Role of C1q & Complement activation deficient IgM in Antibody responses against Thymus independent antigen, Ficoll-NIP
Tasmin Shahnaz
Undergraduate Thesis Report (15 hp) Biomedical Programme
Uppsala University 2011-06-17
Supervisors:
Anna Bergman, Christian Rutemark
Department of Medical Biochemistry and Microbiology Uppsala University
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ABSTRACT
Complement components C1q, C2, C3, C4 (i.e. components of the classical pathway) and complement receptors 1 and 2 (CR1/2) are crucial for the generation of a normal antibody response. Often the classical pathway cascade is activated by IgM + antigen (immune complex, IC). The cascade will result in the cleavage of the complement components
mentioned above, thereby generating the ligands for CR1/2. In mice CR1/2 are expressed on B cells and follicular dendritic cells (FDC). Since the classical pathway is of great importance and IgM is a very good initiator of this pathway, it was hypothesized that, binding of natural IgM to an antigen would initiate the classical pathway and thereby generate the ligands for CR1/2. To test this hypothesis a knock-in mouse strain, Cmu13, was created. Cmu13 has a point mutation inserted making its IgM unable to activate complement. Studies with Cmu13 using thymus dependent antigen have not shown an impaired antibody response. Therefore, in this study the response against a thymus independent antigen were analyzed. First it was concluded that C1qA-/- mice had impaired response against very low doses of the T-
independent antigen, Ficoll-NIP. Secondly the response in Cmu13 against the same doses of Ficoll-NIP was tested. As a negative control Cr2-/- mice were used. The IgG anti NIP
response in Cmu13 were different depending on immunized dose; with 0.1 µg Ficoll-NIP the response was surprisingly higher but only significant at one time point, with 0.5 µg it was lower at two time points (initiation), and with 1 µg there were no difference. As expected, Cr2-/- mice had impaired antibody response although, 1 µg Ficoll-NIP seemed to bypass the dependency of CR1/2. In conclusion, the classical pathway was crucial for antibody response against Ficoll-NIP but IgM alone could not be the C1q activator.
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ABSTRACT ...1
INTRODUCTION ...3
The Complement System ...3
The Complement System and antibody responses ...4
Aim ...6
MATERIALS AND METHODS ...6
Mice ...6
Immunizations and blood sampling ...7
Antigens...7
Antibodies ...7
Chemicals ...7
Equipment for ELISA ...8
ELISA ...8
Statistical Analysis ...8
RESULTS ...8
Impaired antibody responses in C1qA-/- against Ficoll-NIP ...8
Cmu13 mice, lacking complement activating IgM, do not have impaired antibody responses to Ficoll-NIP...9
DISCUSSION ... 10
REFERENCES ... 12
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INTRODUCTION
The Complement System
The Complement system is a potent mediator of our immune defence against pathogens. The effects are mediated by lysis, opsonization, phagocytosis, inflammation and clearance of immune complexes from the blood circulation. The complement system can be divided into three different pathways: the classical pathway, the mannose binding lectin pathway (MBL) and the alternative pathway (Fig 1).
Figure 1. The Complement system (1)
4 The classical pathway can be activated by immune complexes, i.e., ≥ 2 IgG or one IgM
pentamer binding an antigen. The binding of IgM to an antigen will result in a conformational change of the pentamer, revealing the binding site for C1q. When C1q binds and becomes activated it ill cleave other complement components, C4 to C4a and C4b, and C2 to C2a and C2b. C4b and C2 is united to C4b2b complex which is known as C3 convertase. The
additional cleavage of C3 results in the formation of C5 convertase and thereafter the
formation of the membrane attack complex (MAC) (common in all three pathways). It is the MAC that is responsible for the lysis of pathogens. The other two pathways are initiated by interaction with microbial cell-wall or surfaces. Mannose, a sugar on microbial cell-wall initiates the MBL cascade by activating mannose associated serine proteases (MASP), which in turn cleaves C4. After this cleavage the cascade follows as the classical pathway. The alternative pathway, as MBL, is also antibody independent and is initiated by microbial surfaces. The pathway starts by cleavage of C3 into C3b and C3a. C3b binds microbial surfaces and are stabilized by binding of factor B. Factor B is then cleaved into Ba and Bb forming a C3bBb complex also known as C3 convertase. This C3 convertase will result in more cleavage of C3, forming a C3bBbC3b (C5) convertase and finally formation of MAC.
C3 and C5 are cleaved into different components, some that are involved in the formation of C3/C5 convertases and other components that diffuse away. The components which diffuse away are powerful anafylotoxins, e.g., which stimulate mast cells to release histamine and enhance contraction of smooth muscles. The components of C3, C4, and C5 are also ligands for CR1/2.
