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Am J Physiol Heart Circ Physiol 282: H636-H644, 2002. First published October 18, 2001; doi:10.1152/ajpheart.00090.2001
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Vol. 282, Issue 2, H636-H644, February 2002

Mechanisms of decreased leukocyte localization in the developing host

M. Michele Mariscalco*, Wilfredo Vergara*, Jia Mei, E. O'Brian Smith, and C. Wayne Smith

Department of Pediatrics, Sections of Leukocyte Biology and Critical Care Medicine, Baylor College of Medicine, Houston, Texas 77030-2600


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Delays in leukocyte localization likely contribute to diminished host defense in neonates. Understanding the processes that may be affected has been hampered by the lack of suitable developmental models. Using intravital microscopy, we directly examine leukocyte recruitment in a rabbit pup model. In response to intraperitoneal interleukin (IL)-1beta , there were one-third as many leukocytes that arrested in pup mesenteric vessels and emigrated compared with adult vessels, although leukocyte flux was not different. Leukocyte rolling velocity in pups was one-half that in adults. In response to surgical trauma alone, the number of arrested pup cells was 15% that of adult cells, although again leukocyte flux was not different. An anti-L-selectin antibody inhibited rolling significantly by 60 min for both pups and adults. The effect on arrest and emigration occurred at significantly earlier times, although the effect was less in rabbit pups. A primary defect in leukocyte emigration in the rabbit pup appears to be a failure of the cell to transition efficiently from rolling to arrest. L-selectin-dependent adhesion and emigration are decreased, rolling is not, suggesting that at least part of the defect is due to events downstream of the initial tether.

neonate; cell adhesion; intravital microscopy; leukocyte flux; adhesion; emigration


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

THE SUSCEPTIBILITY of human neonates to both localized soft tissue infections and systemic infections due to bacterial or fungal agents has prompted extensive investigations into neonatal host-defense mechanisms. Among the most consistently observed functional abnormalities in the neonate are those related to leukocyte mobility (6, 27). Early studies using "skin windows" in human neonates have shown altered leukocyte exudation compared with older children and adults (34). Neonatal and immature animals, including nonhuman primates, rats, and rabbits, have delayed neutrophil localization into the peritoneal cavity (12, 32), skin (8), and lung (23).

The current conceptual model for leukocyte extravasation incorporates the idea that leukocyte rolling precedes stationary adhesion and that there is a necessary transition from rolling to arrest on the endothelium under flow conditions. Potential molecular mechanisms for deficits in leukocyte localization in the developing host may be linked to abnormal intercellular adhesion. Current studies using in vitro modeling and whole animal studies where the end point is leukocyte accumulation suggest one or more steps may be affected. Leukocyte and endothelial selectins are critical for capturing of the leukocyte from the blood stream and maintaining rolling. A consistently observed abnormality of cord blood neutrophils is a reduction in surface levels of L-selectin (4, 38). The ability of neutrophil L-selectin to bind to fucosylated, sialylated (or sulfated) carbohydrate structures on cell surface glycoproteins contributes to efficient accumulation of neutrophils on monolayers of activated endothelial cells under flow conditions with venular levels of wall shear stress (2, 4, 41). Cord blood neutrophils exhibit reduced ability to accumulate on activated endothelial cells under flow conditions in vitro, and anti-L-selectin antibodies have little ability to diminish peritoneal neutrophil accumulation in response to thioglycollate in 1-day-old rabbits (4, 12). Endothelial P-selectin may also be limited. Lorant and colleagues (32) found that P-selectin was readily observed on peritoneal venules in adult rats following thioglycollate injury but not on venules in neonatal rats. In addition, human umbilical vein endothelial cells (HUVEC) from preterm infants expressed less P-selectin than HUVEC from term infants (32).

