Salwa A. Elgebaly

Urinary Neutrophil Chemotactic Factors in Interstitial Cystitis Patients and a Rabbit Model of Bladder Inflammation

The present study investigates a possible source of inflammatory mediators involved in the pathogenesis of bladder inflammation characteristics of interstitial cystitis disease. Our tested hypothesis is that in response to injury, tissues of the urinary bladder participate in the initiation of bladder inflammation by releasing inflammatory mediators such as neutrophil chemotactic factors. Bladders of anesthetized rabbits (n = 7) were instilled with an acidic solution (pH 4.5) for 15 minutes, then washed with saline and instilled with sterile phosphate buffered saline (PBS) (pH 7.2) for an additional 45 minutes prior to sacrificing the rabbits. Control rabbits (n = 7) were instilled with sterile PBS (pH 7.2) for 15 minutes, then 45 minutes. The levels of neutrophil chemotactic factors were measured using modified Boyden chambers and rabbit peritoneal neutrophils as indicator cells. Results indicated the release of high levels of neutrophil chemotactic factors (via a checkerboard analysis) from acid-treated bladders after 15 minutes (70 +/- 4% of standard) and 45 minutes (80 +/- 7%). Electron microscopy analysis of these acid-treated bladders revealed the infiltration of a large number of neutrophils, which correlates with the recovery of neutrophil chemotactic factors. Control rabbits, on the other hand, showed low levels of chemotactic activity (less than 10 percent) and exhibited normal bladder morphology with absence of neutrophils. The glycosaminoglycan (GAG) layer was intact in both acid-treated and control bladders. High levels of neutrophil chemotactic factors were also detected in urine samples from eleven patients with interstitial cystitis (113 +/- 25%) (not due to interleukin-1 or leukotriene B4) which were not detected in urine samples from healthy volunteers (n = 9) or from thirteen control patients with bladder diseases other than interstitial cystitis. These preliminary studies indicate the capability of injured bladder tissues to release neutrophil chemotactic factors which contribute to the initiation of bladder inflammation. The presence of neutrophil chemotactic factors in urine samples of interstitial cystitis patients suggests a possible role of these mediators in the pathogenesis of the disease.

Evidence of cardiac inflammation after open heart operations

In this study, 6 anesthetized dogs underwent global cardiac arrest for 1 hour, followed by reperfusion on bypass for 45 minutes. The hearts were then weaned off cardiopulmonary bypass and monitored for an additional 2 hours. Using modified Boyden chambers, high levels of neutrophil chemotactic activity were detected (using a checkerboard analysis) in the coronary sinus effluents obtained during cardiac arrest. The activity tended to decline during reperfusion. Assay of myeloperoxidase (a marker for neutrophils) revealed an accumulation of large numbers of neutrophils in the right (14 +/- 1.1 x 10(4) cells/g wet weight) and left (16 +/- 1 x 10(4) cells/g wet weight) ventricles after 2 hours of reperfusion. Light microscopy evaluation confirmed the presence of neutrophils, not only in the ventricles, but also in a greater number in the right and left atria. Electron microscopy study of these hearts revealed the presence of mild reversible changes, indicating good preservation of the hearts during arrest. Results of this study provide evidence for an acute inflammatory reaction that takes place after cardiac operations and suggest a role for myocardial tissues in the initiation of such a response through their release of neutrophil chemotactic factors.

Increased interleukin-1 activity in the injured vitamin A-deficient cornea.

Injury to a vitamin A-deficient cornea leads to severe acute inflammation often culminating in ulceration. We report on possible regulatory mechanisms involved in the pathogenesis of corneal inflammation in vitamin A deficiency. Thymocyte comitogenic assay and interleukin (IL)-6 induction in corneal fibroblasts have shown that thermally injured and mechanically abraded vitamin A-deficient rat corneas produce much higher levels of an IL-1-like factor as compared with uninjured or injured normal control corneas. This was confirmed by antibody capture enzyme immunoassays, which detected high levels of IL-la and IL-1 (3 in injured vitamin A-deficient corneas. To our knowledge this is the first report describing the induction of IL-1 in the vitamin A-deficient cornea by thermal and mechanical injuries. When mechanically injured corneas were screened for chemotactic activity, they were found to contain significantly higher levels of a chemoattractant as compared with similarly injured, normal control corneas. Chemotactic activity [expressed as a percentage of a known chemotactic tripeptide, formylmethionyl- leucyl-phenylalanine (fMLP), found in medium harvested from vitamin A-deficient corneas] averaged 58.8 ± 8.9% (SEM) as compared with 12.6 ± 5.4% in medium conditioned by normal corneas. Checkerboard analysis confirmed that the activity in vitamin A-deficient cornea conditioned medium was chemotactic and not chemokinetic. These results demonstrate a correlation between IL-1 levels and severity of inflammation in the injured vitamin A-deficient rat cornea.

