mutant mice either in the mixed c57bl/6  129 background or   pure 129 background were maintained in specific pathogen 
the surgical procedure was performed as described   ( 15 ) . in brief , mice were anesthetized by avertin ( 0.2 ml of 2.5%   solution per 10 g body wt ) , and a 0.5-cm midline incision was   made in the peritoneum .
the distal two thirds of the cecum was   ligated with a silk suture , and the cecum was punctured once   with an 18.5-gauge needle , and then gently squeezed to ensure   that the holes were completely open .
the cecum was returned to   the peritoneal cavity , and the body wall was stitched and the incision was closed with 9-mm stainless steel wound clips .
sham controls were operated on in the same manner but without ligation   and puncture . in some experiments
peritoneal lavage was harvested after injection of 3 ml of   pbs with 2% fcs and used for further assays ( see below ) . in   other experiments ,
mice were bled through the tail vein at 1.5 , 3 ,   6 , and 12 h after clp and sera were used for further assays . statistical analysis for survival
was performed using the stata program   ( stata corp . , college station , tx ) .
polyclonal igm was isolated from normal   mouse serum ( sigma chemical co. , st .
sera were precipitated by ammonium   sulfate ( 50% saturation ) , and the precipitate was dissolved in pbs   and dialyzed . recovered solution was filtered through a 0.45-m   filter and applied to a 5-ml protein g  sepharose column ( sigma   chemical co. ) to remove igg .
the flow - through was reconstituted with nacl to a final concentration of 0.5 m and applied to   a 4-ml anti - igm sepharose column ( zymed laboratories , inc .
bound protein was eluted with 0.1 m   glycine/0.15 m nacl , ph 2.5 , and neutralized with 1 m tris ,   ph 8.0 .
protein content in each fraction was determined by spectrophotometry , and positive fractions were pooled , dialyzed , and   concentrated ( amicon , inc .
leonore a.   herzenberg ( stanford university , stanford , ca ) , and 5e11 hybridoma secreting pc - specific igm was obtained from dr .
j.   latham claflin ( university of michigan medical school , ann arbor , mi [ 12 , 16 ] ) .
anti - ptc and anti - pc igm was isolated with   anti - igm affinity column from ascites produced in recombination   activating gene ( rag)-2deficient mice .
western blot   and elisa were used to assess igg contamination and degradation of igm .
all batches of purified igm had no detectable degradation and < 1% igg contamination ( data not shown ) .
endotoxin   levels in purified igm was < 200 eu / ml as determined by limulus amebocyte lysate assay ( associates of cape cod , woods hole ,   ma ) .
in igm reconstitution experiments , sigm - deficient mice   were given 0.5 mg i.v .
peritoneal lavage fluid was assayed for escherichia coli ,   neutrophils , and the levels of endotoxin ( lps ) , tnf- , and il-6 .  
e. coli colony - forming units ( cfu ) were determined by overnight culture of serial dilutions of peritoneal lavage fluid on luria   broth  agar plates at 37c .
colonies of e. coli were identified in a   background of heterogeneous colonies by morphology and confirmed by culture on macconkey ii agar and by assay with the   enteric identification system ( organon teknika , durham , nc ) .  
neutrophils in the peritoneal lavage were analyzed by flow cytometry using pe - conjugated anti - granulocyte antibody and fitc - conjugated anti - cd19 antibody ( pharmingen , san diego , ca ) .  
10 live cells were collected for each sample , and neutrophils   were identified as grcd19 . tnf- and il-6 were measured   by elisa using commercial kits ( endogen , inc . , cambridge ,   ma ; and intergen co. , purchase , ny ) .
concentrations of   tnf- and il-6 in individual samples were determined by comparison with a standard curve derived from the cytokine standard   supplied with the kits .
lps concentrations of individual samples were calculated using a standard curve derived   from the lps standard provided with the kit .
mutant mice either in the mixed c57bl/6  129 background or   pure 129 background were maintained in specific pathogen 
the surgical procedure was performed as described   ( 15 ) . in brief , mice were anesthetized by avertin ( 0.2 ml of 2.5%   solution per 10 g body wt ) , and a 0.5-cm midline incision was   made in the peritoneum .
the distal two thirds of the cecum was   ligated with a silk suture , and the cecum was punctured once   with an 18.5-gauge needle , and then gently squeezed to ensure   that the holes were completely open .
the cecum was returned to   the peritoneal cavity , and the body wall was stitched and the incision was closed with 9-mm stainless steel wound clips .
sham controls were operated on in the same manner but without ligation   and puncture . in some experiments
peritoneal lavage was harvested after injection of 3 ml of   pbs with 2% fcs and used for further assays ( see below ) . in   other experiments ,
mice were bled through the tail vein at 1.5 , 3 ,   6 , and 12 h after clp and sera were used for further assays . statistical analysis for survival
was performed using the stata program   ( stata corp . , college station , tx ) .
