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EXPLORINGTHEINNATE : UsingComplement-Mediated CellLysis in the Classroom

KEVIN G. FULLER

"VVhen teachingbiology, instructors often face expansionof appropriateB and T occursin the lymph the challenge of teachingvarious subdisciplines.One of nodes and other lymphoidtissues and is characterizedby

the subdisciplinesthat often receivesvery little attentionis the familiarlymph node swelling observed during infec- Downloaded from http://online.ucpress.edu/abt/article-pdf/70/2/103/54518/30163212.pdf by guest on 28 September 2021 the immunesystem. Part of the reasonfor this oversightis tion. This -specificcell expansion and subsequent the perceptionthat the immunesystem is complexand the immune attackclears the body of pathogens.Antigen-spe- INVESTIGATION appreciationthat few typicalhigh school or undergraduate cificmemory B and T cells surviveand provide immunologic college laboratorieshave the resources,e.g., tissue culture memory.The rapid re- and expansionof these cells & hoods and incubators,to examine the immune system in futureantigen encounters explains why certainpathogens extensively.When the immune system is addressed,the do not infectus more than once and underliesthe concept lessons often focus on the adaptiveimmune system and of vaccinations. the "charismatic"T cells, B cells, and macrophagecells that compriseit. This article explains how complement-medi- The Innate atedbacterial cell lysis providesan opportunityto introduce studentsto the wondersof the innateimmune and The adaptiveimmune system discussed above is funda- system a second line defense specificallyhighlight the often-overlookedimportance and mentally reactive, againstpathogens. of the Theresults of The firstline of defenseagainst disease is the innateimmune properties complementsystem. comple- Innate is of skin bar- ment-mediatedbacterial destruction are dramatic and, while system. composed , the actual is a rather cascadeof riersto pathogenentry, phagocytic cells, and blood pathway complex thatneutralize beforeor soon after enterthe INQUIRY activation,conceptually the processis both easilyexplained pathogens they instructorsand understood students. body.One partof the innateimmune response is the protein by by Additionally, discussedin this article. the exercisepresented here can be modifiedto examinea complementsystem numberof differentvariables of immune system function. The complement system is a set of nine proteins An examinationof complement-mediatedcell lysis links found in the blood of all mammalsand is thought to have the phenomenaof microbiologicalgrowth, enzymatic and existed in animals as far back as 300 million years ago. stoichiometricchemistry, protein-heat instability ("lability"), (Wood, 2006; Sunyeret al., 2005). It is responsiblefor the immunepattern recognition, protein activation, protein cas- recognitionand destructionof foreignpathogens including cades,and the cooperationbetween the innateand adaptive , (von Lackumet al., 2005), fungi (Speth et immunesystems. The lab presentedhere can act as a spring- al., 2004), and other single-celledorganisms (Inal, 2004). boardinto any of these topics. The complementsystem acts througha protein activation cascadein which individualcomplement proteins bind to the targetedpathogen and subsequentlyrecruit additional Background complement pathway proteins. Proteins involved in the complementprocess (referred to as Cl throughC9) areinac- The Adaptive Immune Response tiveuntil they enterthe cascadewhere they join the complex The more familiar understanding of the immune and become activatedthemselves. This activationinvolves responseinvolves the activityof B and T cells. The capacity cleavageof inactivepro-. In this way, complement for certainB or T cells to be selectedand expandedbased is very similarto the blood-clottingcascade. The ultimate on surfacereceptor/antigen engagement drives the concept fate of this complementcascade is often pathogendestruc- of adaptiveimmunity. B and T cells have surfacereceptors tion by directcell rupture("lysis") or, more often, through that are createdby a similarmechanism and which recog- the induction of release and the recruitmentof nize single,unique foreign . When and other cells that destroythe pathogenthrough surfacereceptors encounter an appropriateforeign antigen, (Abbas,2005). Lysis occurs when an assemblyof comple- the individualcell ("clone")undergoes rapid expansion. ment proteins C5b*6*7*8drive the polymerizationof C9 We term this phenomenon"clonal expansion." The clonal moleculesto forma membranepore. This allowswater and ions to movein and rupturethe cell.In additionto its impor- tancein directmicrobiological destruction, the complement system is also involvedin immune cell recruitment(Paul, KEVING. FULLER,Ph.D., is Professorof ,Columbia College The Scienceand Mathematics IL 2003) such as when phagocyticcells engulf complement- Chicago, Department,Chicago, decorated and the removalof 60605;e-mail: [email protected]. cells,inflammation, dangerous

