The submandibular glands play an important role in regulating systemic and pulmonary inflammatory reactions (Mathison et al, 1994). Although the immunomodulatory effect of the submandibular glands are mediated by numerous biologically active growth factors (Mathison et al, 1994), we identified a small seven-amino acid peptide (TDIFEGG; submandibular gland peptide-T; SGP-T) that modulates the hemodynamic effects of endotoxin (Mathison et al, 1997a), and cardiovascular and intestinal anaphylactic reactions (Mathison et al, 1997b). In other studies, we have established that the three terminal amino acids of SGP-T (Phe-Glu-Gly. FEG), as well as the d-isomeric form of this tripeptide (feG), also had potent anti-anaphylactic actions (Mathison et al, 1998).

Lipopolysaccharide also affects the gastrointestinal tract as evidenced by perturbation of gastrointestinal motility (Hellström et al, 1997) and activation of interstitial macrophages (Eskandari et al, 1997). Thus, the objective of this study was to determine if the salivary gland peptides SGP-T, FEG and feG alter LPS-induced activation of intestinal tissues. 

Methods

Submandibular Gland Peptide-T: The isolation and purification of SGP-T is described elsewhere (Mathison et al, 1997).

Animals: Male Sprague-Dawley rats weighing 250-300 g were housed under controlled lighting conditions (lights on from 7:00H to 19:00H), and provided with food and water ad libitum. All surgical procedures were performed with the animal under halothane anaesthesia.

Recording of Migrating Myoelectric Complexes (MMCs): To record myoelectric activity of the jejunum, rats were surgically prepared under halothane anaesthesia (Scott et al, 1988). Three pairs of Teflon-coated bipolar stainless steel electrodes were fixed in the muscle layer of the jejunum at 2.5 cm intervals, with the first pair placed 2.5 cm from the ligament of Treitz. The electrodes of each pair were sutured 3 mm apart for bipolar recording. Seven days later and after an 18h fast jejunal myoelectric activity was recorded for three cycles of the MMCs before challenge with 20 µg of LPS (Salmonella typhosa) in 0.5 ml of saline introduced into the stomach. The MMCs were recorded for 120 min after the challenge. The duration of the disruption of the MMCs was determined.

Immunohistochemistry: Rats were intraperitoneally injected with LPS (2 mg/kg), feG (100 µg/kg) or both LPS and feG 18 h prior to experimentation. Tissue spreads were prepared by spreading sections of rat mesentery onto chrom-alum coated slides and fixing with acetone. Slides were incubated with the appropriate mouse anti-rat primary antibody (ED1, ED2, CD14 or CD18) for 24 h, washed with PBS, and then incubated with secondary antibody (goat anti-mouse FITC). The number of cells per 40X field was counted using fluorescent microscopy.

Peritoneal cell counts: Rats were pretreated with LPS and/or feG as for immunohistochemistry. Cells were then obtained by peritoneal lavage. 10 cc of 0.9% saline was injected into the peritoneal cavity of the rat. The abdomen was massaged and an incision was made to allow removal of all fluid in the cavity. The cells were spun down and resuspended in lysis buffer (NH₄Cl) for 5 min to lyse red blood cells. Cell suspensions were stained for viability with Trypan Blue and counted using a hemocytometer. For macrophage and neutrophil counts, peritoneal cells were fixed to slides by cytospin and stained with modified Wright stain. 

Results

Effects of LPS and Salivary Gland Peptides on Migrating Myoelectric Complexes (MMCs):
In all fasted rats a stable fasting pattern of MMCs was observed with an interval of 17 +/- 4 min. Upon the intravenous injection of 20 µg/kg of LPS the MMCs maintained the fasting pattern for approximately 20 minutes, and thereafter they were replaced with a continuous and unpatterned myoelectric activity characteristic of the fed state. In untreated rats the MMC's were disrupted for more than 90 min (Table 1) before regular MMCs became readily discernable again. Although FEG and feG did not totally prevent the effects of LPS on the MMCs these peptides reduced the duration of disruption by 50%. SGP-T, on the other hand, did not prevent the LPS induced perturbations of myoelectric activity.
 

Table 1: Effects of SGP-T, FEG and feG on Duration of Disrupted Migrating Myoelectric Complexes (MMCs)

Treatment	Dose & Route of Administration	Duration of MMCs (minutes)
Control	Saline; intravenous	101 +/- 7
feG	350 µg/kg; oral	54 +/- 4*
FEG	100 g/kg; intravenous	59 +/- 11
SGP-T	100 g/kg; intravenous	108 +/- 5
	4 to 10 animals in each group	* P<0.05 relative to saline

 
 
Effects of LPS and feG on mesenteric tissue macrophage surface markers:
Intraperitoneal (i.p.) injection of feG (100µg/kg) along with LPS (2 mg/kg) decreased the number of macrophages expressing CD18 by ~76%, as compared to rats treated with LPS only. feG had no effect on the number of macrophages expressing ED1 or ED2.
 