The beta 2-integrins Mac-1 (CD11b/CD18) and LFA-1 (CD11a/CD18) contribute to the ability of the neutrophil to adhere to foreign surfaces and to endothelial cells. Diminished function may contribute to the observed reductions in chemotactic migration in vitro and emigration into tissues. An observed abnormality of cord blood neutrophils is a diminished cell content of Mac-1 and a diminished ability to mobilize this integrin from intracellular stores to the cell surface (3, 5). A second member of the beta 2-integrin family, LFA-1 (CD11a/CD18), may also be reduced, but this has been inconsistently observed.(5, 18, 38) (Landers C. L. and Mariscalco M. M., unpublished observations).

It is unknown whether these observations translate mechanistically into diminished leukocyte accumulation in the neonate. Addressing this question has been hampered by the lack of suitable models allowing observation of these processes directly in vivo. This study aims to provide insights into the steps required for efficient migration in a developmental rabbit model using intravital microscopy. Furthermore, we seek to elucidate the role of L-selectin in this process, because our previous work suggests that L-selectin-dependent function in neonates is diminished (4, 12).


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Animals

Time-dated New Zealand White rabbits (Ray Nichols Rabbitry; Lumberton, TX) were used. Term pups were studied between 2 and 7 days of age and maintained with lactating does until the day of experiment. Rabbits weighing 1.5-3.0 kg were used for comparative adult studies. Adult rabbits received water only 24 h before the experiment. This study complied with the National Institute of Health Guidelines for the Care and Use of Laboratory Animals (1996) and was approved by the Animal Protocol Review Committee at Baylor College of Medicine.

Reagents

Human interleukin (IL)-1beta was purchased from Boehringer Mannheim Biochemicals (Indianapolis, IN) (43). Monoclonal antibody (mAb) TS2/4, murine anti-human CD11a (IgG1), was obtained through American Type Culture Collection (ATCC; Manassas, VA) and purified from culture supernatants. It does not cross-react with rabbit neutrophils and serves as a control antibody (18). DREG 200 (IgG1), an anti-human L-selectin that cross-reacts with rabbits, was kindly provided by Dr. T. K. Kishimoto (Boehringer Ingleheim Pharmaceuticals; Ridgefield, CT) (12, 42, 43). Endotoxin content was <0.1 ng/mg protein (Limulus Amoebocyte Assay, Cape Cod Associates). Antibodies for flow cytometry included M1/70 (ATCC, rat anti-mouse CD11b, IgG2b) (18) and R15.7, an murine anti-canine CD18 that cross-reacts with rabbits, kindly provided by Dr. R. Rothlein (Boehringer Ingleheim Pharmaceuticals) (12). Control antibodies for flow cytometry included CL18/1 (mouse anti-canine [ICAM]-1, IgG1) (40) and SFR3-DR5 (rat anti-human HLA-DR5, IgG2b, ATCC); neither cross-reacts with rabbit neutrophils. R15.7, M1/70, CL18/1, and SFR3-DR5 were biotinylated using sulfo-NHS-biotin (Pierce Chemical; Rockford, IL) according to manufacturer's instructions.

Flow Cytometry

The expression of L-selectin, CD18, and CD11b (Mac-1) on adult and pup neutrophils was determined by flow cytometry as previously described (12, 18). All mAbs were titrated using flow cytometry (FACS-Scan; Becton Dickinson; Mountain View, CA) to determine the concentration that saturated binding sites of unstimulated and stimulated cells (4). Detection of L-selectin was determined using DREG 200 and FITC-labeled goat anti-mouse IgG antibody (Zymed Laboratories; South San Francisco, CA). Aliquots of blood from animals receiving either TS2/4 or DREG 200 intravenously were reacted with anti-mouse FITC to determine the amount of antibody bound in vivo. Saturation of neutrophil L-selectin by DREG200 received in vivo was confirmed by adding back additional first antibody before the reaction with anti-mouse FITC. CD18 and Mac-1 expression were determined using biotinylated R15.7 and M1/70, respectively, followed by R-phycoerythrin-conjugated streptavidin (Jackson ImmunoResearch Laboratories; West Grove PA). Neutrophils were identified by their characteristic forward and side-scatter pattern. The mean fluorescent intensity for 5,000 particles/sample was obtained using linear amplification. Average mean fluorescent intensity for each sample was normalized against the value of the isotype-matched control.

