Lymphokine-Augmented Activation of Avian Heterophils M. H. KOGUT,1 V. K. LOWRY, R. B. MOYES, L. L. BOWDEN, R. BOWDEN, K. GENOVESE, and J. R. DELOACH USDA-ARS, Food Animal Protection Research Laboratory, 2881 F & B Road, College Station, Texas 77845 ABSTRACT Heterophils are important mediators of innate resistance in poultry, especially in young birds that have not yet developed an acquired immune response. Invasion of the intestinal mucosa by Salmonella spp. initiates the recruitment of large numbers of heterophils to the lamina propria. Thus, the heterophilic response can control, but not eliminate, bacterial numbers in the bird until development of acquired immunity. Unfortunately, chicks and turkey poults are highly susceptible to Salmonella infections during the first 4 d posthatch due to the functional immaturity of both the innate and acquired immune systems. We have previously shown that the administration of Salmonella enteritidis (SE)-immune lymphokines (ILK) into either 18-d-old developing embryos or day-of-hatch chicks and poults conferred increased resistance to SE organ invasion. In this review, we present evidence that the protection induced by ILK is mediated by vigorous
recruitment and activation of heterophils. These activated heterophils migrate rapidly to the site of bacterial invasion where they phagocytize and kill the SE. Specifically, in vitro studies demonstrate an enhancement of functional activities of the heterophils including chemotaxis, adherence, phagocytosis, and bacterial killing. In addition, during the activation process, membrane expression of adhesion molecules rapidly changes from L-selectins to b2 integrins (CB11b/CD18) on the cells that become activated. These results further demonstrate the validity of preventive activation in poultry to induce the migration of large numbers of activated phagocytic cells to the site of infection by a pathogenic organism. Importantly, this immunopotentiation of the inflammatory response by ILK, as described here, induces the functional maturation of heterophils during the first 4 d posthatch.
(Key words: chicken heterophils, Salmonella, lymphokine, preventive activation, inflammation) 1998 Poultry Science 77:964–971
control of these activities. Many assumptions about the function(s) of avian heterophils are based on studies of mammalian neutrophils. However, avian heterophils differ in some important functional characteristics. For example, unlike the neutrophil, heterophils lack myeloperoxidase (Brune et al., 1972; Penniall and Spitznagel, 1975; MacRae and Powell, 1979) and do not produce a significant oxidative burst for use in bactericidal activities (Penniall and Spitznagel, 1975). Further comparative studies on the phenotypical, biochemical, and physiological attributes of the heterophil must be similarly investigated. In mature poultry, resting or native heterophils appear to be effective killers of pathogenic bacteria (Andreasen et al., 1991; Harmon et al., 1992; Stabler et al., 1993; Evans et al., 1994, 1995). Nevertheless, the question remains whether these cells can be readily activated so that their functions are significantly enhanced. The term activation, as used here, is described as the rapid and
INTRODUCTION Heterophils, the avian counterpart to the mammalian neutrophil, are important mediators of natural resistance against bacterial infections in poultry (Powell, 1987a,b). These phagocytic cells act early during an infection to restrict bacterial growth to a level that can be eliminated by the acquired host defenses generated later. Their importance in host defenses against invading pathogens appears to be due to their ability to ingest and kill a wide variety of microbial pathogens. Heterophils are especially important in young chickens that have not yet developed an acquired immunity to pathogens (Ziprin et al., 1989; Lowenthal et al., 1993; Kogut et al., 1993, 1994a). The primary biological functions of phagocytosis and killing of pathogens by heterophils are described (Powell, 1987b), although few mechanistic studies have been conducted to define the induction, regulation, and
Received for publication July 10, 1996. Accepted for publication March 2, 1998. 1To whom correspondence should
[email protected]
be
Abbreviation Key: G-CSF = granulocyte-colony stimulating factor; F+ = fluorescent positive; ILK = immune lymphokines; SE = Salmonella enteritidis.
addressed:
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TABLE 1. Representative mediators of neutrophil and macrophage activation Mediator Cytokines IL-8 G-CSF GM-CSF IFN-g TNF-a RANTES MIP-1A Bacterial products LPS Formyl peptides Peptidoglycans Lipid products Leukotrienes Complement C5a C3a/e
References Schroder and Christophers, 1992 Demetri and Griffin, 1991 Gasson, 1991 Berton and Cassatella, 1992 Akira et al., 1990 Schall, 1991 Murphy, 1994 Cohn and Morse, 1960 Schiffman et al., 1975 Pabst, 1994
FIGURE 1. Effects of immune lymphokines (ILK) on the total and differential peripheral blood leukocyte counts of chickens at 4 h after injection. *Significantly different from the control birds (P < 0.001).