The Complement System and antibody responses
The Complement system plays a crucial role in generating antibody response. In 1974 Pepys first demonstrated the role of C3 in antibody response (2). He showed that C3 depleted mice had an impaired antibody production to T- dependent antigens like SRBC. Similarly other studies have shown that deficiencies in other complement components, C1q in mice (8), C2 in guinea pigs (3), C3 in dogs (4) and C4 in humans (5) resulted in an impaired antibody
response (complements of the classical pathway). On the other hand C5-C9 (6), as well as MBL and factor B (1, 7, 9, 10 and 11) have not shown to affect normal antibody production.
Further, the complement receptors 1 & 2 (CR1/2) are also crucial for antibody production. In mice the receptors are expressed on B-cells (effector cells of antibody production) and FDCs and their ligands are some of the cleavage products from the complement cascade. The involvement of the classical pathway may suggest that antibodies are involved. One
5 interesting finding was done by Ehrenstein (17) which showed that mice lacking natural secretory IgM had impaired antibody responses, but the antibody response was restored after immunization with naive serum from wild type (containing natural IgM). This suggests that natural IgM is of importance for a normal antibody response. However, IgM is also an activator of the classical pathway (reviewed in 15). Thus, both IgM and complement seem to be linked in generation of a normal antibody response.
Several hypotheses have been grounded which try to explain the strong influence of complement in antibody responses.
ICs (Antigen-IgM-Complement) would cross-link the B-cell receptor (BCR) and CR 2/CD19 on antigen specific B-cells, known to lower the threshold for B-cell activation (19, 20).
Membrane bound IgM (mIgM) which is the BCR, would directly bind antigen and activate complement by cross-inking BCR and CR1/CD9 (21)
Enhanced trapping of ICs on CR 1/2 on the follicular dendritic cells (FDC) in the follicles of spleen, which in turn would increase the likelihood for antigen specific B- cells to encounter its antigen (14).
Increased transport of ICs on CR 1/2 on marginal zone B-cells (MZB) to the follicles in the spleen (22, 23).
To test if natural IgM needs to activate complement to generate a normal antibody response, a knock-in mouse Cmu13 was created. Cmu13 has a mutation in the third constant domain of the µ heavy chain, making its IgM unable to activate complement (24). These mice were immunized with SRBC and KLH (1, 24) and the results showed that Cmu13 did not have impaired primary or secondary antibody responses against these T-dependent antigens. To verify the role of the classical pathway for generation of antibody response C1qA-/- and Cr2-/- mice were immunized with SRBC. They showed impaired antibody responses compared to wild type mice. Hence, the classical pathway plays a major role in generation of normal antibody responses against T-dependent antigens, but the ability of IgM to activate complement plays a minor role (1, 24).
So far Cmu13 mice were immunized with T-dependent antigens, although many pathogens express antigens that are known as T-independent, e.g., different sugar molecules. It was
6 therefore of interest to test if IgM-complement activation was crucial for antibody responses against the T-independent antigen, Ficoll-NIP. But, first it was important to study if the classical pathway itself was crucial in generation of normal antibody responses against this antigen.
Aim
i) To elucidate if C1q is crucial for antibody response against Ficoll-NIP.
ii) To elucidate if natural IgM needs to activate complement to generate a normal antibody response against Ficoll-NIP.
MATERIALS AND METHODS Mice
BALB/c were obtained from Bommice (Ry, Denmark). Cr2-/- (mice lacking CR1/2) were backcrossed for 10 generations to BALB/c. C57BL/6 were obtained from Jackson
Laboratories (Bar Harbor, ME) and C1qA-/- from Dr. Marina Botto (London).
Cmu13 mice, knock-in mice, were generated at Harvard University by Birgitta Heyman and Micheal Carroll (data unpublished). ES cells were transfected with a mutation from
information provided by Shulman et al. (16) about the complement activation deficient cell line no.13. The IgM gene segment has a point mutation in position 436 in the constant domain of the µ heavy chain. The mutation was ligated into the pR13µ Ei vector. The vector was then transfected into a mouse embryonic stem cell from line 129/SV by knock-in technology. ES cells carrying the mutated segment were selected and put into blastocysts of female mice.
Males carrying the same mutation were mated to females and positive progeny further backcrossed to the same mouse strain to obtain homozygous mice. This was done initially on C57BL/6 strain. Later the Cmu13 mice were backcrossed for 12 generations to BALB/c.
Animals were age and sex matched within each experiment and all animal experiments were approved by Uppsala Animal Research Ethics Committee.
