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Year : 2002  |  Volume : 20  |  Issue : 4  |  Page : 187-193

Modulatory effects of salmonella lap-lps on murine macrophages

Department of Microbiology, Punjab University, Chandigarh - 160 014, India

Correspondence Address:
Department of Microbiology, Punjab University, Chandigarh - 160 014, India

 ~ Abstract 

PURPOSE: To study the modulatory effects of Salmonella lipid associated protein - lipopolysaccharides (LAP-LPS) on murine macrophages as the intracellular survival within the host macrophages is an important feature for a number of gram-negative pathogens like S.typhi. METHODS: Macrophage functions were studied in two groups of mice immunized with either LPS or LAP-LPS. RESULTS: Comparison of protective efficacy of mice preimmunized with LPS based preparations, against challenge infectious doses, showed higher protection in LAP-LPS complex immunized mice group as compared to the mice group immunized with LPS alone. Aggregation of S.typhi cells was lesser with intestinal mucus extracted from LAP-LPS immunized mice as compared to LPS immunized challenged group. A significant increase in the number of macrophages in LAP-LPS immunized mice was also observed in comparison to control and LPS immunized mice groups. Nitric oxide (NO) and superoxide dismutase (SOD) production were also more in macrophages derived from LAP-LPS immunized mice group. Phagocytic uptake studies showed that there was enhanced uptake of bacteria in the LAP-LPS immunized animals in comparison to LPS immunized and controls. Similar trend was observed in intracellular killing of bacteria by the macrophages. CONCLUSIONS: The results indicated the involvement of protein moiety in LAP on modulation of effects of LPS on macrophages.

How to cite this article:
Rishi P, Batra N, Sood S, Tiwari R P. Modulatory effects of salmonella lap-lps on murine macrophages. Indian J Med Microbiol 2002;20:187-93

How to cite this URL:
Rishi P, Batra N, Sood S, Tiwari R P. Modulatory effects of salmonella lap-lps on murine macrophages. Indian J Med Microbiol [serial online] 2002 [cited 2020 Oct 28];20:187-93. Available from:

One of the major public health problems, throughout the world is the bacterial enteric infection, which still causes considerable morbidity and mortality. Among these, the ones caused by  Salmonella More Detailse are most frequent and serious.[1],[2]  Salmonella More Detailse infect a variety of hosts and cause broad spectrum of diseases, ranging from acute self-limiting diarrhea to bacteraemia and enteric fevers.
One of the most hostile environments, encountered by  Salmonella More Detailse, once they enter the host, is the intracellular environment of the macrophages.[3] It has been proposed that the survival of the pathogen within the macrophage is an essential step in the virulence of the pathogen.[4] In view of the fact that the surface of the gram negative organisms encounters the host, outer structures have been reported to play a role in pathogenesis. These have also been considered important antigens against which the host immune response is directed.[5],[6],[7],[8] Hence, various antigens including Vi polysaccharide and lipopolysaccharides alone or conjugated to proteins have been evaluated in the past for their potential as vaccines.[9],[10]
Though the modulatory effects of LPS, on macrophages have been reported in the presence of exogenously added proteins[11] the information regarding the alterations in macrophage functions by naturally occurring lipid A associated protein LPS complex (LAP-LPS) is still incomplete. Knowing that LPS is tightly bound to the native outer membrane components, we examined the modulatory effects of LAP-LPS and LPS on macrophages derived from infected mice and compared with preimmune challenged mice groups.