Animal Preparation

Surgical trauma. Rabbits were anesthetized with intramuscular injections of ketamine hydrochloride 25 mg/kg (pups) or 35 mg/kg (adult rabbits) and xylazine 5 mg/kg before all injections and surgical manipulations. Two separate protocols were developed for these investigations. In the protocol of mild surgical trauma, animals were sedated and tracheotomized. Adult rabbits breathed spontaneously, whereas pups were ventilated using an animal respirator (SAR-830 Ventilator, CWE; Ardmore, PA). Body temperature was continuously monitored (Physitemp Instruments; Clifton, NJ) and maintained by external warming. The rabbit was then transferred to a heated Plexiglas viewing platform, and a midline abdominal incision was performed. The terminal ileum was exteriorized from the abdominal cavity and spread over the viewing platform. The mesentery was perfused with warmed endotoxin-free, bicarbonate-buffered saline (pH 7.4) and covered with plastic wrap. All parts of exteriorized gut were covered with buffer-soaked gauze pads. The superfusion buffer and viewing platform were maintained at 37°C. The rabbit and platform were then transferred to the microscope stage.

IL-1beta administration. We also examined leukocyte rolling in animals after intraperitoneal administration of IL-1beta . Animals were sedated and received intraperitoneal IL-1beta (1 U/g) (43). Three and one-half hours later animals were resedated. Pups were tracheotomized and ventilated. Adult rabbits breathed spontaneously. Pups received internal jugular catheters, and adult animals had catheters placed in ear veins for fluid and mAb administration. Five minutes before exteriorization of the mesentery, animals received 1 mg/kg of either TS2/4 or DREG 200. Physiological saline was infused intravenously at 4-6 ml · kg-1 · h-1. Exteriorization of the mesentery was performed as outlined above. Time 0 (T0) was the point at which mesenteric manipulation began. Vessel segments were identified and recorded every 5 min. For each adult rabbit and pup, 8-12 separate vessel segments were recorded. The parameters were averaged over 15-min intervals as before. White blood cell counts were determined at the completion of the experiment. In this model, leukocytes can be seen to emigrate through the venule wall into the surrounding mesentery. Leukocytes that had passed through the vessel wall were counted in an area of 3,200 µm2. This area was arbitrarily defined by the length of a 40-µm vessel segment and the region 40 µm above and below this segment.

The observation period of 65 min was chosen in both models because the pup mesentery developed spontaneous hemorrhages when lengthened beyond this time. In addition, neonatal animals were ventilated because nonventilated animals did not survive until the completion of the experimental time. Adult animals did not routinely need ventilation because they were able to maintain adequate minute ventilation with anesthesia. We did not record blood pressures. We compared microhemodynamics in both age animals and both models to ensure that these parameters were similar despite any dissimilarities in the preparation. In the antibody treatment studies we did not use each animal as its own control, because over the first 60 min of vessel exteriorization, adhesive mechanisms change (29).

Intravital Microscopy

Observations of the vessels in the terminal segment of the superior mesenteric artery were made on a Axioskop intravital microscope (Carl Zeiss; Werk Gottingen, Germany) using a NPL Fluotar objective (×25/0.35 numerical aperture, Leitz Wetzler) for transillumination. Continuous video recordings of the microcirculation were obtained from a video camera (model DXC-960MD, 3CCD Color Video Camera, Sony) mounted on the microscope, which projected the image onto a color monitor (Panasonic). The images were recorded (Video Cassette Recorder, VO-9600) for playback analysis. Single unbranched venules with diameters ranging between 20 and 40 µm were selected for the study. Venular diameter (Dv) was measured online using a video caliper (Microcirculation Research Institute; College Station, TX). For each animal, 10-30 separate vessel segments were examined during the 65-min experiment. The venule segments were analyzed, and the data were averaged for each 15-min period. Time 0 was arbitrarily set as the time of the first observation.