COMPLEMENT-MEDIATEDCELLLYSIS 103 -antigencomplexes such as those that can cause the . BactoTMAgar solidifying agent (Difco, Sigma) autoimmunedisorder (Sturfeltet al., 2005; Sjoholmet al., . test tubes 2006). In fact, while this laboratoryexercise demonstrates the . test tube rack power of proteinsto directlylyse bacteria,it should not be construedto suggest that this is the predominantway that . permanentmarkers works.A measureof the of complement large power complement . autoclave/0.2 [1,Msyringe filters comes immune cell recruitment, through phagocytosis,and/or . transfer cytokinerelease. disposable pipets . high RPMshaker The protein complementsystem works in a very different . bovine and moreimmediate manner than the adaptiveimmune system. serum, 10 ml/bottle (Sigma,Cat. #: B-8655) Unlike B and T cells, complementactivation does not rely on . sterilesaline the recognitionof specificantigens by surfacereceptors. Instead . incubator it utilizes the binding of serum proteins to pathogen-associ- . waterbath ated molecularpatterns (PAMPs), essentially patterns that recur variable-temperature throughoutpathogens in nature (Heine, 2005). For example, . calculators have single-celledpathogens particularprotein patterns that are . non-pathogenic,characterized bacteria or not fungus present in mammaliancells. By identifyingthese patterns, (Carolina,BioRad) the can and neutralizebroad classes complementsystem target . of the molecular inoculationloops, bacterialspreaders, or cotton swabs pathogens.Among patternsrecognized by com- Downloaded from http://online.ucpress.edu/abt/article-pdf/70/2/103/54518/30163212.pdf by guest on 28 September 2021 plement are lipopolysaccaride(LPS), a component of the cell . 5%bleach wall in gram-negativebacteria (Agramonte-Hevia et al., 2002); peptidoglycanand lipoteichoicacid from the of gram- Teacher Preparation positivebacteria (Kawasaki et al., 1987); bacterialDNA (Heine Preparethe followingmaterials before the laboratoryclass et al., 2005); bacterialN-formylmethioninemannose, a sugar period: common in bacterialglycolipids and glycoproteinsbut rare in mammals;viral double-stranded RNA (Vandermeer et al., 2004); 1. Sterile liquid growth-media-50ml of media should and glucans,components of fungalcell walls (Ma et al., 2004; be preparedper experiment.This amount should be Zhanget al., 2001). Complementpre-exists in the blood priorto adequatefor a single experiment.Liquid media should invasionof the pathogenand thus acts quicklyupon pathogen be autoclavedor filter-sterilizedbefore use. encounter.Because it pre-existsand acts quickly,complement is 2. Sterilesaline-50 ml of 0.85%sodium chloridesolution. consideredpart of the innateimmune system. 3. Nutrient media agar plates-2% (w/v) LB agar plates When the complementsystem was first discovered,it was must be preparedprior to the laboratory.Each group noted that it involved the initial binding of B cell-produced uses between two to five plates dependingon the scale antibodyto the pathogen followedby binding of complement of the experiment.Sterilely-prepared media plates can be proteins (Borsos et al., 1970). This complementpathway has kept at room temperaturein sealedbags. since come to be termedthe "classicalpathway" as it was later 4. Overnightbacterial culture-Inoculate 3-5 ml of liquid observedthat two otherpathways exist thatdo not requireprior media with bacteriaand grow in a shaking incubator antibody These more discovered are binding. recently pathways on the day before the lab. For best results, the culture known as the " et al., 2005) and the pathway"(Worthley shouldbe grownin a highlyaerobic, warm environment, "alternativepathway." While these two differfrom the pathways but a freshovernight culture grown at room temperature classicalpathway in theirinitial activation, all threecomplement worksalso. pathwaysconverge to similaroutcomes. The laboratorypresented here has severalpurposes in a Basic Protocol biology classroom.It can reproduciblyand rapidlydemonstrate The basic relies on two the powerof the innateimmune system. Placed near the firstof complementlysis protocol major An bacterialculture and serum severaldiscussions on immunity,this laboratorycan be used to components: activelygrowing thatcontains active Serumis whatremains introducethe innateimmune system before moving to a discus- complementproteins. when the cell of blood has been removed.Serum sion of the adaptiveimmune system.A brief discussion of the component containsabout 7-10%total and this classicalcomplement pathway allows an instructorto introduce protein proteincomponent containsall of the factorsinvolved in the cascade functionsof antibodythat can then be expandedin a discussion complement antibodiesand serum of the adaptiveimmune system.Additionally, the study of the including proteins. complementsystem in this lab can be manipulatedto examine The basic protocolinvolves incubating bacteria in the pres- several variables.Several of these variableswill be discussed ence of serum or in the presence of saline (negativecontrol) later. for one hour and then platingthe mixtureon nutrientLB agar plates. The plates are examinedthe followingday (or another The later point) to determine the number of bacteria that were Activity destroyedby the complementsystem comparedto the saline control.To demonstratethat the efficiencyof killingcovers sev- Methods& Materials eral ordersof magnitudebetween the negativecontrol and the serum,we typicallyuse serialdilutions of the overnightbacterial . Petridishes (VWR,Carolina) culture and incubate aliquots of each of these dilutions with . microbiologicalmedia, e.g., LBmedia (Sigma, Carolina) both serumand saline.

