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Exploring the innate immune system: using complement-mediated cell lysis in the classroom.

When teaching biology, instructors often face the challenge of teaching various subdisciplines. One of the subdisciplines that often receives very little attention is the immune system. Part of the reason for this oversight is the perception that the immune system is complex and the appreciation that few typical high school or undergraduate college laboratories have the resources, e.g., tissue culture hoods and incubators, to examine the immune system extensively. When the immune system is addressed, the lessons often focus on the adaptive immune system and the "charismatic" T cells, B cells, and macrophage cells that comprise it. This article explains how complement-mediated bacterial cell lysis provides an opportunity to introduce students to the wonders of the innate immune system and specifically highlight the often-overlooked importance and properties of the complement system. The results of complement-mediated bacterial destruction are dramatic and, while the actual pathway is a rather complex cascade of protein activation, conceptually the process is both easily explained by instructors and understood by students. Additionally, the exercise presented here can be modified to examine a number of different variables of immune system function. An examination of complement-mediated cell lysis links the phenomena of microbiological growth, enzymatic and stoichiometric chemistry, protein-heat instability ("lability"), immune pattern recognition, protein activation, protein cascades, and the cooperation between the innate and adaptive immune systems. The lab presented here can act as a springboard into any of these topics.

Background

The Adaptive Immune Response

The more familiar understanding of the immune response involves the activity of B and T cells. The capacity for certain B or T cells to be selected and expanded based on surface receptor/antigen engagement drives the concept of adaptive immunity. B and T cells have surface receptors that are created by a similar mechanism and which recognize single, unique foreign antigens. When B cell and T cell surface receptors encounter an appropriate foreign antigen, the individual cell ("clone") undergoes rapid expansion. We term this phenomenon "clonal expansion." The clonal expansion of appropriate B and T cell occurs in the lymph nodes and other lymphoid tissues and is characterized by the familiar lymph node swelling observed during infection. This antigen-specific cell expansion and subsequent immune attack clears the body of pathogens. Antigen-specific memory B and T cells survive and provide immunologic memory. The rapid re-activation and expansion of these cells in future antigen encounters explains why certain pathogens do not infect us more than once and underlies the concept of vaccinations.

The Innate Immune Response

The adaptive immune system discussed above is fundamentally a reactive, second line defense against pathogens. The first line of defense against disease is the innate immune system. Innate immunity is composed of skin secretions, barriers to pathogen entry, phagocytic cells, and blood proteins that neutralize pathogens before or soon after they enter the body. One part of the innate immune response is the protein complement system discussed in this article.

The complement system is a set of nine proteins found in the blood of all mammals and is thought to have existed in animals as far back as 300 million years ago. (Wood, 2006; Sunyer et al., 2005). It is responsible for the recognition and destruction of foreign pathogens including viruses, bacteria (von Lackum et al., 2005), fungi (Speth et al., 2004), and other single-celled organisms (Inal, 2004). The complement system acts through a protein activation cascade in which individual complement proteins bind to the targeted pathogen and subsequently recruit additional complement pathway proteins. Proteins involved in the complement process (referred to as C1 through C9) are inactive until they enter the cascade where they join the complex and become activated themselves. This activation involves cleavage of inactive pro-enzymes. In this way, complement is very similar to the blood-clotting cascade. The ultimate fate of this complement cascade is often pathogen destruction by direct cell rupture ("lysis") or, more often, through the induction of cytokine release and the recruitment of other cells that destroy the pathogen through phagocytosis (Abbas, 2005). Lysis occurs when an assembly of complement proteins C5b*6*7*8 drive the polymerization of C9 molecules to form a membrane pore. This allows water and ions to move in and rupture the cell. In addition to its importance in direct microbiological destruction, the complement system is also involved in immune cell recruitment (Paul, 2003) such as when phagocytic cells engulf complement-decorated cells, inflammation, and the removal of dangerous antibody-antigen complexes such as those that can cause the autoimmune disorder lupus (Sturfelt et al., 2005; Sjoholm et al., 2006). In fact, while this laboratory exercise demonstrates the power of serum proteins to directly lyse bacteria, it should not be construed to suggest that this is the predominant way that complement works. A large measure of the power of complement comes through immune cell recruitment, phagocytosis, and/or cytokine release.