The release of a neutrophil chemotactic factor from UV-B irradiated rabbit corneas in vitro

Rabbit corneas were isolated, mounted on plastic rings to form a cup and the endothelium was covered with RPMI tissue culture medium. The preparation was then irradiated with 1 J. cm-2 of 300 nm light over 1 hour and then incubated for a further two hours in the dark. The supernatant fluid was assayed for chemotactic activity toward rabbit neutrophils in an in vitro Boyden chamber assay. The results indicated that medium from irradiated corneas had a chemotactic activity that was 42% of that produced by the standard chemoattractant f-met-leu-phe, (10(-9) M) while medium from unexposed corneas and exposed medium alone had less than 3% activity. An in vivo assay using sub-epidermal injection into the back of a rabbit gave qualitatively similar results, only f-met-leu-phe and the medium from irradiated corneas causing neutrophil infiltration of the tissue. A checkerboard analysis confirmed that the activity was chemotactic rather than chemokinetic. Release of a chemotactic factor following UV-B irradiation provides a mechanism for the recruitment of neutrophils, at specific localized areas of the endothelium, that is seen after discrete in vivo irradiation. The results also confirm the importance of corneal inflammatory mediators in the development of tissue damage subsequent to exposure to toxic agents.

Release of neutrophil chemotactic factors from gastric tissue. Initial biochemical characterization.

This study was designed to characterize neutrophil chemotactic factors released by gastric tissue. Full-thickness rabbit stomach (organ culture) was prepared and incubated in Ringers solution at 37°C. Culture supernatants were collected at 1, 2, 3, and 4 hr and assayed for neutrophil chemotactic activity in modified Boyden chambers. High levels of chemotactic activity were seen at 3 hr of incubation. Antral and fundic tissue were equally capable of producing neutrophil chemotactic activity. In addition, high levels of activity were seen from both the serosal and mucosal surfaces. Initial biochemical characterization of these gastricderived factors revealed that: (1) a majority of the activity (80–90%) exhibited molecular weight values of greater than 300 kDa, (2) the chemotactic activity was heat stable but was partially reduced by treatment with a protease, subtilisin (37% inhibition), and (3) 70–80% of the activity in the supernatants was extracted into organic solvent (ethyl acetate). These factors may prove to be important in recruitment of neutrophils to areas of gastric injury.

Cornea derived neutrophil chemotactic factors: intracellular synthesis and release

The present study was designed to test the hypothesis that the release of neutrophil chemotactic factors (NCF) from isolated corneas following hydrogen peroxide stimulation requires specific intracellular synthesis. For these studies, the epithelial surfaces of isolated rabbit corneas were preincubated with various inhibitors of protein synthesis (cycloheximide, 10 micrograms/ml, and puromycin, 50 micrograms/ml) and transcription (actinomycin D, 5 micrograms/ml) for 60 min prior to exposure of the corneas to glucose (G, 1 mg/ml) and glucose oxidase (GO, 20 U/ml) for 6 h at 37 degrees C. All three inhibitors decreased the levels of NCF recovered in the extracorneal fluids by 80-98%, suggesting that peroxide acts to upregulate NCF production at both the transcription and translation levels. When corneas were incubated with G/GO for 6 h at 12 degrees C or 4 degrees C instead of 37 degrees C, a reduction in the levels of NCF recovered in the supernatants was noted at 12 degrees C (46-91% inhibition) and at 4 degrees C (67-96% inhibition), suggesting that the synthesis of NCF at cold temperature was only reduced but not totally inhibited. To demonstrate that the observed reduction in chemotactic activity recovered from corneas incubated at 12 degrees C or 4 degrees C is not due to a temperature-dependent inhibition of NCF biosynthesis, but rather to a disruption of intracellular vesicular transport, temperature shift experiments were performed. Corneas were incubated with G/GO overnight at 12 degrees C or 4 degrees C prior to shifting to 37 degrees C for an additional 6 h.(ABSTRACT TRUNCATED AT 250 WORDS)

Cardiac derived neutrophil chemotactic factors; preliminary biochemical characterization

The purpose of this study is to investigate whether isolated hearts perfused with cardioplegic solution release inflammatory mediators such as neutrophil chemotactic factors (NCF). Three conditions were tested, including: (1) perfusion of rabbit hearts with crystalloid cardioplegic solution (4 degree C) saturated with air (95% oxygen) and containing dextrose (i.e. complete system), (2) perfusion of rabbit hearts with non-oxygenated cardioplegic solution, containing dextrose (i.e. minus oxygen system), and (3) perfusion of hearts with cold cardioplegic solution saturated with air in the absence of dextrose (i.e. minus dextrose system). At various time intervals (5 min, 1, 2, 3 and 4 h) samples of circulated perfusate were removed and assayed for the presence of NCF using modified Boyden chambers. Rabbit peritoneal neutrophils were the indicator cells. The standard chemoattractant, f-Met-Leu-Phe (f-MLP) was the positive control. High levels of neutrophil chemotactic activity were detected in perfusate of all above described hearts perfused for 4 h (i.e. 194 +/- 22% of f-MLP control--complete system, 126 +/- 13%--minus oxygen and 136 +/- 10%-minus dextrose). Histological evaluation of these hearts showed evidence of global ischemia. We also detected significant levels of NCF in effluent of hearts perfused for 5 min, 1, 2 and 3 h. Similar to perfused hearts, isolated rabbit hearts incubated for 4 h with non-oxygenated cardioplegic solution (in presence and absence of dextrose) released high levels of NCF (132 +/- 18%-intact heart; and 100 +/- 6% myocardial segments). Standard checkerboard analysis revealed that the observed activity released from these hearts is chemotactic.(ABSTRACT TRUNCATED AT 250 WORDS)

Inflammatory mediators in alkali-burned corneas: preliminary characterization.