polyclonal igm was isolated from normal   mouse serum ( sigma chemical co. , st .
sera were precipitated by ammonium   sulfate ( 50% saturation ) , and the precipitate was dissolved in pbs   and dialyzed .
recovered solution was filtered through a 0.45-m   filter and applied to a 5-ml protein g 
the flow - through was reconstituted with nacl to a final concentration of 0.5 m and applied to   a 4-ml anti - igm sepharose column ( zymed laboratories , inc .
bound protein was eluted with 0.1 m   glycine/0.15 m nacl , ph 2.5 , and neutralized with 1 m tris ,   ph 8.0 .
protein content in each fraction was determined by spectrophotometry , and positive fractions were pooled , dialyzed , and   concentrated ( amicon , inc . , beverly , ma ) .
leonore a.   herzenberg ( stanford university , stanford , ca ) , and 5e11 hybridoma secreting pc - specific igm was obtained from dr .
j.   latham claflin ( university of michigan medical school , ann arbor , mi [ 12 , 16 ] )
. anti - ptc and anti - pc igm was isolated with   anti - igm affinity column from ascites produced in recombination   activating gene ( rag)-2deficient mice .
western blot   and elisa were used to assess igg contamination and degradation of igm .
all batches of purified igm had no detectable degradation and < 1% igg contamination ( data not shown ) .
endotoxin   levels in purified igm was < 200 eu / ml as determined by limulus amebocyte lysate assay ( associates of cape cod , woods hole ,   ma ) .
in igm reconstitution experiments , sigm - deficient mice   were given 0.5 mg i.v .
peritoneal lavage fluid was assayed for escherichia coli ,   neutrophils , and the levels of endotoxin ( lps ) , tnf- , and il-6 .  
e. coli colony - forming units ( cfu ) were determined by overnight culture of serial dilutions of peritoneal lavage fluid on luria   broth  agar plates at 37c .
colonies of e. coli were identified in a   background of heterogeneous colonies by morphology and confirmed by culture on macconkey ii agar and by assay with the   enteric identification system ( organon teknika , durham , nc ) .  
neutrophils in the peritoneal lavage were analyzed by flow cytometry using pe - conjugated anti - granulocyte antibody and fitc - conjugated anti - cd19 antibody ( pharmingen , san diego , ca ) .  
10 live cells were collected for each sample , and neutrophils   were identified as grcd19 . tnf- and il-6 were measured   by elisa using commercial kits ( endogen , inc . , cambridge ,   ma ; and intergen co. , purchase , ny ) .
concentrations of   tnf- and il-6 in individual samples were determined by comparison with a standard curve derived from the cytokine standard   supplied with the kits .
lps concentrations of individual samples were calculated using a standard curve derived   from the lps standard provided with the kit .
to examine the role of natural igm in the immediate response to microbial infection , we determined the susceptibility of sigm - deficient mice to acute septic peritonitis induced by clp that releases endogenous bacteria from the   cecum into the peritoneal cavity .
32 h after clp , 70% of   sigm - deficient mice died compared with 20% of the wild - type mice ( fig .
1 ) , indicating that the absence of natural   igm rendered mutant mice much more susceptible to the   acute bacterial infection ( p < 0.0001 ) .
the increased susceptibility of mutant mice to clp was due to the absence   of sigm , because reconstitution of mutant mice with a single dose of 0.5 mg i.v .
of total igm isolated from normal   mouse serum 4 h before clp restored their survival to the   same level as wild - type mice ( fig .
sigm - deficient mice were also more sensitive to challenge by individual species of pathogenic bacteria such as group b streptococcus .
the lethal dose to 50% ( ld50 ) animals challenged   was 10-fold lower for sigm - deficient mice than for wild - type mice ( our unpublished observations ) .
sigm - deficient   mice also showed increased incidence of spontaneous bacterial infection by opportunistic bacteria , including pasteurella pneumotropica , in specific pathogen 
these data show that natural igm is required for the protection against bacterial infection .  
resistance to clp is dependent on complement , mast   cells , and tnf- ( 1720 ) .
the susceptibility of sigm - deficient mice to clp is similar to mice deficient in complement component c3 or c4 or mast cells ( 1719 ) .
the requirement of natural igm for resistance to clp is probably   based on its ability to bind to bacteria and activate complement . to test this possibility
, we determined the immediate response by assaying the levels of tnf- , il-6 , and   lps , neutrophil infiltration , and bacterial load in the peritoneal lavage 3 h after clp .
