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Immune competence and diabetes mellitus
JOURNAL
OF SURGICAL
RESEARCH
Immune
26,
199-205 (1979)
Competence
and Diabetes
II. Experimental
Mouse Studies
MARK A. MANDEL, Department
of Surgery, University
Division Hospitals
Mellitus
M.D.
of Plastic Surgery, Case Western Reserve University Medical of Cleveland, 2065 Adelbert Road, Cleveland, Ohio 44106
School
and
Submitted for publication June 20, 1978 An experimental model utilizing congenitally diabetic mice was established to answer the question whether such animals have altered immune responses and ability to ward off infections. Cellular immunity was suppressed in diabetic mice when the parameters of granuloma formation, altograft rejection, and delayed footpad swelling were tested. Spleen cells from diabetic mice grown in tissue culture had an impaired mitogenic response compared to those from control animals. In addition, when diabetic mice were inoculated with the common human intraoral organisms, B-hemolytic streptococcus and Staphylococcus aareas, they could not contain the infection as well as normal littermates. Thus, immune function and infectious control mechanisms appear to be deficient in diabetic mice.
MATERIALS
INTRODUCTION
AND METHODS
The finding that patients with diabetes Animals mellitus, latent or overt, appear to have a Genetically diabetic (C57BWKsJ-db) mice, marked proclivity to severe hand infections their littermate controls (dbl+), and Balblc [ 1 l] led to the present experimental study. mice were obtained from Jackson LaboraPrevious in vifro investigations have dem- tory, Bar Harbor, Maine. All mice were onstrated that altered host-parasite rela- maintained on a standard laboratory diet tionships were found in chemically induced (Ralston Purina Co., St. Louis, MO.) and diabetic mice infected with Schistosoma water ad libitum. mansoni; the diabetic animals were unable Chemical Procedures to control the disease process as well as their normal counterparts [2]. The current study After an overnight fast, blood from the seeks to answer the question whether the retro-orbital plexus was drawn into heparinaltered immunity is reponsible for the high ized microhematocrit tubes and analyzed rate of infection using a congenital diabetic for glucose using the autoanaiyzer. Diabetic (dbldb) mouse experimental model. Mulanimals which were treated with insulin tiple assays of immune function including were fasted for only 4 hr before blood granuloma formation, skin allograft sur- samples were obtained. vival, delayed footpad swelling, and tritiated thymidine uptake by spleen cell Immune Assays cultures were utilized to assess immunity. Three quantitative assays of delayed hyThe ability of diabetic and normal mice to persensitivity were utilized. control infections was quantitated by their (1) Granuloma formation about Schistoinoculation with pure cultures of commonly soma mansoni eggs [17]. Groups of five mice occurring oral bacteria; the length of were used to study the effect of diabetes survival was correlated with the bacterial mellitus on the ability of the host to maintain dose. a cell-mediated granuloma response. Fifteen 199
0022-4804/79/020199-07$01.00/O Copyright All rights
0 1979 by Academic Press, Inc. of reproduction in any form reserved.
200
JOURNAL
OF SURGKAL
RESEARCH:
hundred Schistosoma mansoni eggs were suspended in 0.5 ml normal saline and injected via a tail vein into the lung microvasculature of mice. Eight and sixteen days later, groups of five mice were anesthetized, and their lungs were removed and fixed in formalin. Tissue sections (three from each lung, 5 pm thick and 250 pm apart) were stained with hematoxylin and eosin, and the granuloma area around single eggs (at least 10 per slide) was measured using a IIMC particle measurement computer system [9] (Millipore Corp., Bedford, Mass,). Approximately 50 lesions from each experimental group were measured and the significance of the difference between the groups was assessed using Student’s t test. Percentage change in the area of inflammation was calculated by subtracting the mean area of the lesions in the experimental animals (area of inflammation minus area of eggs) from that in the control animals, dividing this figure by the mean lesion size in the control mice, and multiplying by 100. (2) Delayed footpad swelling. Groups of diabetic mice (dbldb) and littermate controls (dbl+) were sensitized by the intraperitoneal injections of 1500 Schistosoma mansoni eggs. Soluble egg antigens equal to 10 pg protein in 0.03 ml phosphate-buffered saline were injected into the right hind footpad; an identical volume of buffer was injected into the left hind footpad. Twenty-four hours later the swelling was quantitated with a micrometer (L&in Rule Co., Saginaw, Mich.). The mean difference in thickness between the right and left footpads was recorded as the net swelling. (3) Skin allograft survival. Grafting of Balb/c (H-2b) skin onto dbldb mice and dbl+ (H-2”) controls was done using standard techniques [lo]. Casts were removed on the eighth day and graft survival was recorded daily using criteria established by Monaco et al. [12]. Four weeks after rejection of the first grafts, second grafts were applied onto the test mice and their survival was quantitated.