Palmblad et al., 1981 Morgan, 1990 Morgan, 1990
selective up- or down-regulation of the physiologic and biochemical properties and functions of phagocytes (Adams and Hamilton, 1982). Activation of phagocytic cells initiates a complex process that includes adhesion, chemotaxis, phagocytosis, particulate digestion, degranulation, production of active oxygen species, and mediator synthesis. In mammals, neutrophils have been found to express the increased functional activities normally applied to the activated macrophage (Gallin et al., 1988; Wong and Serhan, 1991). Activation is triggered by a number of different chemical signals, of both host and pathogen origin, produced locally at the site of infection or inflammation that regulate the various functions of neutrophils (Table 1). Macrophage activation has been carefully investigated in poultry (Dietert et al., 1990); whereas, few, if any, reports have documented the activation of the avian heterophil. Cytokines are a diverse group of biologically active protein molecules that act as modifiers of both innate and acquired immunity (Oppenheim and Shevach, 1990). Cytokines, secreted by various immune and nonimmune cells, are some of the foremost mediators of activation of neutrophils in both humans and meatproducing animals (Steinback and Roth, 1989; Brown and Roth, 1991; Cairo, 1991; Coe et al., 1993; Semnani et al., 1993; Kabbur et al., 1995). Recent findings from our laboratory demonstrated a connection between the heterophil and early resistance to salmonellae infections in chickens (Kogut et al., 1993; 1994a). Selection depletion of heterophils in young chicks caused otherwise subclinical Salmonella enteritidis (SE) infections to rapidly become clinical with severe extraintestinal organ infections; whereas, the control chickens had infections that were primarily restricted to the intestine with no morbidity nor mortality. We further demonstrated that the prophylactic administration of T-lymphocyte-derived cytokines; i.e., immune lymphokines (ILK), from SE-immune chicks to either
18-d-old embryos, day-of-hatch chicks, or day-of-hatch turkey poults conferred a significant resistance to SE organ infectivity (McGruder et al., 1993, 1995; Ziprin et al., 1995). This resistance was found to be associated with a dramatic increase in the number of circulating peripheral blood heterophils (Figure 1; Kogut et al., 1994b). The concept of “preventive activation” of phagocytic cells as a means of promoting nonspecific defense of poultry against pathogens has recently been introduced (Toth et al., 1987; 1988). Preventive activation involves the introduction of an immunostimulant into chicks which will induce the migration of increased numbers of activated phagocytic cells to the site of infection by a pathogenic organism. We have adopted this concept for use with ILK as a means of inducing a T cell-induced activation of heterophils in poultry resulting in protection against Salmonellae infections (Kogut et al., 1995a,b; 1997a). This paper will focus on our current research on describing the activation of avian heterophils. Here we summarize the up-regulation of various heterophil functions and properties following the in vivo administration of ILK into day-old chickens. The role of the activated heterophil as the effector cell in the ILKinduced protection against SE organ infectivity is also discussed.
FUNCTIONS 1. Chemotaxis. Modified Boyden blind well chemotaxis chambers were used to quantitate heterophil random migration and chemotactic movement (Table 2). Random migration of heterophils from the ILK-treated chickens was significantly greater (P < 0.001) than the control treatment. A 10-fold increase in the number of heterophils from ILK-treated chickens that migrated to the chemotactic stimulus was found when compared with heterophils from the PBS-injected controls. 2. Phagocytosis. Phagocytosis of SE by heterophils from ILK-injected chicks was greatly enhanced (84% of heterophils containing SE) when compared to PBS
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FIGURE 2. Phagocytosis of Salmonella enteritidis by heterophils isolated from chickens injected 4 h previously with either PBS or immune lymphokines (ILK). *Significantly different from the PBSinjected control heterophils (P < 0.001). Bars with different lower case letters above are significantly different (P < 0.001).
controls (28%) (Figure 2). Furthermore, each heterophil from the ILK-treated group contained more than twice as many bacteria (P < 0.001) than the control heterophils. 3. Bactericidal Activity. The ability of heterophils isolated from either the PBS-treated or ILK-treated groups to kill both paratyphoid and typhoid serovars of Salmonella are summarized in Figure 3. The peripheral blood heterophils from chickens administered ILK were found to have significantly enhanced bactericidal activity against all Salmonella serovars relative to the heterophils from the PBS-treated control chickens. 4. Nonspecific Adherence. Adherence to nylon wool of heterophils from chickens injected with ILK was significantly (P < 0.001) increased over the adherence of heterophils from the control birds (Figure 4). 5. Receptor-Mediated Adherence. We utilized an in vitro heterophil adhesion model of BSA-coated chamber slides. Adherence to complex protein matrices is integrin-mediated by the heterodimeric leukocyte adhesion receptor (CD11b/CD18) in mammals (LundgrenAkerland et al., 1992, 1993; Hansell et al., 1994). The heterophils from the ILK-treated chickens were found to increase their adherence by 300% over the control heterophils (Figure 5A). The enhanced ability to adhere was observed as early as 2 h postinjection of the ILK (Figure 5B). In order to determine the specific adherence molecule(s) involved in the adherence of the heterophils to the BSA matrix, polyclonal antibodies were added to the cells prior to their addition to the chamber slides.