The number of adherent leukocytes was determined offline during playback of video-taped images and expressed as the number per 60 µm of vessel segment. A leukocyte was considered adherent if it remained stationary for at least 30 s (43). Rolling leukocytes were defined as those white blood cells (WBC) that moved at a velocity less than that of erythrocytes in the same vessel. Leukocyte rolling velocity (VWBC, µm/s) was determined from the time required for a leukocyte to traverse a given distance along the length of the venule. The flux of leukocytes that rolled past a fixed point of vessel segment in 1 min was determined (FWBC, cells/min). The number of leukocytes that continued to maintain a rolling interaction with the vessel at least 60 µm downstream was also determined (RWBC, cells/min). Because leukocytes may roll for only a section of the vessel before rejoining the flow of blood or adhering to the vessel wall, RWBC is a subset of FWBC.

Centerline red blood cell velocity (VRBC, mm/s) was measured using an optical Doppler velocimeter (Microcirculation Research Institute). Mean bulk velocity (Vmean) was calculated from the red blood cell velocity (Vmean = VRBC/1.6) (22). Venular wall shear rate (WSR) was calculated based on the Newtonian definition: WSR = 8(Vmean/Dv), where Dv is venular diameter (22). The volumetric blood flow in the vessel segment (Q) was estimated from the product of venular cross-sectional area and Vmean, Q = (pi Dv2/4)Vmean (11). The total number of leukocytes that pass the vessel segment in a defined period of time is estimated from the product of the volumetric blood flow and systemic leukocyte count, QWBC = Q(WBC). At the completion of the experiment, blood was drawn via cardiac puncture from the pups, and systemic WBC count was determined using a Coulter Counter (Coulter Electronics; Hialeah, FL). Manual differential counts were performed on Wright-stained smears. Adult animals had blood obtained every 15 min from 15 to 60 min via ear arterial puncture. Leukocyte rolling flux fraction at T60 was defined as the percentage of the total number of leukocytes traversing the vessel segment at time 60 (QWBC at T60) that are rolling past a fixed point; i.e., leukocyte flux (FWBC at T60) (leukocyte rolling flux fraction = FWBC/QWBC) (11).

Statistical Methods

Data are presented as means ± SE. Statistics were performed using SPSS software (SPSS; Chicago, IL). Variables were analyzed using repeated-measures analysis of variance with time as the within-group factor and the age of the animal and treatment as the between-group factors. If no time effect was noted, data are presented as the estimated marginal means ± SE for that age animal and treatment group. Cell counts and expression of adhesion molecules were analyzed using two-way analysis of variance. Post hoc multiple comparisons were performed using the Bonferroni test if statistical significance was reached. In variables that could be affected by circulating cell counts, data were also analyzed using WBC as a covariant. Statistical significance was set at P < 0.05.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Depressed Leukocyte Emigration in Rabbit Pups in Response to IL-1beta is Primarily Due to Decreased Ability of Immature Leukocytes to Arrest