VOL.
26, NO,
13H]Thymidine
2, FEBRUARY
1979
Uptake by Spleen Cells
Spleens were harvested from dbldb and dbl+ mice. Cell suspensions were cultured with various concentrations of the non-specific T-cell mitogen, concanavalin A [ 141. After a 60-hr incubation at 37°C 0.5 &i of [3H]thymidine (Searle Analytics, Inc., Des Plaines, Ill.) was added to each well. Twelve hours later the cultures were harvested, the radioactivity was counted, and the results were expressed as mean counts per minute. Insulin (Isophane Insulin suspension USP, M-40, E.R. Squibb and Sons, New York) was administered in a daily dose of 3 units by subcutaneous inoculation for 4 days to groups of dbldb mice. Control mice received subcutaneous injections of citrate buffer. Blood sugar levels were determined after a 4-hr fast, and then the animals were sacrificed and their spleens used in the [3H]thymidine uptake studies. A control group consisted of spleen cell suspensions from six untreated dbldb mice that were cultured in the presence of insulin (100 punits/ml). Control of Induced infections and Normal Mice
of Diabetic
The ability of diabetic and normal mice to combat pyogenic infections was assessed by the intraperitoneal inoculation of bacteria and quantitation of mean length of animal survival, The mice received a wide dose range of either Staphylococcus aureus (coagulase +), or B-hemolytic streptococci (Lancefield, Group A); these are commonly occurring human intraoral pathogenic organisms [5, 8, 161. Pure bacterial cultures were obtained from the Microbiology Department (Case Western Reserve University Medical School). Bacterial counts were made using standard techniques [B, 161, and then doses ranging from lo5 to IO9 bacteria were injected intraperitoneally into each test animal. The death 50% endpoint (LD5,,) was computed by the method of Lennette [6], and the mean animal survival quantitated.
MARK A. MANDEL:
IMMUNE
COMPETENCE
an increase (P < 0.001).
RESULTS
Granuloma
AND DIABETES
Formation
of
only
201
MELLITUS
0.009 + 0.001
ml
Uptake by Spleen Cells Fasting blood glucose (FBS) levels in dbl i3H]Thymidine db mice and their littermate controls, dbl+ , The ability of spleen cells from dbl+ and were 481 ? 27 and 97 ? 5 mg/lOO ml, re- dbldb mice to respond to the mitogenic agent spectively (Table 1). Minimal variation in concanavalin A is outlined in Table 2. Spleen the FBS occurred when the schistosome cells from diabetic mice consistently failed eggs, utilized in the immune assay, were in- to respond to this mitogenic agent or to phyjected. Determination of granuloma size at tohemagglutinin; this indicates inhibition in 8 days revealed a mean of 35.4 pm2 in conDNA synthesis by these cells. Prior treattrol mice, with marked suppression in inment of the mice, in vivo, with insulin or flammation noted in diabetic mice (P by the addition of insulin to the cultures < 0.001). Representative granulomas are in vitro did not change the ability of these shown in Fig. 1. cells to synthesize DNA following mitogenic stimulation. The data suggest, but do not Skin Allograft Survival confirm, that the defect in insulin utilization Allografts from Balb/c mice on dbl+ con- in congenitally diabetic mice is probably at trols survived for 9.7 -t 0.3 days. Significant the peripheral cellular level. prolongation of graft survival (17.8 ? 1.2 days) was noted on the dbldb mice (P Infection Control < 0.001). When second-set grafts were apBacterial inoculations into normal (dbl+) plied they survived for 6.0 & 0.3 days in mice were performed over a wide dose range control mice and 10.1 2 0.5 days in the diafor both Staphylococcus aureus ( 106- log) betic mice (P < 0.01). and B-hemolytic streptococci (5 x 105l(Y). These organisms were chosen since Delayed Footpad Swelling they are frequent pathogens in human oral The injection of soluble antigens into the bacterial flora [5,16]. The initial experiment, footpads of the mice following sensitization outlined in Table 3, serves to establish the level for the diabetic (dbldb) animal testing. is a direct correlate of delayed hypersensitivity. The control mice (dbl+) showed a A marked difference in survival is seen in net increase in swelling of 0.038 & 0.009 ml, Fig. 2 when diabetic and normal mice are while the diabetic mice (dbldb) showed inoculated with either staphylococci (5 TABLE GRANULOMA
FORMATION
IN CONGENITALLY
1 DIABETIC
MICE
(CS7BLiKsJdb)”
Fasting blood glucose (mg/lOO ml + SE) Experimental dbl+ dbldb
group
Day 0
Day 8
97 + 5
110 5 5
481 + 27
454 k 28
Mean granuloma area (wm2 + SE x lOa)
Change in percentage area of inflammation
35.4 c 3.4 10.8 2 1.0
-77
0
a Congenitally diabetic mice (dbldb) and their littermate controls (db/+) were injected with 1500 S. munsoni eggs. The fasting blood glucose levels in the mice are noted. At 8 days the granuloma areas were calculated, and averaged, for at least 50 lesions. The change in percentage area of inflammation is noted.