FIGURE 3. Bactericidal activity of heterophils following the injection of either PBS or immune lymphokines (ILK) into day-old chickens. Each bar represents the mean percentage of surviving bacteria of three replicate experiments with each Salmonella serovar. *Significantly different from PBS-injected controls (P < 0.001).
Both anti-CD11b (37 to 74%) and anti-CD18 (15 to 62%) antibodies reduced the BSA adherence of the heterophils from the ILK-treated birds in a concentration-dependent manner (Figure 6). When the anti-CD11b and anti-CD18 antibodies were added together, an effective adherence inhibitory effect was found with the lower antibody concentrations (0.5 to 2 mg/mL). The optimal additive inhibitory effect (87%) was found with 2 mg/mL of each antibody. Adherence of the heterophils from the PBSinjected control birds was not inhibited by either the anti-CD11b or anti-CD18 antibodies (Table 3). An isotype-matched antibody (10 to 30 mg/mL) preincubated with heterophils from either treatment group did not inhibit the binding of the cells to the BSA-coated slides (data not shown).
PROPERTIES: CELL SURFACE MOLECULES/CELL MORPHOLOGY 1. L-Selectin. L-Selectin is a surface molecule constitutively expressed and functional on unactivated granulocytes. L-Selectin is involved in the initial contact of the granulocyte with endothelium near sites of inflammation and mediates leukocyte rolling (margination). Activation of neutrophils causes a rapid decrease
TABLE 2. Chemotaxis of heterophils1
Treatments
Random Migration (HBSS)
Chemotoxic movement (chicken serum)
PBS-injected ILK-injected
6.84 ± 1.26A 30.46 ± 5.99B
116.74 ± 11.31A 1,120 ± 154.81B
A,BMeans in a column with no common superscript differ significantly (P < 0.001). 1Chemotaxis of heterophils following injections 4 h previously of either PBS or ILK. Values expressed as mean number of heterophils ± standard error.
FIGURE 4. Heterophils collected 4 h following the injection of either PBS or immune lymphokines (ILK). Asterisk represents significant differences (P < 0.001) between adhesion of the heterophils from the ILK-injected chickens and the PBS-injected chickens. Each bar represents the mean of three replicate experiments.
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SYMPOSIUM: NONLYMPHOID CELLS AND THEIR FACTORS IN IMMUNE RESPONSES TABLE 3. Percentage inhibition of receptor-mediated heterophil adherence1 Heterophil sources Antibody to adhesion molecule
PBS-injected ILK-injected
CG11b (2 mg) CD18 (2 mg) CD11b + CD18 (2 mg)
0 4 4
38*** 52*** 87***
1Maximal additive effect of anti-CD11b and anti-CD18 antibodies on adherence of heterophils isolated from immune lymphokine (ILK)injected chickens. ***Signficantly different for each adhesion molecule (P < 0.001).
in the number of surface L-selectin molecules (Cronstein and Weismann, 1993). Heterophils from either the PBSinjected or ILK-injected chickens were incubated with FITC-labeled anti-L-selectin antibody and fluorescence measured by flow cytometry (Table 4). Heterophils from the control birds were approximately 80% fluorescent positive (FL+) throughout the 4 h period of these experiments. However, within 1 h postinjection of ILK, heterophils expressed virtually no L-selectin (Table 4). 2. CD11b/CD18. Activation of granulocytes upregulates functionally active CD11b/CD18 adhesion molecules. The CD11b/CD18 molecules mediate firm adhesion of granulocytes to endothelium, essential for granulocyte extravasation. From our trials, heterophils from the PBS-injected control chickens expressed few CD11b/CD18 molecules (approximately 5% FL+; Table
FIGURE 5. A) Heterophils collected 4 h following the injection of either PBS or ILK. Results are expressed as the number of cells per five counting fields upon microscopic examination of adherent cells in chamber slides. B) Time course of receptor mediated adherence to BSA-coated chamber slides of heterophils isolated from chickens following the injection of either PBS or immune lymphokines (ILK) at time 0 h. Results are expressed as the total number of heterophils per five fields. *Significantly different from the heterophils isolated from the PBS-injected chickens (P < 0.001).