as in c3-deficient mice , the   levels of tnf- and il-6 in mutant mice were approximately half those in wild - type mice ( table 1 ) . without   clp , both sigm - deficient and wild - type mice had very   few neutrophils in the peritoneum ( < 1% ) and similar numbers of cells in the lavage after clp ( data not shown ) .   again , as in c3-deficient mice , the percentage of neutrophils in the peritoneal lavage recovered from sigm - deficient mice was significantly reduced compared with that   from wild - type mice ( 55 vs. 82% ; table 1 , and data not   shown ) .
furthermore , 10 times more e. coli were recovered from the peritoneal lavage of sigm - deficient mice   than from wild - type mice ( table 1 ) .
associated with the   higher bacterial load , approximately twice the amount of   endotoxin ( lps ) was detected in sigm - deficient mice as in   wild - type mice .
reconstitution of sigm - deficient mice   with total igm restored the levels of tnf- and neutrophils and reduced e. coli load in the peritoneal lavage ( table 1 ) , consistent with the increased survival .
these data   show that the effects resulting from the absence of sigm on   the induction of tnf- , neutrophil infiltration , and bacterial load in the peritoneum are very similar to those seen in   the absence of c3 , indicating that natural igm functions   through the complement pathway .  
a single bound igm molecule is sufficient   to activate complement to lyse a red blood cell ( 21 ) .
binding of natural igm to bacteria immediately after infection   likely results in the activation of complement through the   classical pathway .
since serum from sigm - deficient mice   lysed antibody - opsonized red blood cells just as efficiently   as serum from wild - type mice in a hemolytic assay ( data   not shown ) , the increased susceptibility of sigm - deficient   mice to clp is probably associated with the absence of   igm - mediated complement activation .
c3- or c4-deficient mice appear to be even more sensitive to clp than   sigm - deficient mice as indicated by 100% mortality within   24 h ( 17 )
. this may be because complement can also be activated through the alternative and lectin pathways , and   complement is important in the efficient clearance of bacteria .
in addition , sigm - deficient mice have relatively normal levels of iggs ( 13 ) , some of which are probably natural   antibodies .
although the igg proteins may contribute to   the survival of sigm - deficient mice , they are clearly not   sufficient to compensate fully the absence of natural igm .
uncontrolled bacterial infection in the peritoneum leads   to a fatal systemic infection and inflammation . to determine the systemic inflammatory response to clp in the absence of natural igm
, we collected serum from both sigm - deficient and wild - type mice at 1.5 , 3 , 6 , and 12 h after   clp and assayed for the levels of lps , tnf- , and il-6 .  
we divided the sera into four groups based on the genotype of the mice and whether the mice died or survived at   32 h after clp . as shown in fig .
2 , the levels of serum lps   were similar to those of sham controls 3 h after clp but   significantly higher at 6 and 12 h after clp . among the   four groups of mice , sigm - deficient mice that died had a   significantly higher level of lps by 6 h after clp , consistent with previous findings indicating that natural igm is   involved in the clearance of lps ( 22 ) .
similarly , sigm - deficient mice that died had a significantly elevated level of   tnf- 3 h after clp ( fig .
although the initial local   release of tnf- is beneficial to the containment of bacterial infection , systemic release of tnf- is usually associated with wide - spread inflammation .
the level of proinflammatory cytokine il-6 was also elevated in the serum 6   and 12 h after clp in sigm - deficient mice as well as in   wild - type mice that died ( fig .
il-6 is a major cytokine   that induces the production of acute phase proteins , such as   c - reactive protein , that are thought to have similar functions as natural antibodies ( 2325 ) .
the similar levels of   serum il-6 in both sigm - deficient and wild - type mice suggest that acute phase proteins were similarly elevated in   both types of mice .
thus , natural igm appears to have   some unique functions in the immediate response against   bacterial infection that can not be replaced by acute phase   proteins . in the absence of natural igm
reconstitution of sigm - deficient mice with polyclonal   igm from normal mouse serum restored their resistance to   clp ( fig .
1 ) . therefore , it was of obvious interest to test   whether the resistance can be restored by reconstitution   with monoclonal igm specific to ptc or pc .
ptc is a common membrane component and anti - ptc is the most   widely expressed specificity by natural igm ( 11 , 12 ) .
pc is   chemically related to ptc and is found as a determinant of   the pneumococcal cell wall ( 26 ) .
anti - pc igm has been   shown to confer protection against certain types of streptococcus pneumoniae ( 27 , 28 ) .