104 THEAMERICAN BIOLOGY TEACHER, VOLUME 70,NO. 2, FEBRUARY2008 Experimental Procedure 9. Aftera one-hourincubation, obtain your tubes and invert them to resuspendthe bacteriathat may have settled. 1. To the each (two to four stu- begin experiment, group Using a small transferpipet, add one drop or approxi- dents) receivesindividual test tubes containingaliquots mately20 ill fromTube E5 onto one Petridish contain- of the media, undiluted following: liquid overnight ing LB nutrientagar. Using a cell spreader,inoculating bacterialculture, serum, and saline.Each should group loop, or cotton swab, spread the drop evenly on the also receive 15 smalltest tubes (or 1.5 ml approximately plate.Do this forsamples E4, E3, E2, and El on separate tubes),10 transfer (or if centrifuge pipets pipettips using nutrientagar plates. By workingwith the most dilute a a tuberack, and a marker. pipetman), permanent sample and moving to the most concentratedsample, 2. Using transferpipets or pipettors,students dispense 1 students do not need to change or clean their spread- ml of the liquid bacterialmedia into five of their test ing apparatus.Label your plates to reflectwhat is being tubes.Label these tubes "#1"through "#5." plated. Label the bottom of the plates with both the name and tube etc. as the 3. Transferone drop (if using transferpipet) or 10 microli- group's name, e.g., E5, E2, lids can be On a ters (if using pipettor)of undiluted,overnight bacterial accidentallyswapped among plates. differentset of the same amountof control cultureinto the tube labeled "#1."Cap the tube, and plates,plate invertto mix. This servesas the firstin a seriesof bacte- reactionfrom C5 throughCl similarto thatdone in Step rialdilutions. 7. Labelthese platessimilarly. 10. After the incubatethem for 24 hours 4. Using a clean transferpipet or new pipet tip, transfera oinoculating plates,

at 37 C. If an incubatoris incubatethem at Downloaded from http://online.ucpress.edu/abt/article-pdf/70/2/103/54518/30163212.pdf by guest on 28 September 2021 singledrop (pipet) or 10 microliters(pipettor) from Tube unavailable, room for 24-48 hours.Incubate until #1 as preparedin Step 3 into Tube #2 containingthe temperature plates bacterialcolonies are obvious and identifiablebut before liquidmedia. This representsthe second dilutionin the the of individualcolonies to into one series. margins begin grow o another.After the colonies appear,store the plates at 4 5. Continuethis serialdilution method (using a new pipet C until needed. at each dilution) through Tube #5. These tubes will containthe bacteriadilutions that will be incubatedwith 11. On the second day of the experiment,count and record the number of colonies on each and construct serum or saline and plated on agarplates. Given that plate a the of each dilutionis approximately1:100 of the prior tube, graph showing killing ability complement. Rememberthat a 20 amount of the mixture have your studentscalculate the effective,final dilution only pl of all five tubes. was added to the plate. Calculatethe values to reflect "bacteria/ml."Graph the numberof bacteriaper ml on 6. Labelthe remainingtubes in two differentways. Label the Y-axisand the bacterialdilution on the X-axis.The one set of tubes "El" "E5."These will through represent axes can be linear;however the of the the tubes that will receive serum and a using negativelog experimental bacterialdilutions on the X-axismore easily and better portion of the bacterialdilutions prepared in Steps 2-5 above. Label another set of tubes "Cl" through "C5."These tubes will receive saline and bacteria and represent the