The protein complement system works in a very different and more immediate manner than the adaptive immune system. Unlike B and T cells, complement activation does not rely on the recognition of specific antigens by surface receptors. Instead it utilizes the binding of serum proteins to pathogen-associated molecular patterns (PAMPs), essentially patterns that recur throughout pathogens in nature (Heine, 2005). For example, single-celled pathogens have particular protein patterns that are not present in mammalian cells. By identifying these patterns, the complement system can target and neutralize broad classes of pathogens. Among the molecular patterns recognized by complement are lipopolysaccaride (LPS), a component of the cell wall in gram-negative bacteria (Agramonte-Hevia et al., 2002); peptidoglycan and lipoteichoic acid from the cell wall of gram-positive bacteria (Kawasaki et al., 1987); bacterial DNA (Heine et al., 2005); bacterial N-formylmethioninemannose, a sugar common in bacterial glycolipids and glycoproteins but rare in mammals; viral double-stranded RNA (Vandermeer et al., 2004); and glucans, components of fungal cell walls (Ma et al., 2004; Zhang et al., 2001). Complement pre-exists in the blood prior to invasion of the pathogen and thus acts quickly upon pathogen encounter. Because it pre-exists and acts quickly, complement is considered part of the innate immune system.

When the complement system was first discovered, it was noted that it involved the initial binding of B cell-produced antibody to the pathogen followed by binding of complement proteins (Borsos et al., 1970). This complement pathway has since come to be termed the "classical pathway" as it was later observed that two other pathways exist that do not require prior antibody binding. These more recently discovered pathways are known as the "lectin pathway" (Worthley et al., 2005) and the "alternative pathway." While these two pathways differ from the classical pathway in their initial activation, all three complement pathways converge to similar outcomes.

The laboratory presented here has several purposes in a biology classroom. It can reproducibly and rapidly demonstrate the power of the innate immune system. Placed near the first of several discussions on immunity, this laboratory can be used to introduce the innate immune system before moving to a discussion of the adaptive immune system. A brief discussion of the classical complement pathway allows an instructor to introduce functions of antibody that can then be expanded in a discussion of the adaptive immune system. Additionally, the study of the complement system in this lab can be manipulated to examine several variables. Several of these variables will be discussed later.

The Activity

Methods & Materials

* Petri dishes (VWR, Carolina)

* microbiological media, e.g., LB media (Sigma, Carolina)

* Bacto[TM] Agar solidifying agent (Difco, Sigma)

* test tubes

* test tube rack

* permanent markers

* autoclave/ 0.2 [micro]M syringe filters

* disposable transfer pipets

* high RPM shaker

* bovine serum, 10 ml/bottle (Sigma, Cat. #: B-8655)

* sterile saline

* incubator

* variable-temperature water bath

* calculators

* non-pathogenic, characterized bacteria or fungus (Carolina, BioRad)

* inoculation loops, bacterial spreaders, or cotton swabs

* 5% bleach

Teacher Preparation

Prepare the following materials before the laboratory class period:

1. Sterile liquid growth-media--50 ml of media should be prepared per experiment. This amount should be adequate for a single experiment. Liquid media should be autoclaved or filter-sterilized before use.

2. Sterile saline--50 ml of 0.85% sodium chloride solution.

3. Nutrient media agar plates--2% (w/v) LB agar plates must be prepared prior to the laboratory. Each group uses between two to five plates depending on the scale of the experiment. Sterilely-prepared media plates can be kept at room temperature in sealed bags.

4. Overnight bacterial culture--Inoculate 3-5 ml of liquid media with bacteria and grow in a shaking incubator on the day before the lab. For best results, the culture should be grown in a highly aerobic, warm environment, but a fresh overnight culture grown at room temperature works also.

Basic Protocol

The basic complement lysis protocol relies on two major components: An actively growing bacterial culture and serum that contains active complement proteins. Serum is what remains when the cell component of blood has been removed. Serum contains about 7-10% total protein and this protein component contains all of the factors involved in the complement cascade including antibodies and serum proteins.