 ~ Materials and Methods Top

Balb/c mice weighing 17-22 grams (6-8 weeks old) were procured from the central animal house, Punjab University, Chandigarh. These were kept in clean, well ventilated rooms and given food and water ad libitum.
Bacterial Culture
 Salmonella More Details typhi Ty2 was procured from Central Research Institute, Kasauli, India and was maintained in nutrient agar stabs at 4oC. The strain was checked biochemically and serologically prior to storage and use.
Extraction of immunogens
For the extraction of the immunogens (i.e. the LAP-LPS complex and LPS), two procedures were tried. The conventional hot phenol extraction method of Westpal and Jann[12] as modified by Morrison and Leive[13] and the aqueous butanol extraction method given by the same workers. Phenol method was selected for further preparation of LPS based on better LPS yield.
Pure LPS and LAP-LPS preparations were electrophoresed on 12% linear sodium dodecyl sulphate polyacrylamide (SDS) slab gels by the procedure described by Laemmli.[14] For detection of LPS and proteins in the preparation, modified silver staining and Coomassie blue procedures were employed, respectively. The standard protein marker (14-97 kDa) and LPS (Bacto Laboratories, Difco Company) were also used for comparison.
Immunization of mice
Mice in different groups of six each were injected LPS based preparations to find the immunizing dose of each preparation. Based on the observations, mice were grouped into four groups consisting of six mice in each group for immunoprotection studies. (i) Group I- control group in which the mice were injected with normal saline, (ii) Group II - infected group, (iii) Group III - immunized intraperitoneally (i.p.) with 50 mg of LAP - LPS per mouse, and (iv) Group IV - immunized with 3 mg of pure LPS.
LD50 of S.typhi Ty2 in mice was determined using 5% hog gastric mucin. LD50 and challenge dose was calculated by the method of Reed and Muench.[15] Mice were challenged 28 days after the immunization dose and were then sacrificed after 72 hours of challenge to study various parameters.
Aggregation of S.typhi
This assay was carried out as described by Ensgarber and Loss.[16] 50 mL of bacterial suspension (cultured to log phase) was sedimented after low speed centrifugation. The pellet obtained was suspended in 50 mL of mucus solution obtained from intestines of different groups of mice. Bacterial pellet incubated with saline was used as a control. Immediately and then at 30, 60, 90 min. of incubation, 5 mL of the sample was taken for determining the number of aggregated and non-aggregated bacteria under microscope.
Macrophage studies
Isolation of macrophages from the mice of four groups was done from the peritoneal cavity using the RPMI - 1640 medium. Their number was estimated by mixing equal amounts of macrophage cell suspension and 0.1% trypan blue and taking 10 mL of this mixture on a haemocytometer.
For determining the levels of different enzymes in the macrophages isolated from the various groups of mice, the peritoneal macrophages were first sputtered by sonication (3 cycles of 15 seconds each with 30 seconds interval between 2 cycles). The disrupted cells were removed by centrifugation (3400 rpm, 10 min) and the cell free supernatant was preserved for various assays.
Nitric oxide assay
The amount of nitric oxide (NO) in the cell free supernatant was determined by Griess reaction, as described by Green et al.[17] Briefly, 100 mL aliquots of supernatant mixed with equal volume of Griess reagent (1% sulfanilamide, 0.1% napthylethylenediamine dihydrochloride, 2% H3PO4) was incubated at room temperature for 10 minutes after which the optical density was measured at 550 nm. NO2 was quantitated using NaNO2 as standard.
Superoxide dismutase estimation
Levels of SOD in the cell free supernatant was measured by the method of Kono.[18] Briefly, 1.3 mL of solution A (0.1 nM EDTA containing 50 mM Na2CO3, pH 10.5), 0.5 mL of solution B (90 M NBT - nitro blue tetrazolium dye) and 0.1 mL of solution C (0.6% TritonX-100 in solution A), 0.1 mL of solution D (20 mM Hydroxylamine hydrochloride, pH 6.0) were mixed and the rate of NBT reduction was recorded for one minute at 560 nm. 20 mL of the supernatant was added to the test cuvette as well as reference cuvette, which do not contain solution D. Finally, the percentage inhibition in the rate of reduction of NBT was recorded as described above. One enzyme unit was expressed as inverse of the amount of protein (mg) required inhibiting the reduction rate by 50% in one minute.
Acid phosphatase estimation
For the measurement of AP in the supernatant, the method of Linhart and Walte[19] was employed. Briefly, 1.0 mL of assay volume containing 0.9 mL of 1.0 M acetate buffer (pH) 4.8, 0.05 mL of p-nitrophenyl-phosphate (PNPP) and 0.05 mL of cell free supernatant, was incubated for 30 minutes at 37oC. Reaction was terminated by addition of 0.4 N NaOH. Yellow color developed was read at 405 nm. One unit of enzyme was expressed as the amount of p-nitrophenol (mol) liberated in 30 minutes at 37oC.
Phagocytic uptake and intracellular killing assay
Uptake of bacteria and intracellular killing by macrophages was assessed by the method described by us earlier.[20]