controltubes. 2. Saline 250 ill (Control) 7. Add 250 ul or approximately20 drops of serum to each tube labeled "El" to "E5."Similarly, add 250 ill of salineto the C1 C2 C3 C4 C5 3 tubeslabeled "Cl" to "C5."If using 1.5 ml 10111 101,1l 10111 10111 10ul microfugetubes, use the graduationson 100x 100x 100x 100x the tubes to judge how much to add. To dilution dilution dilution dilution each add 250 of the Serial Dilution tube, ul appropriate of bacterialdilution. For Dilution Bacteria 1 ml 1 ml 1 ml 1 ml 1 ml example, media media media media media #1 should be added to Cl and El while 1 3 Dilution#2 shouldbe used forC2 andE2, 250111 Serum etc. To economize and Overnight 2 equipment plastic culture (Experimental) waste,if studentsdispense bacterial dilu- of bacteria tions to the control and experimental E1 E2 E3 E4 E5 tubesbeginning with the most dilutesolu- of 100x dilutions of tion (Tube#5) andmoving to #4, then#3, 0 Series overnight bacterial culture. etc., they do not need to use a new pipet CI Distribution of 250 IIIof either with each bacterialtransfer. 1 is a saline or serum into individual tubes. 2501.1.1 Figure 0 Dispensing of 250 pi (equal volumes) schematicof the laboratoryprocess. of diluted bacteria into individual tubes. 8. After combiningbacterial dilutions with saline and serum, the tubes should be Figure1. Schematicof experimentaldesign. Serial dilution of bacteriaisshown pro- closed and invertedto mix. At this point, gressingfrom left to right across the figure's middle with progressive 100-fold dilutions of note the on the tubes.Initials can be group bacteria.Thisisfollowed by addition ofsaline or serum to control (C) and experimental (E) on the sides or the of the tube placed top tubes,respectively, inStep #2. Equal volumes of dilutedbacteria are added to either control with a permanentmarker. At this point, tubesare incubated for one hourat 37 C. tubes or experimental tubes in Step #3. Illustrationcreated by Colin Whitehurst.

COMPLEMENT-MEDIATEDCELLLYSIS 105 illustratesthe typicaloutcomes. For instance, the firstdilution (100-fold dilution) becomes Complement killing across bacterial dilutions 2; second dilution(10,000-fold) becomes 4, 40000 etc., with each dilution approximately100

times more dilute than the previous.A lin- 35000 ear Y-axismore dramaticallydemonstrates the growthdifferences observed across the 30000 Legend seriesof dilutions. Trial 1 Experimental 25000 Control Trial 2 (colonies/m1) Experimental Results 20000 Control

This laboratory exercise produces reliable, Growth reproducibleresults. The resultsof severalindepen- 15000 dent this article 2). experimentsaccompany (Figure Bacterial10000 Figure2 shows the survivalcurve for two different Students find the of experiments. typically efficiency 5000 bacterialkilling to be dramaticand undeniablewith the powerof the serumcomplement system readily 0 0 1 2 3 4 5 6 7 8 9 10 apparent.Typically, anywhere from 95 to 99.9%of Downloaded from http://online.ucpress.edu/abt/article-pdf/70/2/103/54518/30163212.pdf by guest on 28 September 2021 log of complement dilution dilutedbacteria are killed by complement-mediated in this The is lysis experiment. complementkilling 2. acrossbacterial dilutions. of also rapid,as shown in Figure3. While the experi- Figure Complementkilling Representation bacterial bacterialcolonies numbers ment is designedfor a one-hourincubation, a large twoseparate lysisexperiments.Y-axis represents numberof cells are killed within the first 20 min- after24-hour growth. X-axis represents the negativelog of the bacterialdilution. utes and almostall aredead within 40 minutes.This Experiment#1 is representedby diamonds on eitherdashed (serum) or solid (saline) mightaid those teacherswho arelimited in time. lines.Experiment #2 is representedby inverted triangles on similarlines. When looking at bacterialcell counts from the first dilution,it is not uncommonto see large numbersof colonieson both the and experimental Effect of complement improves with incubation time controlplates. This is due to the largenumber of 50000 bacteriathat quenchthe lytic powerof the comple- ment proteins in the serum. It is not unusual to 45000 observebacterial lawns in the initial dilutions of 40000 saline treatedbacteria. Typically, students see the best results(20-500 colonies/plate)in the 10-4,10-6 35000 and 10-8dilutions. 30000