The basic protocol involves incubating bacteria in the presence of serum or in the presence of saline (negative control) for one hour and then plating the mixture on nutrient LB agar plates. The plates are examined the following day (or another later point) to determine the number of bacteria that were destroyed by the complement system compared to the saline control. To demonstrate that the efficiency of killing covers several orders of magnitude between the negative control and the serum, we typically use serial dilutions of the overnight bacterial culture and incubate aliquots of each of these dilutions with both serum and saline.

Experimental Procedure

1. To begin the experiment, each group (two to four students) receives individual test tubes containing aliquots of the following: liquid media, undiluted overnight bacterial culture, serum, and saline. Each group should also receive approximately 15 small test tubes (or 1.5 ml centrifuge tubes), 10 transfer pipets (or pipet tips if using a pipetman), a tube rack, and a permanent marker.

2. Using transfer pipets or pipettors, students dispense 1 ml of the liquid bacterial media into five of their test tubes. Label these tubes "#1" through "#5."

3. Transfer one drop (if using transfer pipet) or 10 microliters (if using pipettor) of undiluted, overnight bacterial culture into the tube labeled "#1." Cap the tube, and invert to mix. This serves as the first in a series of bacterial dilutions.

4. Using a clean transfer pipet or new pipet tip, transfer a single drop (pipet) or 10 microliters (pipettor) from Tube #1 as prepared in Step 3 into Tube #2 containing the liquid media. This represents the second dilution in the series.

5. Continue this serial dilution method (using a new pipet at each dilution) through Tube #5. These tubes will contain the bacteria dilutions that will be incubated with serum or saline and plated on agar plates. Given that each dilution is approximately 1:100 of the prior tube, have your students calculate the effective, final dilution of all five tubes.

6. Label the remaining tubes in two different ways. Label one set of tubes "E1" through "E5." These will represent the experimental tubes that will receive serum and a portion of the bacterial dilutions prepared in Steps 2-5 above. Label another set of tubes "C1" through "C5." These tubes will receive saline and bacteria and represent the control tubes.

7. Add 250 [micro]l or approximately 20 drops of serum to each tube labeled "E1" to "E5." Similarly, add 250 [micro]l of saline to the tubes labeled "C1" to "C5." If using 1.5 ml microfuge tubes, use the graduations on the tubes to judge how much to add. To each tube, add 250 [micro]l of the appropriate bacterial dilution. For example, Dilution #1 should be added to C1 and El while Dilution #2 should be used for C2 and E2, etc. To economize equipment and plastic waste, if students dispense bacterial dilutions to the control and experimental tubes beginning with the most dilute solution (Tube #5) and moving to #4, then #3, etc., they do not need to use a new pipet with each bacterial transfer. Figure 1 is a schematic of the laboratory process.

8. After combining bacterial dilutions with saline and serum, the tubes should be closed and inverted to mix. At this point, note the group on the tubes. Initials can be placed on the sides or the top of the tube with a permanent marker. At this point, tubes are incubated for one hour at 37[degrees]C.

9. After a one-hour incubation, obtain your tubes and invert them to resuspend the bacteria that may have settled. Using a small transfer pipet, add one drop or approximately 20 [micro]l from Tube E5 onto one Petri dish containing LB nutrient agar. Using a cell spreader, inoculating loop, or cotton swab, spread the drop evenly on the plate. Do this for samples E4, E3, E2, and E1 on separate nutrient agar plates. By working with the most dilute sample and moving to the most concentrated sample, students do not need to change or clean their spreading apparatus. Label your plates to reflect what is being plated. Label the bottom of the plates with both the group's name and tube name, e.g., E5, E2, etc. as the lids can be accidentally swapped among plates. On a different set of plates, plate the same amount of control reaction from C5 through C1 similar to that done in Step 7. Label these plates similarly.

10. After inoculating the plates, incubate them for 24 hours at 37[degrees]C. If an incubator is unavailable, incubate them at room temperature for 24-48 hours. Incubate plates until bacterial colonies are obvious and identifiable but before the margins of individual colonies begin to grow into one another. After the colonies appear, store the plates at 4[degrees]C until needed.