Table 2: Effect of feG on Expression of ED1, ED2 and CD14 by Mesenteric Tissue Macrophages

Marker	LPS (2mg/kg) i.p. (Cells per 40X field)	LPS (2mg/kg) and feG (100µg/kg) i.p. (Cells per 40X field)
ED1	31.3 +/- 7.2	27.2 +/- 2.5
ED2	32.9 +/- 5.6	23.2 +/- 3.2
CD18	45.6 +/- 10.7	10.8 +/- 4.2*
	4 to 5 animals in each group	* P<0.05 relative to LPS

Effect of feG on total cell count in peritoneum:
LPS (2 mg/kg i.p.) caused an increase in total cell count in the peritoneum as well as increasing differential counts of macrophages and neutrophils. Treatment with LPS and feG (100µg/kg i.p.) attenuated the increase in total cell count caused by LPS, and also decreased macrophage and neutrophil counts to below control levels.
 

Table 3: Effect of feG on total and differential counts of peritoneal cells

Treatment	Total Number of Cells	Number of Neutrophils	Number of Macrophages
Control	8.55 x106 +/- 1.8 x106	1.66 x106 +/- 3.42 x 105	3.93 x106 +/- 9.06 x 105
LPS (2 mg/kg i.p.)	1.83 x107 +/- 7.03 x106	4.12 x106 +/- 1.38 x106	1.14 x107 +/- 4.57 x 06
LPS and feG (100µg/kg i.p.)	6.12 x106 +/- 2.99 x106	8.88 x105 +/- 4.23 x105*	3.77 x106 +/- 2.00 x106
	6 to 8 animals in each group	* P<0.05 relative to LPS

 
Discussion
 
Through their profound activation of macrophages and neutrophils, bacterial toxins such as LPS elicit the release of a wide variety of biologically active molecules that if not properly counterbalanced can result in the development of sepsis and multiple organ failure. Gastrointestinal dysfunction may precede and is often associated with these severe clinical conditions. The present study and others (Hellström et al, 1997) show that the gastrointestinal tract of the rat is very sensitive to LPS at doses as low as 20 µg/kg, whereas much larger doses (near 2 to 5 mg/kg) are required to produce measurable changes in mean arterial blood pressure (Mathison et al, 1997a). A detailed examination of the doses of LPS required to activate rat intestinal and mesenteric macrophages has not been done, but we have found that 2 mg/kg of LPS cause activation of interstitial and peritoneal macrophages, even though this dose is 5 to 10 times lower than those used by Eskandrai et al (1997).

The present study extends the anti-inflammatory effect of the tripeptide FEG and its d-isomeric form feG, as observed in animal models of cardiovascular and intestinal anaphylaxis (Mathison et al, 1997b; 1998), into septic events associated with the peritoneum, mesentery and intestine. These peptides not only act rapidly (within 60 min) in that they significantly reduced the length of time that LPS disrupted the normal interdigestive myoelectric pattern (the MMCs; Table 1), but also they act over an extended period of time since they prevented the activation of interstitial macrophages (Table 2) and the migration of cells into the peritoneal cavity (Table 3); events that occur over an 18 h period.

The tripeptides FEG and feG may be useful in preventing some of the untoward effects of bacterial endotoxins on gastrointestinal function.

References

1. Eskandari MK, Kalff JC, Billiar TR, Lee KK, Bauer AJ. (1997) Lipopolysaccharide activates the muscularis macrophage network and suppresses circular smooth muscle activity. Am. J. Physiol. 273:G727-34.
   
2. Hellström PM, al-Saffar A, Ljung T, Theodorsson E. (1997) Endotoxin actions on myoelectric activity, transit, and neuropeptides in the gut. Role of nitric oxide. Dig. Dis. Sci. 42:1640-51.
   
3. Mathison RD, Befus AD, Davison JS. (1997a) A submandibular gland peptide protects against endotoxin induced hypotension. Amer J Physiol 273: R1017-R1023.
   
4. Mathison RD, Daimen T, Oliver M, Befus AD, Davison JS, Scott B. (1997b) A novel peptide from submandibular glands inhibits intestinal anaphylaxis. Dig Dis Sci 442: 2378-2383.
   
5. Mathison RD, Lo, P., Davison JS, Scott B, Moore G. (1998) Attenuation of intestinal and cardiovascular anaphylaxis by the salivary gland tripeptide FEG and its D-isomeric analogue feG. Peptides 19: 1037-1042.
   
6. Scott RB, Daimant SC, Gall DG. (1988) Motility effects of intestinal anaphylaxis in the rat. Am. J. Physiol. 255: G505-G511.
   

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