202
JOURNAL
OF SURGICAL
RESEARCH:
VOL.
26, NO.
2, FEBRUARY
1979
MARK
A. MANDEL:
IMMUNE
COMPETENCE
x lo7 bacteria), or B-streptococci (lo6 bacteria). This indicates the inability of diabetic mice to control the infectious process as well as normal control mice (P < 0.01).
A relationship between altered immunity, diabetes mellitus, and infection control was suggested by the clinical study of hand infections [ 111. An experimental model, using congenitally diabetic mice, was established to study whether the above parameters are related. Marked suppression in cellular immunity was found in congenitally diabetic mice using three quantitative assays of delayed hypersensitivity, namely, granuloma formation around parasite eggs, skin allograft rejection, and footpad swelling. These findings are similar to those previously reported in mice rendered diabetic by either streptozotocin or alloxan [9], chemical agents that destroy the pancreatic p cells and lead to insulin deficiency. Tissure culture studies, using the T-cell mitogen concanavalin A, revealed that spleen cells from congenitally diabetic mice
Trssu~
2
CULTURE STUDIES: TRITIATED THYMIDINE UPTAKE IN SPLEEN CELLS’ Concanavalin
Experimental grow dbldb dblt dbldb Insulin in vivo dbldb Insulin in vitro
A concentration 1
(m&f)
0
0.1
0.3
3
NR” NR
NR NR
NR 3,000
NR 20,000
NR 14,000
NR
NR
NR
NR
NR
NR
NR
NR
NR
NR
n Spleen cells from congenitally diabetic mice (dbidb) and their littermate controls (dbl+) were treated in tissue culture with different concentrations of the mitogenie agent, concanavalin A. The effect of treating the dbidb mice in vivo with insulin for 4 days prior to the assay and of adding insulin to the culture media is shown. b Values expressed as cpm. Values less than 2000 cpm were scored as no response (NR).
DIABETES
203
MELLITUS TABLE
3
SURVIVAL OF NORMAL MICE INOCULATED WITH DIFFERENT DOSES OF BACTERIAL
Bacteria
DISCUSSION
TABLE
AND
Number of organisms
Mean
survival (days)
Staphylococcus aureus
109 5 x 10s 108 5 x 10’ 10’ 5 x 106 106
B-hemolytic streptococci
10” 5 x 10’ 10’ 5 x 106 106 5 x 105
time
4.5 7 12 All survived
n Groups of 10 normal mice were injected by the intraperitoneal route with different doses of either Staphylococcus aureus or B-hemolytic streptococci. The mean survival times are listed.
had a markedly reduced response when compared to littermate controls. A similar situation was found in clinical studies where lymphocytes from poorly controlled diabetic patients failed to respond to mitogenic agents as well as cells from patients with well-controlled disease [7]. Congenitally diabetic mice differ from those with chemically induced diabetes in their response to insulin, both in viva and in vitro. Mice with alloxan- or streptozototin-induced diabetes regained their ability to manifest delayed hypersensitivity when treated with insulin [9]; this did not occur to the same degree in dbldb mice. Furthermore, the mitogenic response of cultured dbldb spleen cells was not altered by prior animal treatment with insulin, or by the addition of insulin to the culture medium. This indicates that the defect in dbldb mice is in the peripheral utilization of insulin. Diabetic mice were unable to contain infections as well as their littermate controls when tested with two common intraoral organisms, B-hemolytic streptococci and
204
JOURNAL Staph db+ db/db
OF SURGICAL
RESEARCH:
Aureus -
VOL. 26, NO. 2, FEBRUARY
El- hemolytic db+ db/db x-x
1979
Strep
100
z K w a
25 250or,,
, , I234
,
,
, 5
(
,
,
,
6
7
0
9
SURVIVAL
, , IO IIII IO
x-x-x-x-x x-x-x-x-x , , , , , , , I2 12 13 13 I4 14 15 15 16 16 17 17 I8
,
(
19 20
(DAYS)
FIG. 2. Groups of 20 normal (db/+) or diabetic (&Mb) mice were injected by the intraperitoneal route with either Staphylococcus aureus or B-hemolytic streptococci. The percentage of surviving animals is plotted on a daily basis. The mean survival time for staphylococcus-inoculated mice is 10 and 4 days for the dbl+ and &Mb groups, respectively, while for the streptococcal animals it is 19 and 5 days for the dbl+ and dbldb groups.