(colonies/Inn25000 A Comment on Safety growth20000 Aswith all science careshould labs,appropriate - be takenwith this First,care should be 15000 laboratory. Bacterial takenwhen preparingsterile liquid and solid nutri- 10000 ent media as can improperautoclaving technique 5000 resultin injury.When conductingthis lab, charac- terized,non-pathogenic strains of bacteriashould 0 0 20 40 60 80 100 120 140 be utilized.Even so, studentsshould take carenot Time of incubation to contaminatethemselves or surfaceswith the bac- teria. bovine serum is assumed Although generally Figure3. Effectof complementimproves with incubationtime. Abacterial safe frominfectious agents, users arereminded that dilution(101 of anovernight culture is incubatedwith an equal volume of saline it is an animalblood productand to treatit accord- (diamond)or serum Atvarious times, incubated tubes are removed and ingly.Finally, when the lab is finished,bacteria-con- (square). theircontents amountof seenbetween the first taminatedmaterial and agarplates should be either plated.Theincreasing bacteria/ml autoclavedor soakedin a 5%bleach solution to kill andsecond hour in the controltube represents cell growth during the incubation bacteria. period.Timepoints plotted represent 0, 5, 25,35, 45, 60, 90, and 120 minutes. Other Variables, Numbersof of Considerations & dilutions/Numbers plates Questions As a way to introducedifferent observations among the This experimenthas manypossibilities for modificationor differentgroups and save plates and resources,reduce expansion.While the experimentas writtenexamines comple- the numberof dilutionsthat each group tests. While ment killingof bacteriaacross a dilutionrange of severalorders each groupshould still prepareall five dilutions,allow of magnitude,there are a numberof variablesthat one can exam- individualgroups to choose two of the dilutionsand ine. Theseinclude but arenot limitedto: set up experimentaland controltubes using only those

106 THEAMERICAN BIOLOGY TEACHER, VOLUME 70, NO.2, FEBRUARY2008 two. Of course, confirm that the choices of the class the lytic abilityincrease? Is the lytic abilityin several cover the entire dilution range. Combine the results proteinfractions or just one? fromthe class and prepareand graphcomplement-kill- ing curvesfrom the combinedresults. Assessment & Student Reaction Time As a lab exercise,I ask students to formulatea hypothesis Althougha one-hourincubation is standard,one can before we begin as to what they expect to see on the control shorten or extend incubation time. Allow different and experimentalplates acrossthe rangeof dilutions.I then ask student groups to examine the effects of different them to incorporatethat hypothesis into a lab report that asks incubationtimes. Results of this experimentare shown the students the followingquestions. in 3. that at bacterialcon- Figure Note, however, high . Based on the volume added to each from the the time will not plate centrations,extending indefinitely reaction tubes and the number of colonies lead to of bacteria.This can be used to (dilutions) completekilling observedon each calculatethe numberof bacteria demonstratethe stoichiometricnature of the plate, comple- ml in each dilution for both controland ment reaction. per experimen- tal tubes at each dilution.Graph the numberof bacteria Temperature per ml (Y-axis)versus the -log of the bacterialdilution for and controltubes. What trends Changing the incubation temperaturealters the rate (X-axis) experimental do observein each series of dilutions? of bacterialkilling. Examine a range of temperatures you to see which kills the bacteriamost efficiently.Discuss . Comparingyour control and experimentalplates for Downloaded from http://online.ucpress.edu/abt/article-pdf/70/2/103/54518/30163212.pdf by guest on 28 September 2021 why this observationis physiologicallyrelevant. each dilution, what percentage of bacteria survived at each dilution? ratioof Heat/Inactivation complementkilling (Determine living cells on experimentalversus controlplate). Show on Manytypes of mammaliantissue culturemedia rely your work. heat-inactivatedserum as a nutrientsource. o Incubating observationsand the class the serumat 56 C forone hourinactivates the . Based on your pooled data, comple- results If what ment in the serum. To demonstrateto students the did the support your hypothesis? not, observationsdid not your hypothesis and how heat-sensitivenature of proteinsand the principlethat support do explain them? protein activity can be inactivatedby heat (among you other things), have your student groups pre-incubate . Was there complete killing of all the bacteriafrom the aliquots of their serum at differentindividual tem- undilutedbacteria tube and the firstdilution of bacteria? peratures(and for differenttimes) to see the effect on complementactivity. Observe what happens when serumis exposed to increasingheat priorto incubation with the bacteria.Discuss and test otherways that one could inactivatethe proteinsin the complement. Grampositive vs. gram-negativebacteria The DNA Store To examine the efficiency of complement killing in differentscenarios, use differenttypes of bacteriain the experiments.Which types of bacteriawere most efficiently killed by serum? Were there differences betweenthe killingof gram-negativeand gram-positive bacteria?Why? LPSquenching LPS,a majorcomponent of the bacterialcell wall and a majorcontributor to human sepsis, can be purchased and reconstituted.What happens when LPSis 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 the increasesthe amount of LPSadded to a fixed number of cells? Discuss and test what happens when other bacterialand fungalserum-binding proteins are added to the incubationreaction. Protein size Dialysis tubing and spin columns are availablethat allow one to removeproteins below certainmolecular thresholds.Allow students to remove weight (kD) pro- DNA ITEMS: DNA teins below certain thresholds and determinewhich Including toys, pins, magnets,lamps ties, jewelry,models - if you can think it, we've got it. fraction(s) contains complement as judged by lytic ability.What molecularweight range contains the lytic http://www.thednastore.com ability?What happens if we combine fractions?Does