 ~ Results Top

Out of the two methods used to isolate the immunogens, the hot phenol extraction method was finally used, as it resulted in better yield of LPS estimated by the KDO (2-keto-3-deoxyoctonate) method. This yield was 0.13 mg/mL as compared to the 0.04 mg/mL in case of aqueous butanol-1 method.
In the silver stained gel, the LPS portion of the LAP-LPS complex demonstrated an electrophoretic mobility pattern similar to that of the protein free LPS preparation, however the number and clarity of bands appearing in lane 1 was better. Both antigens demonstrated the characteristic ladder like structure of  Salmonella More Details LPS, which results from the sequential additions of oligosaccharide O antigen subunits to the LPS molecule [Figure - 1]. However, on staining the gel with Coomassie blue, proteins could only be detected in the LAP-LPS preparation and not in the pure LPS preparation. The molecular weights of the proteins in the LAP-LPS preparation are in the range of 14-43 kDa.
The lethality data for various LPS preparations has been shown in [Table - 1]. Variations were observed in the immunoprotection among various groups to challenge infectious dose. Mice in control group did not show any protection. Immunoprotection was 100% in mice immunized either with 50 mg of LAP-LPS antigen (containing 18 mg LPS and 32 mg of protein) or with 3 mg of protein free LPS (data indicated in text only).
More aggregation of the S.typhi Ty2 cells with the intestinal mucus extracted from the infected group of mice was found by 90 minutes as compared to LAP-LPS immunized group [Figure - 2], [Figure:3].
Mice from the two groups giving 100% protection and those from the infected group were scarified after three days of infection, along with those from the control group, to obtain the peritoneal macrophages. The number of macrophages and their protein content showed a significant increase (p<0.001) in the LAP-LPS immunized group as compared to the control group of mice. In case of the protein free LPS immunized group, the number was less than that in the LAP-LPS immunized group and the infected group but it was still higher than in the control group [Table - 2].
A significant increase in the production of nitric oxide was observed in macrophages from the infected as compared to that in the control group. The nitric oxide production was still higher in the LAP-LPS and LPS immunized groups as compared to the control and the infected group (p<0.01). However, the increase was more significant in comparison to the control group. Between the two immunized groups themselves, the difference was not statistically significant, though LAP-LPS immunized group showed an increased nitric oxide production as compared to LPS immunized group [Table - 3].
The superoxide dismutase level was also found to be high in the infected group as compared to the control group. However, the levels were significantly higher in the LAP-LPS and the protein free LPS immunized groups as compared to the control group (p<0.01). The SOD levels were also found to be significantly higher in the LAP-LPS immunized group as compared to the protein free LPS. Both, infected as well as immunized mice showed a decrease in the levels of acid phosphatase (AP) but the decrease was more pronounced in immunized group (p<0.001). The decrease in the level of AP was more in the LAP-LPS immunized group, though not significant compared to the LAP immunized group [Table - 3].
In phagocytic uptake studies, it was observed that within 30 minutes of exposure of S.typhi to peritoneal macrophages, there was enhanced uptake of the bacteria in the LAP-LPS immunized group as compared to the control, infected and protein free LPS immunized group. By 60 minutes the percent uptake was found to be the highest in the LAP-LPS immunized mice. With an increase in the incubation time, the percent uptake was comparable in all the four groups [Table - 4].
It was also observed that within first hour of exposure of the bacteria to the macrophages, percent killing in protein free LPS immunized group was comparable to that of the control group that was found to be the least in case of infected group. Nevertheless, thereafter, appreciable increase in percentage killing by macrophages of LAP-LPS immunized group was noticed which remained the highest upto three hours [Table - 5].