Staphylococcus aureus. Significant differences in the survival patterns indicate that the mechanisms by which pyogenic organisms are defensed by the diabetic host are deficient when compared to normal mice. Although exact extrapolation of this experimental data to the clinical findings [I I] is not possible, it does suggest a relationship between infection control and diabetes. The implication exists that patients with poorly controlled diabetes, known to have altered cellular immunity [ 1, 71, may well have an inability to control inflammatory processes and that this predisposes them to a higher rate of clinical infections. In addition to alterations in immunity, the mechanism of infection may also be due to changes in the phagocytic activity of polymorphonuclear leukocytes and changes induced in the lymphocytes by the different metabolic states [l, 4, 13, 151. The experimental data intimate that adequate functioning of the insulin-glucose system is necessary for full expression of cellular immunity. Diabetic mice had impaired immune function as quantitated by
several assays, as well as an inability to control experimentally induced infections. Although conjectural at this time, the data suggest that patients with diabetes may have similar subtle immune defects that may predispose them to severe infections. Adequate control of diabetes is therefore necessary to diminish the rate of clinical hand infections. REFERENCES 1. Bagdade, J. D., Root, R. K., and Bulger, R. J. Impaired leucocyte function in patients with poorly controlled diabetes. Diabetes 23: 9, 1974. 2. Billingham, R. E., and Silvers, W. K. Transplantation of Tissues and Cells. Philadelphia: Wistar Inst. Press, 1961. P. 149. 3. Boros, D. L., and Warren, K. S. Delayed hypersensitivity-type granuloma formation and dermal reaction induced and elicited by a soluble factor isolated from Schistosoma mansoni eggs. J. Exp. Med. 132: 488, 1970. 4. Bybee, J. D., and Rogers, D. R. The phagocytic activity of polymorphonuclear leucocytes obtained from patients with diabetes mellitus. J. Lab. Clin. Med. 64: 1, 1964. 5. Gibbons, R. J. Significance of the bacterial flora indigenous to man. Amer. Inst. Oral Biol. Symp. 26: 27, 1%9. 6. Lennette, E. H. General principles underlying lab-
MARK A. MANDEL:
IMMUNE
COMPETENCE
oratory diagnosis of virus and rickettsial infections. In E. H. Lennette and N. J. Schmidt (Eds.), Diagnostic
7.
8.
9.
10.
11. 12.
Procedures
of Virus
and
Rickettsial
Dis-
ease. New York: Amer. Public Health Assoc., 1964. P. 45. MacCuish, A. C., Urbaniak, S. J., Campbell, C. J., et al. Phytohemagglutinin transformation and circulating lymphocyte subpopulations in insulin-dependent diabetic patients. Diabetes 23: 708, 1974. McCarty, M. Streptococci. In B. D. Davis, R. Dulbecco, H. H. Eisen, H. S. Ginsberg, and W. F. Wood, Jr. (Eds.), Microbiology. Hagerstown: Harper & Row, 1973. P. 707. Mahoud, A. A. F., Rodman, H. M., Mandel, M. A., et al. Induced and spontaneous diabetes mellitus and suppression of cell-mediated immunologic responses. J. C/in. Invest. 57: 362, 1976. Mandel, M. A., and Asofsky, R. The effects of heterologous anti-thymocyte sera in mice. I. The use of a graft vs host assay as a measure of homograft reactivity. /. Immunol. 100: 1319, 1968. Mandel, M. A. Immune competence and diabetes mellitus I. Pyogenic human hand infections. J. Hund. Surg. 3: 458, 1978. Monaco, A. P., Wood, M. L., Gray, J. G., and
13. 14.
15.
16.
17.