To provide mechanistic insights into neonatal leukocyte transmigration, we examined leukocyte behavior directly. Here 20- to 40-µm vessels and the surrounding mesentery of 1-wk-old and adult animals were evaluated 4 h after receiving intraperitoneal human IL-1beta (42, 43). Four hours after IL1beta administration and 5 min after receiving control mAb (TS2/4), the skin was incised (T0), and the mesentery was isolated and placed on the viewing platform. We quantified the number of transmigrated leukocytes in a 3,200-µm2 area surrounding a 40-µm vessel. In both 1-wk-old and adult animals, the number of emigrated cells increased significantly over time after mesentery exteriorization (P < 0.05 and P < 0.001, respectively, Fig. 1). The rate of increase was greater in the adult than the neonatal animals (i.e., there is a within-subject interaction between time and age, P < 0.001; Fig. 1). At all times after T15 there were significantly more transmigrated leukocytes in the adult animal than in 1-wk-old animals. Adult animals had significantly higher WBC and absolute neutrophil counts (ANC) than 1-wk-old rabbits by T60 (Table 1). As the number of transmigrated leukocytes could be affected by circulating WBC counts, we reanalyzed the number of transmigrated leukocytes at T60 using WBC as the covariant. Again, there were still significantly more emigrated cells in the adult animals compared with the 1-wk-old rabbits (P < 0.05).


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  		Fig. 1.   Leukocyte emigration into mesentery of 1-wk-old and adult rabbits 4 h after intraperitoneal interleukin (IL)1beta . Number of leukocytes that had emigrated into the mesentery in a 3,200-µm2 area superior and inferior to the 40-µm vessel segment was assessed. T0 is the time (T) of abdominal incision at time 0. Number of transmigrated leukocytes were counted every 5 min beginning at T10 and averaged every 15 min for each animal. Leukocyte emigration increased significantly over time in the adult (P < 0.001) and 1-wk-old animals (P < 0.05), although it was greater in adult animals (interaction between age group and time, P < 0.001). *P < 0.05 vs. 1 wk. **P < 0.001 vs. 1 wk. Results are given as means ± SE; n = 7 and 5 for 1-wk-old and adult rabbits, respectively.


                              
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  		Table 1.   Leukocyte and hemodynamic characteristics of rabbits treated with intraperitoneal IL-1beta at T60

Leukocyte rolling was characterized several ways in this model. FWBC is the number of leukocytes rolling passed a fixed point on the vessel wall. FWBC did not change over time in either adult or 1-wk-old rabbits. FWBC was significantly greater in adult rabbits than neonatal rabbits (131 ± 9 vs. 43 ± 8 cells/min, P < 0.001). RWBC is the number of leukocytes that continued to maintain a rolling interaction 60 µm downstream past a second fixed point along the vessel wall. FWBC was not significantly different from RWBC in either age group. Therefore, neonatal leukocytes that were captured from the flow stream rolled at least 60 µm further downstream. Because circulating WBC may also impact FWBC, we determined leukocyte rolling flux fraction at T60 (11). Leukocyte rolling flux fraction was not significantly different between 1-wk-old and adult animals (13.2 ± 3.3% vs 15.0 ± 2.8%, respectively). In contrast, VWBC was 50% less in 1-wk-old rabbits compared with the adults (Table 1). The lower VWBC in 1-wk-old rabbits was not the result of changes in microhemodynamics because there were no significant differences in centerline VRBC, vessel diameter, Q, or venular WSR between the two age groups (Table 1).

Although rolling flux fraction was equal between the age groups and leukocytes rolled slower in younger animals, adhesion was dramatically reduced in the 1-wk-old rabbits compared with the adults (P < 0.001, Fig. 2). Additionally, the number of adherent cells increased over time after vessel exteriorization in the adult but not in the 1-wk-old animals (P < 0.05, Fig. 2). When the number of adherent cells at T60 were covaried for WBC count, adherent cell number was still greater in the adult rabbits (P < 0.05).


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  		Fig. 2.   Leukocyte adhesion (cells/60 µm of vessel length) in mesenteric vessels after intraperitoneal IL-1beta . Leukocyte adhesion significantly increased over time only in adult animals (P < 0.05) but not 1-wk-old rabbits. *P < 0.05 vs. 1 wk. **P < 0.01 vs. 1 wk.