3 h after clp , mice were killed and peritoneal   lavage was harvested to assay for the levels of tnf- and   lps , neutrophil infiltration , and bacterial load . as in reconstitution with polyclonal igm , reconstitution with
anti - ptc   igm resulted in elevated levels of tnf- and neutrophils   and a reduced e. coli load and lps in the peritoneal lavage   ( table 1 ) .
the difference between the two monoclonal igm preparations was probably due to their differences in specificity , because both were > 98% pure , had   minimal endotoxin contamination , and reconstituted to a   similar level as assayed by elisa of serum taken 3 h after   clp ( data not shown ) .
the resistance conferred by anti - ptc igm was clearly evident but it appeared not to be as   effective as polyclonal igm , probably because polyclonal   igm bind bacteria through many different antigenic determinants . that anti - ptc igm had a clear effect suggests that   it is involved in the immediate defense against bacterial infection under physiological conditions .
activation of complement is essential for the initial containment of systemic bacterial infection as demonstrated by   the susceptibility of complement - deficient humans , guinea   pigs , and mice to bacterial infection ( 17 , 2931 ) .
c3a   and c5a peptides function as chemoattractants to recruit   leukocytes to the site of infection .
opsonization of bacteria   by c3b and ic3b promotes efficient phagocytosis by neutrophils . in addition
, complement is required for the efficient activation of mast cells to release tnf- for the initiation of a local inflammatory response ( 17 ) .
however , the   optimal clearance of bacteria by complement requires the   presence of natural antibodies .
gram - positive bacteria are   generally resistant to complement - mediated lysis because   the thick peptidoglycan layer in their cell wall prevents insertion of the membrane - attack complex ( 32 ) .
although   gram - negative bacteria are effectively lysed by complement , these bacteria contain lps in their cell walls which   when released in substantial amount can be fatal within   hours ( 33 , 34 ) .
furthermore , sialic acids of the pneumococcal capsular polysaccharides can inhibit the activation of   the alternative complement pathway ( 35 ) .
the capsular   polysaccharides also inhibit phagocytosis by macrophages   and neutrophils ( 36 ) .
effective responses to bacterial infection require antibodies to opsonize bacteria for efficient   phagocytosis and to neutralize the released endotoxin ( 22 ) .   what is the source of antibodies before the onset of a specific   humoral response to a particular infectious agent ?
natural   igm is ideally suited for these purposes because of its polyreactivities and the exquisite ability to activate complement ,   and because normally it is already present . the susceptibility   of sigm - deficient mice to clp and restoration of resistance   by reconstitution with polyclonal igm and to a lesser extent   by anti - ptc igm clearly demonstrate a critical role of natural   igm in the immediate defense against bacterial infection
sigm - deficient   ( / ) and wild - type ( + /+ )   mice at 6 - 8 wk of age were subject to clp .
igm - reconstituted   sigm - deficient mice ( / igm )   were given as a single dose of 0.5   mg i.v .
mice   were monitored for survival   within the first 32 h. analyses of peritoneal lavage 3 h after clp   sigm - deficient mice ( / ) , wild - type mice ( + /+ ) , and igm - reconstituted sigm - deficient mice were subject to clp .
peritoneal lavage was carried   out by injection of 3 ml i.p . of pbs with 2% fcs 3 h after clp .
of purified polyclonal igm from normal mouse serum or monoclonal igm specific to ptc or pc 4 h before clp .
peritoneal lavages of sigm - deficient and wild - type   mice were each pooled from seven mice , and the levels of tnf- , il-6 , and lps , e. coli counts , and neutrophils were assayed ( see materials and   methods ) .
the levels of tnf- and lps , e. coli counts , and neutrophils were assayed in peritoneal lavage from individual igm - reconstituted mice .  
comparison of the   levels of lps , tnf- , and il-6   in the serum of sigm - deficient   and wild - type mice at different   time points after clp .
sera were   collected at 1.5 , 3 , 6 , and 12 h   after clp and divided into four   groups based on the genotype of   the mice and whether the mice   died or survived at 32 h after   clp .
, the numbers of   mice used in each category are as   follows : / dead , 12 ; /   survived , 10 ; + /+ dead , 10 ; and   + /+ survived , 16 .
numbers of sham controls for   tnf- , il-6 , and lps were 2 , 1 ,   and 1 , respectively .
 to evaluate the role of natural immunoglobulin ( ig)m in the immediate response against microbial infection , we tested mutant mice that are deficient in secreted ( s)igm in an acute peritonitis model induced by cecal ligation and puncture ( clp ) . 
 20% of wild - type mice died   within 32 h of clp , whereas 70% of sigm - deficient mice died within the same time period .   
 the increased susceptibility was associated with a reduced level of tumor necrosis factor   ( tnf)- , a decreased neutrophil recruitment and an increased bacterial load in the peritoneum , and elevated levels of endotoxin and proinflammatory cytokines in the circulation . 
 resistance to clp by sigm - deficient mice was restored by reconstitution with polyclonal igm   from normal mouse serum . 
 reconstitution with a monoclonal igm specific to phosphatidylcholine , a conserved cell membrane component , has a modest effect but a monoclonal igm   specific to phosphocholine is not protective . 
 these findings demonstrate a critical role of natural igm in the immediate defense against severe bacterial infection . 
Materials and Methods
Mice.
CLP.
IgM Purification.
Assays.
Results and Discussion
Figures and Tables