 ~ Discussion Top

The role of bacterial endotoxins (LPS) in several gram-negative infections has been well studied and it is known that the effect of LPS are modulated in the presence of serum lipoproteins.[11],[21] This aspect hence led us to investigate the modulatory effects, if any, of naturally occurring lipid A associated protein (LAP) complexes present in the outer membrane. LPS doses more than 3 mg were found to be lethal. However, LPS complexed with protein as LAP-LPS (50 mg) comprising even much higher dose of 18 mg LPS, was nonlethal for mice. This data suggested that 100% survival observed is due to the presence of protein moiety in LAP-LPS preparation. Less aggregation observed in case of immunized mice indicates that LAP-LPS may modulate the ligand receptor interaction in order to inhibit the association of S.typhi with host cells. It is possible that LAP acts as a relevant antigen in cellular or humoral immune response, which is supported by some of the reports.[9],[22],[23],[24] However, the precise mechanism by which LAP modulates this protection, remains to be elucidated.
LPS is known to be one of the classical activator of macrophages[2] and stimulates these cells to produce interleukins, TNFs, leukotrienes, prostaglandin.[25],[26] and reactive oxygen as well as nitrogen intermediates, which can regulate immune responses dramatically. Therefore, we carried out studies to compare the responses of macrophages in response to LPS and LAP-LPS complexes.
Increase in the macrophages number observed in the infected group, may be due to the presence of heavy bacterial polypeptides, which are known to be potent chemoattractants for phagocytes. Enhanced macrophage count observed in the LAP-LPS immunized than the protein free LPS immunized group could be explained by these findings, which suggests that the outer membrane proteins (of which LAP is a part) in association with LPS evoke higher level of protective immunity in mice against  Salmonellosis More Details.[27],[28]
For better understanding of immunomodulatory properties of LPS, it is crucial to elucidate the host reaction mechanism against  Salmonella More Detailse after immunization with LPS with a focus on defense oriented molecules such as reactive oxygen intermediates (ROIs) and reactive nitrogen intermediates (RNIs) produced by activated macrophages. The macrophages of the immunized group of mice showed significant up regulation of NO as compared to control and infected group. These results are supported by the studies done by Alder et al[29] who have reported that there is induction of NOS by activation with LPS and IFN- either alone or in combination. The NO produced either by itself or via reaction with O2 imposes cytotoxic effects.[30],[31] Thus, to have a more definite knowledge of O2 production, the levels of superoxide dismutase were estimated in macrophages from mice of all groups.
The higher level of SOD in the macrophages from immunized group may be due to enhanced production of O2 radicals that are produced because of host defense response.[32] LPS activated macrophages release various cytokines like IL-1, IL-6, which cause up regulation of xanthine oxidase (XO) which in turn is required for O2 dependent host mechanism against  Salmonella More Detailse[33]. This further causes higher production of O2 radicals, toxic to the pathogen. Significantly higher levels of SOD in the LAP-LPS immunized compared to LPS immunized group may be because the proteins in association with the LPS enhance the cellular and humoral immune response, which then through lymphokines release, bring about macrophages activation.
In addition to oxygen dependent mechanisms, macrophages employ non-oxidative mechanisms of bactericidal activity, which include the presence of a number of enzymes including lysozyme, acid phosphatase and glucoronidase. The lower levels of acid phosphatase observed in the immunized as compared to control and infected group may be due to the altered physiological activity of the macrophages leading to the generation of additional amount of ROIs and RNIs, thereby indicating preferential oxygen dependent killing process. ROI and RNI combine to form peroxynitrite, which is known to be more cytotoxic.
Enhanced stimulation of macrophages by the presence of LAP moiety with LPS might have resulted in the enhanced uptake of bacteria in case of LAP-LPS immunized group compared to control and LPS immunized group. Macrophages from the immunized group especially from the LAP-LPS immunized group also showed comparatively enhanced intracellular killing which may be explained on the basis of the report where it was shown that proteins elicit T-cell response and these activated T-cells confer protection by increasing the killing ability of macrophages through the release of lymphokines.[28]
Present study clearly indicates that the association of proteins (in the range of 14-43 kDa) with the LPS does have an immunomodulatory effect on macrophages in mice. 

 ~ References Top

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25.Hoffeld JT. Oxygen derived metabolites as suppressors of immune response in vitro. In: Lymphokines, volume 2. Pick E, Ed. (Academic Press Inc., New York) 1981:63-68.  Back to cited text no. 25    
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28.Gonzalez CR, Isibasi A, Ortiz-Navarrete V, Paniaqua J, Garcia JA, Blanco F, Kumata J. Lymphocyte proliferative response to OMPs isolated from Salmonella. Microbiol Immunol 1993;37:793-799.  Back to cited text no. 28    
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30.Miles AM, Bohle DS, Glassbrenner PA, Hansert B, Wink DA, Grisham MB. Modulation of superoxide dependent oxidation and hydroxylation reaction by nitric oxide. J Biol Chem 1996;271:40-47.  Back to cited text no. 30    
31.Rubbo H, Usmar DU, Freeman BA. Nitric oxide regulation of tissue free radical injury. Chem Res Toxic 1996;9:809-820.  Back to cited text no. 31    
32.Farr SB, Kogoma T. Oxidative stress in E. coli and S.typhimurium. Microbiol Rev 1991;55:561-585.  Back to cited text no. 32    
33.Umezawa K, Akaike T, Fujii S, Suga M, Setoguchi K, Ozawa A, Maeda H. Induction of nitric oxide synthesis and xanthine oxidase and their roles in the antimicrobial mechanism against Salmonella typhimurium infection in mice. Infect Immun 1997;65:2932-2940.  Back to cited text no. 33    
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