11. On the second day of the experiment, count and record the number of colonies on each plate and construct a graph showing the killing ability of complement. Remember that only a 20 ?l amount of the mixture was added to the plate. Calculate the values to reflect "bacteria/ml." Graph the number of bacteria per ml on the Y-axis and the bacterial dilution on the X-axis. The axes can be linear; however using the negative log of the bacterial dilutions on the X-axis more easily and better illustrates the typical outcomes. For instance, the first dilution (100-fold dilution) becomes 2; second dilution (10,000-fold) becomes 4, etc., with each dilution approximately 100 times more dilute than the previous. A linear Y-axis more dramatically demonstrates the growth differences observed across the series of dilutions.

[FIGURE 1 OMITTED]

[FIGURE 2 OMITTED]

Results

This laboratory exercise produces reliable, reproducible results. The results of several independent experiments accompany this article (Figure 2). Figure 2 shows the survival curve for two different experiments. Students typically find the efficiency of bacterial killing to be dramatic and undeniable with the power of the serum complement system readily apparent. Typically, anywhere from 95 to 99.9% of diluted bacteria are killed by complement-mediated lysis in this experiment. The complement killing is also rapid, as shown in Figure 3. While the experiment is designed for a one-hour incubation, a large number of cells are killed within the first 20 minutes and almost all are dead within 40 minutes. This might aid those teachers who are limited in time.

When looking at bacterial cell counts from the first dilution, it is not uncommon to see large numbers of colonies on both the experimental and control plates. This is due to the large number of bacteria that quench the lytic power of the complement proteins in the serum. It is not unusual to observe bacterial lawns in the initial dilutions of saline treated bacteria. Typically, students see the best results (20-500 colonies/plate) in the [10.sup.-4], [10.sup.-6] and [10.sup.-8] dilutions.

[FIGURE 3 OMITTED]

A Comment on Safety

As with all science labs, appropriate care should be taken with this laboratory. First, care should be taken when preparing sterile liquid and solid nutrient media as improper autoclaving technique can result in injury. When conducting this lab, characterized, non-pathogenic strains of bacteria should be utilized. Even so, students should take care not to contaminate themselves or surfaces with the bacteria. Although bovine serum is generally assumed safe from infectious agents, users are reminded that it is an animal blood product and to treat it accordingly. Finally, when the lab is finished, bacteria-contaminated material and agar plates should be either autoclaved or soaked in a 5% bleach solution to kill bacteria.

Other Variables, Considerations & Questions

This experiment has many possibilities for modification or expansion. While the experiment as written examines complement killing of bacteria across a dilution range of several orders of magnitude, there are a number of variables that one can examine. These include but are not limited to:

Numbers of dilutions/Numbers of plates

As a way to introduce different observations among the different groups and save plates and resources, reduce the number of dilutions that each group tests. While each group should still prepare all five dilutions, allow individual groups to choose two of the dilutions and set up experimental and control tubes using only those two. Of course, confirm that the choices of the class cover the entire dilution range. Combine the results from the class and prepare and graph complement-killing curves from the combined results.

Time

Although a one-hour incubation is standard, one can shorten or extend incubation time. Allow different student groups to examine the effects of different incubation times. Results of this experiment are shown in Figure 3. Note, however, that at high bacterial concentrations, extending the time indefinitely will not lead to complete killing of bacteria. This can be used to demonstrate the stoichiometric nature of the complement reaction.

Temperature

Changing the incubation temperature alters the rate of bacterial killing. Examine a range of temperatures to see which kills the bacteria most efficiently. Discuss why this observation is physiologically relevant.

Heat/Inactivation

Many types of mammalian tissue culture media rely on heat-inactivated serum as a nutrient source. Incubating the serum at 56[degrees] C for one hour inactivates the complement in the serum. To demonstrate to students the heat-sensitive nature of proteins and the principle that protein activity can be inactivated by heat (among other things), have your student groups pre-incubate aliquots of their serum at different individual temperatures (and for different times) to see the effect on complement activity. Observe what happens when serum is exposed to increasing heat prior to incubation with the bacteria. Discuss and test other ways that one could inactivate the proteins in the complement.

Gram positive vs. gram-negative bacteria

To examine the efficiency of complement killing in different scenarios, use different types of bacteria in the experiments. Which types of bacteria were most efficiently killed by serum? Were there differences between the killing of gram-negative and gram-positive bacteria? Why?