TABLE 4. Expression of CD11b/CD18 and L-selectin on heterophils1 L-selectin
CD11b/CD18
Hours after injection
PBS
ILK
PBS
1 2 3 4
79.91*** 80.07*** 80.11*** 79.84***
2.12 2.18 2.22 2.28
(% FL) 4.88 5.13 5.51 5.55
ILK 75.21*** 70.72*** 66.13*** 63.91***
1Flow cytometric analysis of expression of L-selectin and CD11b/ CD18 adhesion molecules on heterophils from chickens injected 4 h previously with either PBS or ILK. %FL = percentage fluorescing heterophils. ***Significantly different for each adhesion molecule (P < 0.001).
4). At the same time points, heterophils from ILKinjected chickens expressed little L-selectin molecules (about 2% FL+), which were replaced with the expression of CD11b/CD18 adhesion molecules. Maximal expression of CD11b/CD18 (75% FL+) occurred within an hour after injection of ILK. The heterophils from the ILK-treated birds remained greater than 60% FL+ throughout the 4 h study. 2. IL-8 Receptor. We previously found that an IL8-like molecule is produced by the host locally at the site of bacterial invasion following ILK-treatment (Kogut et al., 1996a). The production of the IL-8-like molecule apparently is involved in the site-directed migration of heterophils to this inflamed site. For the heterophils to migrate to this inflamed site, IL-8 receptors must be present on the heterophil membrane. Using flow cytometry, we measured the presence of an IL-8 receptor (IL-8R) on the heterophils from control and ILK-treated chickens (Table 5). Within 1 h, approximately 50% of the heterophils from the ILK-treated birds were FL+. By 2 h postinjection, maximal IL-8R expression was observed on the heterophils from the ILK-treated birds (59% FL+). The number of heterophils positive for the IL-8R
FIGURE 6. Concentration dependence of antibody neutralization of CD11b, CD18, or combined CD11b/CD18 adherence molecules on heterophils isolated from immune lymphokine (ILK)-injected chickens.
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KOGUT ET AL. TABLE 5. Expression kinetics of IL-8R on heterophils1
Variable % FL
LMFC
Time postinjection
PBS-injected heterophils
ILK-injected heterophils
(h) 1 2 3 4 1 2 3 4
4.37B 7.37B 7.42B 14.63A 71.82B 53.15B 56.71B 125.63A
57.41A 59.62A 50.61A 23.11A 158.37A 186.42A 203.59A 123.07A
A,BMeans in a row with no common superscript differ significantly (P < 0.001). 1Flow cytometric analysis of the kinetics of the IL-8 receptor expression of peripheral blood heterophils. Heterophils were isolated from chickens injected 4 h previously with either PBS or immune lymphokines (ILK). %FL = percentage of fluorescing heterophils. MFC = mean fluorescent channel.
decreased rapidly over the next 2 h as presumably the IL-8R-positive cells left the blood and migrated to the site of bacterial infection (Kogut et al., 1995b, 1996a). Few IL-8 receptors were found (4 to 14% FL+) on the heterophils from the PBS-injected control chickens. 3. Cell Morphology. Extensive studies with mammalian neutrophils have demonstrated that the activation of these cells results in a dramatic change in cell shape (reviewed in Qu et al., 1995). Interestingly, the changes in neutrophil shape correlates to the increased chemotactic responsiveness and adhesion (Smith et al.,
1979; Haston and Shields, 1985). Assessment of heterophil shape was made by scanning electron microscopy following incubation of control and ILK-treated heterophils on BSA-coated slides (Figures 7A and 7B). The vast majority (> 70%) of the control heterophils were round and did not display membrane ruffling or extensive pseudopod formation (Figure 7A). However, the heterophils (> 90%) from the ILK-treated chickens had undergone dramatic shape change characterized by flattening with extensive membrane ruffling and pseudopod formation (Figure 7B).