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Immunizations and blood sampling
Mice were immunized i.v in one of the lateral tail veins with 0.1 µg, 0.5 µg or 1 µg Ficoll- NIP in 0.2 ml PBS. All blood samples were taken from one of the tail arteries at indicated time points. The blood was centrifuged for 5 min at 11000 rpm, 4 ° C and sera were collected in eppendorf tubes and stored at -18°C until they were analyzed.
Antigens
Ficoll-NIP was purchased from Biosearch Technologies (San Francisco, CA, USA) and OVA from Sigma, St Louis, MO, USA. OVA-NIP was used as antigen for coating in ELISA. NIP was conjugated to OVA by mixing 1 volume of NIP-CAP-Osu [Cambridge Research Biochemicals Ltd, Cambridge, UK (3mg/ml in DMSO) Sigma] with 3 volumes of OVA [3mg/ml dissolved in 3% NAH-CO3 (30g in 1000ml distilled H20]. The mixture was then incubated in a dark chamber at room temperature for 1 hour, with several stirring moments in between. After incubation OVA-NIP was dialysed against PBS followed by sterile filtration and stored in a dark chamber.
Antibodies
Sheep anti- mouse IgG conjugated to alkaline phosphatase (Jackson ImmunoResearch
Laboratories, West Grove,PA,USA) was used as secondary antibody in ELISA. IgG anti BSA (Bovine Serum Albumin)-NIP was obtained by hyper immunization of BALB/c with BSA- NIP. The IgG antibodies in the sera were affinity purified on a protein A Sepharose column according to manufacturer’s instructions.
Chemicals
Phosphatase Substrate 5 mg tablets were purchased from Sigma Life science. ELISA buffer was prepared by mixing 800 ml dH2O with 97 ml diethanolamine and 100 mg MgCl2X6H2O.
The Substrate buffer for ELISA was prepared by dissolving phosphatase substrate tablets (1 tbl/ 5 ml) in ELISA buffer in the dark. A dilution buffer [PBS containing 0.05 % Tween (SIGMA), 0.25 % dry milk (Semper) and 0.02 % NaN3] was made for the dilution series.
PBS and PBS containing 0.05 % Tween was used for washing the microtiterplates.
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Equipment for ELISA
i) Microtiterplates from Immunolon 2HB, Thermo Electron Corporation, Milford,MA
ii) Dilution plates from Corning Life Science, Corning Incorporated, NY.
ELISA
Microtiterplates were coated with 100 µl OVA-NIP (10 µg/ml) dissolved in PBS containing 0.02 % NaN3 and incubated overnight at 4°C. The plates were then blocked with 100 µl dry milk (50 mg/ml) dissolved in PBS containing 0.04 % NaN3, and further incubated overnight at 4°C. Sera and IgG anti BSA-NIP (Standard) were diluted in PBS containing 0.05 % Tween, 0.25 % dry milk and 0.02 % NaN3 in dilution plates. The coated plates were washed twice in PBS and 50 µl of diluted sample were added, and the plates incubated overnight at 4°C. Next day the plates were washed thrice in PBS containing 0.05 % Tween. Fifty micro litres of alkaline phosphatase-conjugated sheep anti-mouse IgG (diluted 1:1000) in the same buffer as the sera were then added. The plates were then incubated for 3 hours at room temperature.
After incubation the plates were washed thrice in PBS + 0.05 % Tween, and twice in PBS.
100 µl Phosphatase substrate dissolved in ELISA buffer was added to the plates, and the absorbance was measured at 405 nm after 30 min incubation at room temperature in a dark chamber. Results are obtained as absorbance. Data were analyzed using SOFTmax software (Molecular Devices, Sunnyvale, CA, USA) at 405 nm.
Statistical Analysis
Statistical differences between groups were determined by Student’s T-test. Statistical significance levels were set at: ns = p0.05; * = p0.05; ** = p 0.01; *** = p 0.001.
RESULTS
Impaired antibody responses in C1qA-/- against Ficoll-NIP
Several studies have shown the importance of complement component C1q in classical pathway for normal antibody responses against West Nile virus (8), SRBC (25), or Plasmodium chabaudi parasites (26). To analyze if C1q also was important against T- independent antigen, B6 and C1qA-/- mice were immunized with three different doses of Ficoll-NIP (0.1 µg, 0.5 µg and 1 µg). The mice were bled at indicated time points and their IgG anti NIP responses were analyzed in ELISA. The C1qA-/- mice showed severely impaired IgG anti NIP response compared to B6 mice (Fig.2). With the lowest dose of antigen, 0.1 µg,
9 only one time point was significant. But with increasing dose the difference was more
pronounced. These results confirm that the classical pathway is of crucial importance for generation of normal antibody responses to Ficoll-NIP.