AND DIABETES
MELLITUS
205
Russell, P. S. Studies in heterologous antilymphocyte sera in mice. II. Effect on the immune system. J. Immunol. 96: 229, 1966. Mowat, A. G., and Baum, J. Chemotaxis of polymorphonuclear leucocytes from patients with diabetes mellitus. N. Engl. J. Med. 284: 621, 1971. Pelley, R. P., Ruffier, J. J., and Warren, K. S. Suppressive effect of a chronic helminth infection, Schistosomiasis mansoni, on the in vitro responses of spleen and lymph node cells to the T cell mitogens phytohemagglutinin and concanavalin A. Inf. and Imm. 13: 1176, 1976. Robertson, H. D., and Polk, H. C., Jr. The mechanism of infection in patients with diabetes mellitus: a review of leukocyte malfunction. Surgery 75: 123, 1974. Sonnenwirth, A. C., Gibbons, R. V., and Socransky, S. Bacteria indigenous to man. In B. D. Davis, R. Dulbecco, H. H. Eisen, H. S. Ginsberg, and W. F. Wood, Jr. (Eds.), Microbiology. Hagerstown: Harper & Row, 1973. P. 954. Warren, K. S., Domingo, R., and Cowan, R. B. T. Granuloma formation around schistosoma eggs as a manifestation of delayed hypersensitivity. Amer. J. Pathol. 51: 735, 1967.
OF SURGICAL
RESEARCH
Immune
26,
199-205 (1979)
Competence
and Diabetes
II. Experimental
Mouse Studies
MARK A. MANDEL, Department
of Surgery, University
Division Hospitals
Mellitus
M.D.
of Plastic Surgery, Case Western Reserve University Medical of Cleveland, 2065 Adelbert Road, Cleveland, Ohio 44106
School
and
Submitted for publication June 20, 1978 An experimental model utilizing congenitally diabetic mice was established to answer the question whether such animals have altered immune responses and ability to ward off infections. Cellular immunity was suppressed in diabetic mice when the parameters of granuloma formation, altograft rejection, and delayed footpad swelling were tested. Spleen cells from diabetic mice grown in tissue culture had an impaired mitogenic response compared to those from control animals. In addition, when diabetic mice were inoculated with the common human intraoral organisms, B-hemolytic streptococcus and Staphylococcus aareas, they could not contain the infection as well as normal littermates. Thus, immune function and infectious control mechanisms appear to be deficient in diabetic mice.
MATERIALS
INTRODUCTION
AND METHODS
The finding that patients with diabetes Animals mellitus, latent or overt, appear to have a Genetically diabetic (C57BWKsJ-db) mice, marked proclivity to severe hand infections their littermate controls (dbl+), and Balblc [ 1 l] led to the present experimental study. mice were obtained from Jackson LaboraPrevious in vifro investigations have dem- tory, Bar Harbor, Maine. All mice were onstrated that altered host-parasite rela- maintained on a standard laboratory diet tionships were found in chemically induced (Ralston Purina Co., St. Louis, MO.) and diabetic mice infected with Schistosoma water ad libitum. mansoni; the diabetic animals were unable Chemical Procedures to control the disease process as well as their normal counterparts [2]. The current study After an overnight fast, blood from the seeks to answer the question whether the retro-orbital plexus was drawn into heparinaltered immunity is reponsible for the high ized microhematocrit tubes and analyzed rate of infection using a congenital diabetic for glucose using the autoanaiyzer. Diabetic (dbldb) mouse experimental model. Mulanimals which were treated with insulin tiple assays of immune function including were fasted for only 4 hr before blood granuloma formation, skin allograft sur- samples were obtained. vival, delayed footpad swelling, and tritiated thymidine uptake by spleen cell Immune Assays cultures were utilized to assess immunity. Three quantitative assays of delayed hyThe ability of diabetic and normal mice to persensitivity were utilized. control infections was quantitated by their (1) Granuloma formation about Schistoinoculation with pure cultures of commonly soma mansoni eggs [17]. Groups of five mice occurring oral bacteria; the length of were used to study the effect of diabetes survival was correlated with the bacterial mellitus on the ability of the host to maintain dose. a cell-mediated granuloma response. Fifteen 199
0022-4804/79/020199-07$01.00/O Copyright All rights
0 1979 by Academic Press, Inc. of reproduction in any form reserved.