DISCUSSION The studies reported here were undertaken to ascertain the effect of in vivo administration of ILK on heterophil function and the possible relationship between these cells and host resistance to salmonellosis. The impetus for these studies was provided by our earlier studies showing ILK-induced resistance of dayold chickens and turkeys to SE infections (McGruder et al., 1993, 1995; Ziprin et al., 1995) and several published reports demonstrating preventive activation of phagocytic cells as a means of promoting nonspecific defense of poultry against pathogens (Toth et al., 1987, 1988). Most recent research efforts on immunoprophylactic measures to control or prevent intestinal and tissue colonization of invasive Salmonella in poultry have primarily focused on the development of killed and live, avirulent Salmonella vaccines (Nagaraja et al., 1991; Cooper et al., 1994). Regardless of the efficacy of a
FIGURE 7. A) Scanning electron micrograph of heterophils from control chickens are predominately of spherical morphology (Arrows; magnification 2,616×). Bar = 10 mm. b) Scanning electron microgram of heterophils from chickens injected with immune lymphokines (ILK). Heterophils were flattened with increased membrane ruffling (Large arrows). These cells possessed extended pseudopodia which frequently terminated with areas of cytoplasmic expansion (Small arrows; magnification 1,762×). Bar = 10 mm.
SYMPOSIUM: NONLYMPHOID CELLS AND THEIR FACTORS IN IMMUNE RESPONSES
vaccine, at least 7 to 10 d are required for the stimulation of the acquired immune response for protection. However, neonatal chicks are most highly susceptible to Salmonella infections during the first 4 d posthatch, after which they become increasingly more resistant to infection (Ziprin et al., 1989). It is during this first 4 d posthatch that we hypothesized that immunopotentiation of the innate defense mechanism(s) would prevent Salmonella from multiplying to numbers capable of overwhelming any response that is generated. The data presented here demonstrate that ILK exerts just such a rapid effect on heterophil function. These results represent some of the first demonstrations of the in vivo activity of ILK on heterophil functions and corroborates our hypothesis on the practicality of preventive activation as an industry applicable method for controlling Salmonella infections. Effective host defense against bacterial invasion is characterized by the vigorous recruitment and activation of inflammatory cells. The most common histologic feature of salmonellosis in the avian intestine is the recruitment of large numbers of heterophils to the lamina propria (Pomeroy and Nagaraja, 1991). However, the heterophils of chickens and turkeys are functionally immature for the first 4 to 5 d posthatch (Bowden et al., 1996; Lowry et al., 1997). In addition, it is doubtful that day-old chicks possess the ability to activate these heterophils (Lowenthal et al., 1993). In this review, we present evidence indicating that the SE-ILK is an effective immunopotentiator of SE infections in day-old chickens. Specifically, in vitro studies indicate components of the ILK augment the ability of heterophils to phagocytose and kill SE. Cytokine networks between nonimmune and immune cells are necessary for cellular communication during inflammatory response. These interactions are central to the initiation and propagation of the protective inflammatory response induced by the administration of ILK. The results described here emphasize an association of ILK, adhesion molecules, and IL-8 that coordinate the recruitment of heterophils to the site of salmonellae invasion. The working paradigm that we have developed to describe the induction of the protective heterophilic inflammatory response by the administration of ILK consists of at least five steps. First, the administration of ILK and challenge with salmonella results in an emigration of heterophils from the bone marrow into the blood stream (Figure 1; McGruder and Kogut, unpublished results). The emigration of peripheral blood heterophils is due to the presence of granulocyte-colony stimulating factor in the ILK (McGruder and Kogut, unpublished results; Kogut et al., 1996c). Second, production of an IL-8-like chemotactic protein in the local environment by the host in response to SE invasion appears to be responsible for the specific migration of heterophils to the site of bacterial invasion (Kogut et al., 1995b, 1997b). Administration of ILK appears to enhance the production of this IL-8-like
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chemotactic factor (Kogut et al., unpublished data). Additionally, at the time of the local IL-8 production, a population of peripheral blood heterophils begins to express IL-8 membrane receptors that are required for their continued migration to the inflamed site (Table 5). Third, the heterophils from birds injected with ILK have a significant enhancement (activation) of their biological effector functions including adherence, chemotaxis, phagocytosis, and bacterial killing (Figures 2 to 5; Tables 2 to 5). Fourth, a 10-fold increase in the expression of the PMN-derived adhesion molecule, CD11b/CD18, which during the recruitment process is essential for extravasation and sequestration of the heterophils at the inflamed site (Table 4). Fifth, a massive influx of the activated inflammatory heterophils from the blood to the site of infection by Salmonella (Kogut et al., 1995b). Thus, the administration of ILK induces a rapid and dynamic reaction in the bird that results in the activation of heterophils with a subsequent resistance to systemic salmonella infections. In conclusion, our studies indicate that ILK contains specific cytokines that important mediators of leukocyte recruitment and activation in avian salmonellosis. Immunologic manipulation (preventive activation) of avian innate resistance may serve as an important industry applicable method for the prevention and control of intestinal and organ colonization by paratyphoid and typhoid salmonellae infections in broiler and table egg producing flocks.
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