COMPLEMENT-MEDIATEDCELLLYSIS 107 If not, explainthat observation. Nakagawa, T., Ma, B.Y., Uemura, K., Oka, S., Kawasaki, N. & Kawasaki,T. (2003). Role of MBP,in . What did this experimentteach you about the innate mannan-bindingprotein, innate immunity.Anticancer Research, 23(6a), 4467-71. immune system and specifically about complement lysis of bacteria?Consider speed and efficiencyin your Paul, W. (2003). Fundamental .Philadelphia, PA: answer. Lippincott,Williams, and Wilkens. . Would this be an example of a pre-existingimmune Sjoholm,A.G., Jonsson, G., Broconier,J.H., Sturfelt, G. & Truedsson, responseor an adaptiveresponse? Why? L. (2006). and disease: An update (Review).Molecular Immunology, 43(1-2), 78-85. . Why is the observationof complementbacterial lysis an importantphenomenon? Speth, C., Rambach,G., Lass-Florl,C., Dierich,M.P. & Wurzner,R. (2004). The role of complementin invasivefungal Overall, students are successfulin this my accomplishing (Review).Mycoses, 47(3-4), 93-103. lab and areimpressed by the dramaticresults. The "flowchart" of the lab shown in Figure 1 gives an indispensableoverview Sturfelt,G. & Truedsson,L. (2005). Complementand its breakdown of how this lab operates.Students enjoy the compilationand products in SLE(Review). Rheumatology, 44(10), 1227-32. discussion of the class data. The fact that the bacteriain the Sunyer,J.0., Boshra,H. & Li,J. (2005). Evolutionof , firstdilution are not alwayscompletely killed gives the students their diversityand novel roles in innateimmunity: Insights from pause but also underscoresthe limitsof any system. the study of fish complement (Review).Veterinary Immunology & Immunopathology,108(1-2), 77-89. Conclusion Vandermeer,J., Sha, Q., Lane,A.P. & Schleimer,R.P. (2004). Innate Downloaded from http://online.ucpress.edu/abt/article-pdf/70/2/103/54518/30163212.pdf by guest on 28 September 2021 immunity of the sinonasal cavity: Expression of messenger. The innate immune neutralizes RNA for complement cascade components and toll-likerecep- system effectively many - pathogensbefore the adaptiveimmune response is triggered. tors.Archives of OtolaryngologyHead & NeckSurgery, 130(12), Thisstraightforward and highly adaptable lab exerciseunambig- 1374-80. demonstratesthe of serum uously power complementspecifi- von Lackum, K., Miller,J.C., Bykowski,T., Riley, S.P., Woodman, callyand the innateimmune system generally. We have used it M.E.,Brade, V., Kraiczy,P., Stevenson,B. & Wallich,R. (2005). repeatedlywith success and to the amazementof our students. Borreliaburgdorferi regulates expression of complement regula- surface 1 the mammal-tickinfec- It is a usefuladdition to a classwhen placedbefore a discus- tor-acquiring protein during tion & 7398-405. sion of the adaptiveimmune system as it foreshadowsconcepts cycle. Immunity,73(11), such as antibody,, and B cells, and reinforcesthe