increase in cytotoxicity up to 120 min (Fig. 4C). To determine whether Ca2+ and Mg2+ were required for the cytotoxic effects of the antisera, RRBCs-stimulated antisera were incubated with serial dilution of EDTA, followed by incubation with RRBCs. The cytolytic effects of the antisera had a dose-dependent response to the chelators of EDTA (Fig. 4D). 3.5. Agglutinate effects of lamprey antisera The multivalent structure of VLRB suggested that it could function as a potent agglutinin. To examine this potential, we observed the ability of the antisera to agglutinate various antigens. In the group of RRBCs, E. coli, S. aureus, M. smegmatis and NB4 cells (Fig. 5), the agglutinphore group could be observed in the dilution

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64 1: 12 8

1:

1: 1

28

64 1:

1:

12 8

32

NB4 cells

2

Number of agglutinphore group

Hela cells

1:

1: 64

2 1: 3

1: 16

1: 8

1: 4

20 18 16 14 12 10 8 6 4 2 0 1: 32

D

S. aureus S. aureus M. smegmatis M. smegmatis

16

1: 2

1: 12 8

1: 64

1: 32

16 1:

8 1:

18 16 14 12 10 8 6 4 2 0

1: 2

Number of agglutinphore group

C

1: 4

1: 2

0

1:

2

1:

6 4

1: 16

8

1: 8

10

1: 8

12

E. E. coli coli B. proteus proteus B.

1: 4

16 14

20 18 16 14 12 10 8 6 4 2 0

4

B

RRBCs RRBCs SRBCs SRBCs

18

1:

20

Number of agglutinphore group

Number of agglutinphore group

A

Fig. 5. Agglutination assay of lamprey antisera against various antigens. The antisera were diluted in serial concentration. (A) The antigens for agglutination assay were RRBCs and SRBCs. (B) The antigens for agglutination assay were E. coli and B. proteus. (C) The antigens for agglutination assay were S. aureus and M. smegmatis. (D) The antigens for agglutination assay were Hela cells and NB4 cells. Data represent mean numbers ± SE, Error bars indicated (s.e.m), n = 3.

B Percentage of cell lysis (%)

70

specific antisera for E. coli

60 50 40 30 20 10

Number of agglutinphore group

A

E. coli RRBCs

20 18

B. proteus Hela cells

S. Aureus

16 14 12 10 8 6 4 2 0

ce l ls H el a

BC s RR

eu s S. A ur

s B. pr ot eu

E. co l

i

0

2 1:

4 1:

1:

8

1:

16

1:

32

6 1:

4 1:

12

8

Fig. 6. Analysis of antigen specificity. (A) The cytolytic effects of lamprey antisera were analyzed. (B) The agglutination effects of lamprey antisera were analyzed. Data represent mean numbers ± SE, Error bars indicated (s.e.m), n = 3.

ratio of 1:64. In the group of SRBCs, B. proteus and Hela cells, the dilution ratio was 1:32 when the agglutinphore group could be observed (Fig. 5). It’s worth noting that, the antisera had no signally cytotoxicity against those Gram-positive bacteria (S. aureus and M. smegmatis) (Fig. 2), while their agglutination against Gram-positive bacteria was of the same magnitude as it against Gram-negative bacteria (Fig. 5B). We supposed the dense cell wall of Grampositive bacteria which was made of peptidoglycan, lipoteichoic

acid, wall teichoic acid and surface proteins did a great job in preventing the attack of lamprey antisera [11]. 3.6. Antigen specificity of lamprey antisera The E. coli, B. proteus, S. Aureus, RRBCs and Hela cells were incubated with the antiserum stimulated with E. coli. As the data indicated, the lamprey antisera against E. coli showed more significant

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P. Su et al. / Cellular Immunology 280 (2012) 85–91 Table 2 Immunization schedule of various antigens. Antigens

Dose of antigens

Way of immunization

Time of immunization

Intraperitoneal injection

Immunization at 10 day intervals by 4 intraperitoneal injections

8

E. coli B. proteus S. Aureus

10 each 108 each 108 each

M. smegmatis

108 each

RRBCs SRBCs NB4 cells Hela cells

107 107 106 106

each each each each

cytotoxicity than it against others (Fig. 6A). The similar results were found in agglutination assay. In the group of E. coli, the agglutinphore group could be observed in the dilution ratio of 1:64. In the group of B. proteus, S. Aureus, RRBCs and Hela cells, the dilution ratio was 1:16 when the agglutinphore group could be observed (Fig. 6B). 4. Discussion There is much left to be learned about the VLR-based adaptive immune system before we can understand the survival advantage of agnathans defense against pathogens in their living surroundings. But even more primary, the standard immunization schedule for lamprey is indispensable to study lamprey’s resistance to pathogenic microorganisms. Herein, we provided immunization schedule for different kinds of antigens, such as Gram-negative bacteria (E. coli and B. Proteus), Gram-positive bacteria (S. Aureus and M. smegmatis), RBCs (RRBCs and SRBCs) and tumor cells (NB4 and Hela cells) (Table 2). And the antisera have been proved to possess activity. Additionally, the lamprey antisera showed a certain degree of antigen specificity. As it is reported that the highest antigen specific VLRB titer obtained after postimmunization is 1:10,000 approximately which is similar to the IgM responses in cartilaginous (e.g. nurse shark) [12]. The researches on antigen specificity of VLRs illuminated the potential function of the unique adaptive immunity in early vertebrates. Due to their unique features, VLRs may hold considerable potential as alternatives to conventional Ig-based antibodies for various biotechnology applications [13–15]. In this study, we found that there were significant differences in bacteriolytic activity between the first and the last three times of immunizations (Fig. 1). We hypothesized that the innate immune system defended against infection by relatively nonspecific recognition of pathogen patterns. Then the VLR-based adaptive immune system recognized and activated the protective response against bacterial invasion. Moreover, the VLR-based adaptive immunity got the capacity to remember previously specific infectious agents, which allowed more efficient immune response to the invader. During the preparation of antiserum, one of the most important elements is temperature. As we know, the best incubation temperature for antisera from rats, rabbits and other mammals is 37 °C. Interestingly, we testified that the best incubation temperature for antisera from lamprey is 4 °C. That might be related to the parasitic life and low-temperature living environment of lamprey [16] though it still needs to be testified. There were some interesting phenomena captured our attention in the research. The cytotoxicity of lamprey antisera was depended on the dose of antisera, time of incubation, temperature of the reaction and dose of EDTA. These provided supportive evidences for the involvement of the complement system. In lamprey, the lectin and alternative complement activation pathways have been described owing to the discovery of key complement components such as MBL, Bf, C3 and other complement molecules

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