LPS quenching

LPS, a major component of the bacterial cell wall and a major contributor to human sepsis, can be purchased and reconstituted. What happens when LPS is added to bacterial dilutions that receive serum? Students might expect that there would be less killing. Why would this be the case? What is observed as one increases the amount of LPS added to a fixed number of cells? Discuss and test what happens when other bacterial and fungal serum-binding proteins are added to the incubation reaction.

Protein size

Dialysis tubing and spin columns are available that allow one to remove proteins below certain molecular weight (kD) thresholds. Allow students to remove proteins below certain thresholds and determine which fraction(s) contains complement as judged by lytic ability. What molecular weight range contains the lytic ability? What happens if we combine fractions? Does the lytic ability increase? Is the lytic ability in several protein fractions or just one?

Assessment & Student Reaction

As a lab exercise, I ask students to formulate a hypothesis before we begin as to what they expect to see on the control and experimental plates across the range of dilutions. I then ask them to incorporate that hypothesis into a lab report that asks the students the following questions.

* Based on the volume added to each plate from the reaction tubes (dilutions) and the number of colonies observed on each plate, calculate the number of bacteria per ml in each dilution for both control and experimental tubes at each dilution. Graph the number of bacteria per ml (Y-axis) versus the -log of the bacterial dilution (X-axis) for experimental and control tubes. What trends do you observe in each series of dilutions?

* Comparing your control and experimental plates for each dilution, what percentage of bacteria survived complement killing at each dilution? (Determine ratio of living cells on experimental versus control plate). Show your work.

* Based on your observations and the pooled class data, did the results support your hypothesis? If not, what observations did not support your hypothesis and how do you explain them?

* Was there complete killing of all the bacteria from the undiluted bacteria tube and the first dilution of bacteria?

If not, explain that observation.

* What did this experiment teach you about the innate immune system and specifically about complement lysis of bacteria? Consider speed and efficiency in your answer.

* Would this be an example of a pre-existing immune response or an adaptive response? Why?

* Why is the observation of complement bacterial lysis an important phenomenon?

Overall, my students are successful in accomplishing this lab and are impressed by the dramatic results. The "flow chart" of the lab shown in Figure 1 gives an indispensable overview of how this lab operates. Students enjoy the compilation and discussion of the class data. The fact that the bacteria in the first dilution are not always completely killed gives the students pause but also underscores the limits of any system.

Conclusion

The innate immune system effectively neutralizes many pathogens before the adaptive immune response is triggered. This straightforward and highly adaptable lab exercise unambiguously demonstrates the power of serum complement specifically and the innate immune system generally. We have used it repeatedly with success and to the amazement of our students.

It is a useful addition to a class when placed before a discussion of the adaptive immune system as it foreshadows concepts such as antibody, phagocytes, and B cells, and reinforces the concept that the innate immune system acts so that the adaptive immune system has to work less.

Other Reading

Doffinger, R., Patel, S. & Kumaranratne, D.S. (2005). Human immunodeficiencies that predispose to intracellular bacterial infections. Current Opinion in Rheumatology, 17(4), 440-6.

Holmskov, U., Thiel, S. & Jensenius, J.C. (2003). Collections and ficolins: Humoral lectins of the innate immune defense. Annual Review of Immunology, 21, 547-78.

Hourcade, D., Holers, V.M. & Atkinson, J.P. (1989). The regulators of complement activation (RCA) gene cluster. Advances in Immunology, 45, 381-416.

Kilpatrick, D.C. (2002). Mannan-binding lectin and its role in innate immunity. Transfusion Medicine, 12(6), 335-52.

Lydyard, P.M., Whelan. A. & Fanger, M.W. (2000). Instant Notes in Immunology. New York, New York: Springer-Verlag.

Mayer, M.M. (1967). Complement and complement fixation. In E.A. Kabat, M.M. Mayer & C. Charles (Eds.), Experimental Immunochemistry. Springfield, IL: Thomas Publishing.

Roitt, I.M. & Delves, P.J. (2001). Roitt's Essential Immunology, 10th Edition. Malden, MA: Blackwell Science.

References

Abbas, A. & Lichtman, A. (2005). Cellular and Molecular Immunology, Updated Edition. Philadelphia, PA: W. B. Saunders.