200
JOURNAL
OF SURGKAL
RESEARCH:
hundred Schistosoma mansoni eggs were suspended in 0.5 ml normal saline and injected via a tail vein into the lung microvasculature of mice. Eight and sixteen days later, groups of five mice were anesthetized, and their lungs were removed and fixed in formalin. Tissue sections (three from each lung, 5 pm thick and 250 pm apart) were stained with hematoxylin and eosin, and the granuloma area around single eggs (at least 10 per slide) was measured using a IIMC particle measurement computer system [9] (Millipore Corp., Bedford, Mass,). Approximately 50 lesions from each experimental group were measured and the significance of the difference between the groups was assessed using Student’s t test. Percentage change in the area of inflammation was calculated by subtracting the mean area of the lesions in the experimental animals (area of inflammation minus area of eggs) from that in the control animals, dividing this figure by the mean lesion size in the control mice, and multiplying by 100. (2) Delayed footpad swelling. Groups of diabetic mice (dbldb) and littermate controls (dbl+) were sensitized by the intraperitoneal injections of 1500 Schistosoma mansoni eggs. Soluble egg antigens equal to 10 pg protein in 0.03 ml phosphate-buffered saline were injected into the right hind footpad; an identical volume of buffer was injected into the left hind footpad. Twenty-four hours later the swelling was quantitated with a micrometer (L&in Rule Co., Saginaw, Mich.). The mean difference in thickness between the right and left footpads was recorded as the net swelling. (3) Skin allograft survival. Grafting of Balb/c (H-2b) skin onto dbldb mice and dbl+ (H-2”) controls was done using standard techniques [lo]. Casts were removed on the eighth day and graft survival was recorded daily using criteria established by Monaco et al. [12]. Four weeks after rejection of the first grafts, second grafts were applied onto the test mice and their survival was quantitated.
VOL.
26, NO,
13H]Thymidine
2, FEBRUARY
1979
Uptake by Spleen Cells
Spleens were harvested from dbldb and dbl+ mice. Cell suspensions were cultured with various concentrations of the non-specific T-cell mitogen, concanavalin A [ 141. After a 60-hr incubation at 37°C 0.5 &i of [3H]thymidine (Searle Analytics, Inc., Des Plaines, Ill.) was added to each well. Twelve hours later the cultures were harvested, the radioactivity was counted, and the results were expressed as mean counts per minute. Insulin (Isophane Insulin suspension USP, M-40, E.R. Squibb and Sons, New York) was administered in a daily dose of 3 units by subcutaneous inoculation for 4 days to groups of dbldb mice. Control mice received subcutaneous injections of citrate buffer. Blood sugar levels were determined after a 4-hr fast, and then the animals were sacrificed and their spleens used in the [3H]thymidine uptake studies. A control group consisted of spleen cell suspensions from six untreated dbldb mice that were cultured in the presence of insulin (100 punits/ml). Control of Induced infections and Normal Mice
of Diabetic
The ability of diabetic and normal mice to combat pyogenic infections was assessed by the intraperitoneal inoculation of bacteria and quantitation of mean length of animal survival, The mice received a wide dose range of either Staphylococcus aureus (coagulase +), or B-hemolytic streptococci (Lancefield, Group A); these are commonly occurring human intraoral pathogenic organisms [5, 8, 161. Pure bacterial cultures were obtained from the Microbiology Department (Case Western Reserve University Medical School). Bacterial counts were made using standard techniques [B, 161, and then doses ranging from lo5 to IO9 bacteria were injected intraperitoneally into each test animal. The death 50% endpoint (LD5,,) was computed by the method of Lennette [6], and the mean animal survival quantitated.
MARK A. MANDEL:
IMMUNE
COMPETENCE
an increase (P < 0.001).
RESULTS
Granuloma
AND DIABETES
Formation
of
only
201
MELLITUS
0.009 + 0.001
ml