Leukocyte chemotactic factors (LCF) are important inflammatory mediators which activate and recruit leukocytes from the circulation into sites of tissue damage. These factors were recently detected in the tear fluid of inflamed eyes induced by alkali burn. It remains unclear, however, whether the detected LCF are released from injured corneal tissues or leaked from the circulation. Using a corneal cup model developed in our laboratory, we began examining the capability of corneal tissues to produce LCF in response to alkali injury. We also evaluated the influence of citric acid on the production of LCF from corneas preinjured by the alkali sodium hydroxide (NaOH). For these studies, the epithelial surfaces of corneas isolated from bovine and human eyes were exposed to 1N NaOH for 35 seconds at room temperature. The NaOH was then removed and the epithelial surfaces washed once with buffer and incubated with culture medium for 1, 2, 4, and 6 hours at 37 degrees C/5% CO2 atmosphere. Our results showed that (1) NaOH2 induced corneal epithelial cell injury ranging from cell discoloration and moderate damage of the upper half of the epithelium (1-4 hrs) to total destruction of the epithelium (6 hrs); (2) NaOH-injured corneas (2 hr incubation post injury) produced significant levels of chemotactic activity (via checkerboard analysis) specific for neutrophils (115% maximum chemotactic response [MCR]) and mononuclear cells (94% MCR); (3) preliminary characterization of these factors revealed that they are protease and heat sensitive, extractable by organic solvents, and possess molecular weight values greater than 100,000 daltons; and (4) incubation of NaOH-pretreated corneas with 0.01% citric acid for 2 hours markedly inhibited the production of LCF for both neutrophils (98% inhibition) and mononuclear cells (91% inhibition). Results of these studies indicate that alkali-burned bovine and human corneas generate leukocyte chemoattractants which differ in their biochemical characteristics from previously known low molecular weight chemotactic factors such as C5a, interleukin-1, or leukotriene B4.

An in vitro model of leukocyte mediated injury to the corneal epithelium

To enhance efforts directed at unraveling the role and mechanisms of leukocytes in mediating injury to corneal epithelium, an isolated bovine corneal cup was developed and evaluated. Bovine peripheral leukocytes and lysates were added to the corneal epithelial surface of isolated cornea for various periods after which the degree of morphologic changes and cell damage were assessed using light and electron microscopy. Results of these studies indicate that leukocyte/epithelial cell interactions are characterized by five successive stages: (1) leukocyte adhesion to superficial layer of the epithelium, (2) leukocyte penetration beneath the superficial epithelium, (3) epithelial cell injury, (4) leukocyte phagocytosis of killed epithelial cells and (5) ulceration and total destruction of the full thickness of the epithelial layer. The above sequence appears to be both time and dose dependent; that is epithelial cells exposed to leukocytes for short periods (5-60 minutes) or to low dose levels (10(5) - 10(7) cells/ml) shows leukocyte adhesion and penetration beneath the superficial layer of the epithelium, (stage 1 and 2), while longer exposures (2-3 hours) or higher numbers of leukocytes (10(7) - 10(8) cells/ml), leads to deeper penetration of epithelium by leukocytes and epithelial injury (Stages 3,4,and 5). We also observed that direct contact of intact leukocytes with epithelial cells is apparently necessary to induce this type of injury. These findings demonstrate the ability of leukocytes to destroy corneal epithelial cells and the value of this new ocular model for studies of the basic immunology of ocular inflammation.

Enhancement of the recovery of rat hearts after prolonged cold storage by cyclocreatine phosphate

The present study determined whether the administration of cyclocreatine phosphate (CCrP) prior to ischemia can enhance the recovery of rat hearts hypo-thermically preserved for a prolonged period. Rats (n=6 per group) were injected intravenously with 1 ml saline or CCrP (500 mg/kg). After 2 hr, hearts were excised and arrested by an infusion of University of Wisconsin solution. Saline hearts were then incubated in 40 ml UW, while CCrP hearts were incubated in 40 ml UW containing 100 mg CCrP; a mixture that is now referred to as Hartford Hospital (HH) solution. After 6 hr of storage at 4°C, hearts were reperfused in the Langendorff mode for 15 min and then in the working heart mode for 30 min. Results indicated that the recovery of cardiac function—measured as aortic flow, coronary flow, cardiac output, stroke volume, and stroke work—was significantly better in CCrP group (50–55% baseline) compared with that of saline hearts (20–25%). Although no difference in enzyme leakage (i.e., creatine kinase) or lactate was detected between the two groups, the increase in heart weight after the initial 6-hr storage was significantly higher in saline hearts compared with that of CCrP hearts. Results of this study support the conclusion that CCrP treatment provides improved functional recovery after prolonged hypothermic preservation.