Agramonte-Hevia, J., Gonzalez-Arenas, A., Barrera, D. & Velasco-Velazquez, M. (2002). Gram-negative bacteria and phagocytic cell interaction mediated by complement receptor 3. FEMS Immunology & Medical Microbiology, 34(4), 255-66.

Borsos, T. & Rapp, H.J. (1970). The Molecular Basis of Complement Action. New York, New York: Appleton Century Crafts.

Heine, H. & Ulmer, A.J. (2005). Recognition of bacterial products by toll-like receptors (Review). Chemical Immunology & Allergy, 86, 99-119.

Inal, M. (2004). Parasite interaction with host complement: Beyond attack regulation (Review). Trends in Parasitology, 20(9), 407-12.

Kawasaki, A., Takada, H., Kotani, S., Inai, S., Nagaki, K., Matsumoto, M., Yokogawa, K., Kawata, S., Kusumoto, S. & Shiba, T. (1987). Activation of the human complement cascade by bacterial cell walls, peptidoglycans, water-soluble peptidoglycan components, and synthetic muramylpeptides--studies on active components and structural requirements. Microbiology & Immunology, 31(6), 551-69.

Ma, Y.G., Cho, M.Y., Zhao, M., Park, J.W., Matsushita, M., Fujita, T. & Lee, B.L. (2004). Human mannose-binding lectin and L-ficolin function as specific pattern recognition proteins in the lectin activation pathway of complement. Journal of Biological Chemistry, 279(24), 25307-12.

Nakagawa, T., Ma, B.Y., Uemura, K., Oka, S., Kawasaki, N. & Kawasaki, T. (2003). Role of mannan-binding protein, MBP, in innate immunity. Anticancer Research, 23(6a), 4467-71.

Paul, W. (2003). Fundamental Immunology. Philadelphia, PA: Lippincott, Williams, and Wilkens.

Sjoholm, A.G., Jonsson, G., Broconier, J.H., Sturfelt, G. & Truedsson, L. (2006). Complement deficiency and disease: An update (Review). Molecular Immunology, 43(1-2), 78-85.

Speth, C., Rambach, G., Lass-Florl, C., Dierich, M.P. & Wurzner, R. (2004). The role of complement in invasive fungal infections (Review). Mycoses, 47(3-4), 93-103.

Sturfelt, G. & Truedsson, L. (2005). Complement and its breakdown products in SLE (Review). Rheumatology, 44(10), 1227-32.

Sunyer, J.O., Boshra, H. & Li, J. (2005). Evolution of anaphylatoxins, their diversity and novel roles in innate immunity: Insights from the study of fish complement (Review). Veterinary Immunology & Immunopathology, 108(1-2), 77-89.

Vandermeer, J., Sha, Q., Lane, A.P. & Schleimer, R.P. (2004). Innate immunity of the sinonasal cavity: Expression of messenger RNA for complement cascade components and toll-like receptors. Archives of Otolaryngology--Head & Neck Surgery, 130(12), 1374-80.

von Lackum, K., Miller, J.C., Bykowski, T., Riley, S.P., Woodman, M.E., Brade, V., Kraiczy, P., Stevenson, B. & Wallich, R. (2005). Borrelia burgdorferi regulates expression of complement regulator-acquiring surface protein 1 during the mammal-tick infection cycle. Infection & Immunity, 73(11), 7398-405.

Wood, P. (2006). Understanding Immunology. Essex, England: Pearson Educators Ltd.

Worthley, D.L., Bardy, P.G. & Mullighan, C.G. (2005). Mannose-binding lectin: Biology and clinical implications. Internal Medicine Journal, 35(9), 548-55.

Zhang, M.X., Brandhorst, T.T., Kozel, T.R. & Klein, B.S. (2001). Role of glucan and surface protein BAD1 in complement activation by Blastomyces dermatitidis yeast. Infection & Immunity, 69(12), 7559-64.

Kevin G. Fuller, Ph.D., is Professor of Biology, Columbia College Chicago, The Science and Mathematics Department, Chicago, IL 60605; e-mail: kgfuller@colum.edu.
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Title Annotation:INQUIRY & INVESTIGATION
Author:Fuller, Kevin G.
Publication:The American Biology Teacher
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Date:Feb 1, 2008
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