Uptake by Spleen Cells Fasting blood glucose (FBS) levels in dbl i3H]Thymidine db mice and their littermate controls, dbl+ , The ability of spleen cells from dbl+ and were 481 ? 27 and 97 ? 5 mg/lOO ml, re- dbldb mice to respond to the mitogenic agent spectively (Table 1). Minimal variation in concanavalin A is outlined in Table 2. Spleen the FBS occurred when the schistosome cells from diabetic mice consistently failed eggs, utilized in the immune assay, were in- to respond to this mitogenic agent or to phyjected. Determination of granuloma size at tohemagglutinin; this indicates inhibition in 8 days revealed a mean of 35.4 pm2 in conDNA synthesis by these cells. Prior treattrol mice, with marked suppression in inment of the mice, in vivo, with insulin or flammation noted in diabetic mice (P by the addition of insulin to the cultures < 0.001). Representative granulomas are in vitro did not change the ability of these shown in Fig. 1. cells to synthesize DNA following mitogenic stimulation. The data suggest, but do not Skin Allograft Survival confirm, that the defect in insulin utilization Allografts from Balb/c mice on dbl+ con- in congenitally diabetic mice is probably at trols survived for 9.7 -t 0.3 days. Significant the peripheral cellular level. prolongation of graft survival (17.8 ? 1.2 days) was noted on the dbldb mice (P Infection Control < 0.001). When second-set grafts were apBacterial inoculations into normal (dbl+) plied they survived for 6.0 & 0.3 days in mice were performed over a wide dose range control mice and 10.1 2 0.5 days in the diafor both Staphylococcus aureus ( 106- log) betic mice (P < 0.01). and B-hemolytic streptococci (5 x 105l(Y). These organisms were chosen since Delayed Footpad Swelling they are frequent pathogens in human oral The injection of soluble antigens into the bacterial flora [5,16]. The initial experiment, footpads of the mice following sensitization outlined in Table 3, serves to establish the level for the diabetic (dbldb) animal testing. is a direct correlate of delayed hypersensitivity. The control mice (dbl+) showed a A marked difference in survival is seen in net increase in swelling of 0.038 & 0.009 ml, Fig. 2 when diabetic and normal mice are while the diabetic mice (dbldb) showed inoculated with either staphylococci (5 TABLE GRANULOMA
FORMATION
IN CONGENITALLY
1 DIABETIC
MICE
(CS7BLiKsJdb)”
Fasting blood glucose (mg/lOO ml + SE) Experimental dbl+ dbldb
group
Day 0
Day 8
97 + 5
110 5 5
481 + 27
454 k 28
Mean granuloma area (wm2 + SE x lOa)
Change in percentage area of inflammation
35.4 c 3.4 10.8 2 1.0
-77
0
a Congenitally diabetic mice (dbldb) and their littermate controls (db/+) were injected with 1500 S. munsoni eggs. The fasting blood glucose levels in the mice are noted. At 8 days the granuloma areas were calculated, and averaged, for at least 50 lesions. The change in percentage area of inflammation is noted.
202
JOURNAL
OF SURGICAL
RESEARCH:
VOL.
26, NO.
2, FEBRUARY
1979
MARK
A. MANDEL:
IMMUNE
COMPETENCE
x lo7 bacteria), or B-streptococci (lo6 bacteria). This indicates the inability of diabetic mice to control the infectious process as well as normal control mice (P < 0.01).
A relationship between altered immunity, diabetes mellitus, and infection control was suggested by the clinical study of hand infections [ 111. An experimental model, using congenitally diabetic mice, was established to study whether the above parameters are related. Marked suppression in cellular immunity was found in congenitally diabetic mice using three quantitative assays of delayed hypersensitivity, namely, granuloma formation around parasite eggs, skin allograft rejection, and footpad swelling. These findings are similar to those previously reported in mice rendered diabetic by either streptozotocin or alloxan [9], chemical agents that destroy the pancreatic p cells and lead to insulin deficiency. Tissure culture studies, using the T-cell mitogen concanavalin A, revealed that spleen cells from congenitally diabetic mice
Trssu~
2
CULTURE STUDIES: TRITIATED THYMIDINE UPTAKE IN SPLEEN CELLS’ Concanavalin
Experimental grow dbldb dblt dbldb Insulin in vivo dbldb Insulin in vitro
A concentration 1
(m&f)
0
0.1
0.3
3
NR” NR
NR NR
NR 3,000
NR 20,000
NR 14,000
NR
NR
NR
NR
NR
NR
NR
NR
NR
NR
n Spleen cells from congenitally diabetic mice (dbidb) and their littermate controls (dbl+) were treated in tissue culture with different concentrations of the mitogenie agent, concanavalin A. The effect of treating the dbidb mice in vivo with insulin for 4 days prior to the assay and of adding insulin to the culture media is shown. b Values expressed as cpm. Values less than 2000 cpm were scored as no response (NR).
DIABETES
203
MELLITUS TABLE
3
SURVIVAL OF NORMAL MICE INOCULATED WITH DIFFERENT DOSES OF BACTERIAL
Bacteria
DISCUSSION
TABLE
AND
Number of organisms
Mean
survival (days)
Staphylococcus aureus
109 5 x 10s 108 5 x 10’ 10’ 5 x 106 106
B-hemolytic streptococci
10” 5 x 10’ 10’ 5 x 106 106 5 x 105
time
4.5 7 12 All survived
n Groups of 10 normal mice were injected by the intraperitoneal route with different doses of either Staphylococcus aureus or B-hemolytic streptococci. The mean survival times are listed.