Wood, P. (2006). UnderstandingImmunology. Essex, England: conceptthat the innateimmune system acts so thatthe adaptive PearsonEducators Ltd. immune has to workless. system Worthley, D.L., Bardy,P.G. & Mullighan, C.G. (2005). Mannose- binding lectin: Biology and clinical implications. Internal References MedicineJournal, 35(9), 548-55. Zhang,M.X., Brandhorst, T.T., Kozel, T.R. & Klein,B.S. (2001). Role Abbas,A. & Lichtman,A. (2005). Cellularand MolecularImmunology, of glucan and surface protein BAD1in complement activation UpdatedEdition. Philadelphia, PA: W. B. Saunders. by Blastomycesdermatitidis yeast. Infection& Immunity,69(12), 7559-64. Agramonte-Hevia,J., Gonzalez-Arenas,A., Barrera,D. & Velasco- Velazquez,M. (2002). Gram-negativebacteria and phagocytic cell interaction mediated by 3. FEMS Other Immunology& MedicalMicrobiology, 34(4), 255-66. Reading Borsos, T. & Rapp, HJ. (1970). TheMolecular Basis of Complement Doffinger,R., Patel,S. & Kumaranratne,D.S. (2005). Human immu- Action.New York,New York:Appleton CenturyCrafts. nodeficiencies that predispose to intracellularbacterial infec- tions. Current in 440-6. Heine, H. & Ulmer, AT (2005). Recognition of bacterialproducts Opinion Rheumatology,17(4), by toll-likereceptors (Review). ChemicalImmunology & , Holmskov, U., Thiel, S. & Jensenius, J.C. (2003). Collections and 86, 99-119. :Humoral of the innate immune defense.Annual Review 547-78. Inal, M. (2004). Parasiteinteraction with host complement:Beyond of Immunology,21, attack Trendsin 407- regulation (Review). Parasitology,20(9), Hourcade, D., Holers, V.M. & Atkinson, J.P. (1989). The regula- 12. tors of complement activation(RCA) gene cluster.Advances in 381-416. Kawasaki,A., Takada,H., Kotani,S., Inai, S., Nagaki,K., Matsumoto, Immunology,45, M., K., Kawata, S., Kusumoto, S. & T. Yokogawa, Shiba, Kilpatrick,D.C. (2002). Mannan-bindinglectin and its role in innate Activation of the human cascade (1987). complement by immunity.Transfusion Medicine, 12(6), 335-52. bacterial cell walls, peptidoglycans,water-soluble peptidogly- can components, and synthetic muramylpeptides-studieson Lydyard,P.M., Whelan. A. & Fanger,M.W. (2000). InstantNotes in active components and structuralrequirements. Microbiology Immunology.New York,New York:Springer-Verlag. & Immunology,31(6), 551-69. Mayer, M.M. (1967). Complement and complement fixation. In Ma, Y.G.,Cho, M.Y.,Zhao, M., Park,J.W., Matsushita, M., Fujita,T. E.A. Kabat, M.M. Mayer & C. Charles (Eds.), Experimental & Lee, B.L.(2004). Humanmannose-binding lectin and L- Immunochemistry.Springfield, IL: Thomas Publishing. function as specific pattern recognition proteins in the Roitt, I.M. & Delves, P.J.(2001). Roitt'sEssential 10th lectin activationpathway of complement.Journal of Immunology, Biological Edition.Malden, MA: Blackwell Science. Chemistry,279(24), 25307-12.

108 THEAMERICAN BIOLOGY TEACHER, VOLUME 70,NO. 2, FEBRUARY2008