Figure 2. Impaired antibody responses in C1qA-/- against Ficoll-NIP. B6 and C1qA-/- mice were
immunized i.v. with 0.1 µg, 0.5 µg or 1 µg Ficoll-NIP and they were bled at indicated time points. Sera from all the bleeding moments were diluted 1:25. IgG anti Ficoll-NIP responses were measured in ELISA. P values represent a comparison between the response in B6 and C1qA-/- mice, where ns = p0.05; * = p0.05; ** = p
0.01; *** = p 0.001.
Cmu13 mice, lacking complement activating IgM, do not have impaired antibody responses to Ficoll-NIP
To analyze if IgM needs to activate complement for normal antibody responses, BALB/c and Cmu13 mice were immunized with three different doses of Ficoll-NIP (0.1 µg, 0.5 µg and 1 µg). Cr2-/- mice were included as negative controls. The mice were bled at indicated time points and their IgG anti NIP responses were analyzed in ELISA. Cmu13 produced close to normal levels of IgG anti NIP, and surprisingly higher than BALB/c (with 0.1 µg Ficoll-NIP) but only significant at one time point. With 0.5 µg antigen IgG anti NIP responses in Cmu13 were low at most of the time points, but not as low as in Cr2-/-. With the highest dose (1 µg Ficoll-NIP) was there no difference between Cmu13 and BALB/c. Cr2-/- like the C1qA-/- in the previous experiment showed impaired antibody responses against the two lower doses of Ficoll-NIP, 0.1 µg and 0.5 µg, (Fig 3). However, against 1 µg antigen Cr2-/- responded with an almost normal antibody response (Fig 3). Thus, lack of complement activation IgM does not generate as impaired antibody response, as deficiency in complement receptors.
10 Figure 3. Cmu13 mice, lacking complement activating IgM, do not have impaired antibody
responses to Ficoll-NIP. BALB/c, Cmu13 and Cr2-/- mice were immunized i.v. with 0.1 µg, 0.5 µg or 1µg Ficoll-NIP and they were bled at indicated time points. Sera from all the bleeding moments were diluted 1:25.
IgG anti NIP responses were measured in ELISA. P values represent a comparison between the response in BALB/c and Cmu13 mice, where ns = p0.05; * = p0.05; ** = p 0.01; *** = p 0.001.
DISCUSSION
Previous studies (12, 13, 25 and 26) have showed the importance of complement and the classical pathway in generating normal antibody responses. This is again verified in our system, where both C1qA-/- and Cr2-/- mice show impaired antibody responses against the T- independent antigen Ficoll-NIP. In 1998 Ehrenstein (17) showed that mice lacking natural secretory IgM had an impaired antibody response, but the response was restored after
immunization with naive serum from wild typwe (containing natural IgM). This suggests that natural IgM is of importance for a normal antibody response, although the ability of IgM to activate complement was not addressed. To analyze if IgM needs to activate complement to generate a normal antibody response, Cmu13 mice lacking complement activating IgM was tested previously by immunizing with T-dependent antigens SRBC and KLH (1, 24).
Unexpectedly Cmu13 did not have impaired primary or secondary antibody responses against these T-independent antigens. However, many pathogens express different polysaccharides that are T-independent antigens. Therefore, it was of interest to test if IgM-complement activation was crucial for antibody responses against T-independent antigens. For this, Cmu13 was immunized with the T-independent antigen, Ficoll-NIP. Results showed that Cmu13 had normal antibody production against Ficoll-NIP and in some cases higher than wild type (with the lowest dose of antigen) though only significant at one time point (Fig 3). A possible
11 explanation for this could be that, IgM mediated clearance of small pathogens/particles by macrophages (18) would be dependent on IgM being able to activate complement. This process would then be effective in the wild type (BALB/c) but not in Cmu13, as it lacks complement activating IgM. But the speed of clearance by the macrophages might be constant, i.e, with increasing antigen dose the macrophages might get saturated leaving sufficient antigen necessary for generating a normal antibody response. Thus, the higher antibody response in Cmu13 with the lowest dose of antigen was not seen against higher doses of Ficoll-NIP. Although IgM is a very good activator of the classical pathway, in this study it does not seem to be the only C1q activator. There are however three other known endogenous activators of C1q: specific intracellular adhesion molecule-grabbing nonintegrin R1 (SIGN-R1) [27], serum amyloid P component (SAP) [28], and C-reactive protein (CRP) [29], that could be responsible for activation of C1q. Yet, no difference in antibody response was noticed when mice deficient in these components were immunized with SRBC (24).
Finally, there is also a possibility that C1q itself are able to bind Ficoll-NIP and be activated.
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