had a markedly reduced response when compared to littermate controls. A similar situation was found in clinical studies where lymphocytes from poorly controlled diabetic patients failed to respond to mitogenic agents as well as cells from patients with well-controlled disease [7]. Congenitally diabetic mice differ from those with chemically induced diabetes in their response to insulin, both in viva and in vitro. Mice with alloxan- or streptozototin-induced diabetes regained their ability to manifest delayed hypersensitivity when treated with insulin [9]; this did not occur to the same degree in dbldb mice. Furthermore, the mitogenic response of cultured dbldb spleen cells was not altered by prior animal treatment with insulin, or by the addition of insulin to the culture medium. This indicates that the defect in dbldb mice is in the peripheral utilization of insulin. Diabetic mice were unable to contain infections as well as their littermate controls when tested with two common intraoral organisms, B-hemolytic streptococci and
204
JOURNAL Staph db+ db/db
OF SURGICAL
RESEARCH:
Aureus -
VOL. 26, NO. 2, FEBRUARY
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1979
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FIG. 2. Groups of 20 normal (db/+) or diabetic (&Mb) mice were injected by the intraperitoneal route with either Staphylococcus aureus or B-hemolytic streptococci. The percentage of surviving animals is plotted on a daily basis. The mean survival time for staphylococcus-inoculated mice is 10 and 4 days for the dbl+ and &Mb groups, respectively, while for the streptococcal animals it is 19 and 5 days for the dbl+ and dbldb groups.
Staphylococcus aureus. Significant differences in the survival patterns indicate that the mechanisms by which pyogenic organisms are defensed by the diabetic host are deficient when compared to normal mice. Although exact extrapolation of this experimental data to the clinical findings [I I] is not possible, it does suggest a relationship between infection control and diabetes. The implication exists that patients with poorly controlled diabetes, known to have altered cellular immunity [ 1, 71, may well have an inability to control inflammatory processes and that this predisposes them to a higher rate of clinical infections. In addition to alterations in immunity, the mechanism of infection may also be due to changes in the phagocytic activity of polymorphonuclear leukocytes and changes induced in the lymphocytes by the different metabolic states [l, 4, 13, 151. The experimental data intimate that adequate functioning of the insulin-glucose system is necessary for full expression of cellular immunity. Diabetic mice had impaired immune function as quantitated by
several assays, as well as an inability to control experimentally induced infections. Although conjectural at this time, the data suggest that patients with diabetes may have similar subtle immune defects that may predispose them to severe infections. Adequate control of diabetes is therefore necessary to diminish the rate of clinical hand infections. REFERENCES 1. Bagdade, J. D., Root, R. K., and Bulger, R. J. Impaired leucocyte function in patients with poorly controlled diabetes. Diabetes 23: 9, 1974. 2. Billingham, R. E., and Silvers, W. K. Transplantation of Tissues and Cells. Philadelphia: Wistar Inst. Press, 1961. P. 149. 3. Boros, D. L., and Warren, K. S. Delayed hypersensitivity-type granuloma formation and dermal reaction induced and elicited by a soluble factor isolated from Schistosoma mansoni eggs. J. Exp. Med. 132: 488, 1970. 4. Bybee, J. D., and Rogers, D. R. The phagocytic activity of polymorphonuclear leucocytes obtained from patients with diabetes mellitus. J. Lab. Clin. Med. 64: 1, 1964. 5. Gibbons, R. J. Significance of the bacterial flora indigenous to man. Amer. Inst. Oral Biol. Symp. 26: 27, 1%9. 6. Lennette, E. H. General principles underlying lab-
MARK A. MANDEL:
IMMUNE
COMPETENCE
oratory diagnosis of virus and rickettsial infections. In E. H. Lennette and N. J. Schmidt (Eds.), Diagnostic
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Russell, P. S. Studies in heterologous antilymphocyte sera in mice. II. Effect on the immune system. J. Immunol. 96: 229, 1966. Mowat, A. G., and Baum, J. Chemotaxis of polymorphonuclear leucocytes from patients with diabetes mellitus. N. Engl. J. Med. 284: 621, 1971. Pelley, R. P., Ruffier, J. J., and Warren, K. S. Suppressive effect of a chronic helminth infection, Schistosomiasis mansoni, on the in vitro responses of spleen and lymph node cells to the T cell mitogens phytohemagglutinin and concanavalin A. Inf. and Imm. 13: 1176, 1976. Robertson, H. D., and Polk, H. C., Jr. The mechanism of infection in patients with diabetes mellitus: a review of leukocyte malfunction. Surgery 75: 123, 1974. Sonnenwirth, A. C., Gibbons, R. V., and Socransky, S. Bacteria indigenous to man. In B. D. Davis, R. Dulbecco, H. H. Eisen, H. S. Ginsberg, and W. F. Wood, Jr. (Eds.), Microbiology. Hagerstown: Harper & Row, 1973. P. 954. Warren, K. S., Domingo, R., and Cowan, R. B. T. Granuloma formation around schistosoma eggs as a manifestation of delayed hypersensitivity. Amer. J. Pathol. 51: 735, 1967.