We examined the expression of L-selectin, Mac-1, and CD18 on circulating leukocytes at T60 using flow cytometry. As reported by us and others in 1-day-old and premature rabbits (12, 37), L-selectin expression was higher on neutrophils from adult animals compared with 1-wk-old animals (P < 0.02) (Table 1). There was no difference in the expression of Mac-1 or CD18 between neonatal and adult animals.

Thus in 1-wk-old rabbits, a primary defect leading to diminished leukocyte emigration is the decreased ability of the leukocyte to transition from a rolling to an arrested cell. This occurs despite dramatically decreased WBC rolling velocity, "adultlike" levels of Mac-1 and CD18, and a rolling flux fraction equal to that of adult animals. Although intrinsic transmigration defects may also occur, the decreased ability to arrest appears to predominate in this model.

Neonatal Animals Have Diminished Leukocyte Arrest After Surgical Trauma.

A low frequency of constitutively rolling leukocytes has been observed in normal skin (24), whereas rolling is virtually absent in undisturbed mesentery (10). Leukocyte rolling is rapidly induced on surgical manipulation of the rabbit and rat mesentery as well as the mouse cremaster muscle (10, 29). Because leukocyte emigration does not appreciably occur in this model, we examined arrest directly without loss of cells from the blood vessel into the surrounding tissue.

Adult and 1-wk-old rabbits received no IL-beta or mAb but were subjected to surgical manipulation of the bowel only. There was no significant difference between the WBC counts of the 1-wk-old and adult rabbits 60 min after vessel exteriorization (Table 2). The ANC was less in the 1-wk-old rabbits, although it was not significant (P = 0.055). VRBC, WSR, and Q were not significantly different between 1-wk-old and adult rabbits (Table 2).

                              
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  		Table 2.   Leukocyte and hemodynamic characteristics of rabbits with mild surgical trauma

In both age groups, FWBC and RWBC decreased over time (Fig. 3). There were significantly fewer rolling leukocytes that maintained an interaction with a 60-µm segment of vessel in the 1-wk-old group (FWBC vs. RWBC, Fig. 3). This did not occur in the adult animals. Neither FWBC nor RWBC was different between the two age groups. Leukocyte rolling flux fraction at T60 was also not different between 1-wk-old and adult animals (Table 2). In contrast, VWBC was significantly increased in 1-wk-old rabbits (Table 2).


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  		Fig. 3.   Flux of white blood cells (FWBC) rolling past a fixed segment of vessel per minute and number of leukocytes that continue to maintain a rolling interaction with the vessel at least 60-µm downstream past a second fixed point (RWBC) in mesenteric vessels of 1-wk-old and adult rabbits after surgical trauma only (see text for further discussion). FWBC and RWBC decreased over time (P < 0.01). There was no difference between the age groups for either FWBC or RWBC. In neonates only, RWBC was significantly less than FWBC (P < 0.05). n = 5 for each age.

The number of adherent cells did not change over the 60-min observation time. However, the number of adherent leukocytes in the 1-wk-old rabbits was only 15% that of adult rabbits (Table. 2). When the number of adherent leukocytes were covaried for the WBC, there were still significantly fewer arrested cells in the 1-wk-old rabbits compared with the adult rabbits.

In this model, the leukocytes of neonatal animals also failed to transition from rolling to arrest, despite having similar numbers of leukocytes that initially roll as adult animals. However, rolling cells of neonatal animals have a higher rolling velocity, and fewer of them maintain a rolling interaction over at least 60 µm of vessel length compared with adult animals.

L-Selectin-Dependent Adhesion And Transmigration Is Depressed in 1-Wk-Old Animals

In adult rabbits, IL-1beta -induced peritoneal inflammation results in L-selectin-dependent leukocyte rolling at 45-60 min after mesenteric manipulation (43). L-selectin-ligand interactions may also function to activate leukocytes, resulting in increased adhesion and emigration (17, 20). We previously demonstrated that 1-day-old rabbits had decreased localization of leukocytes to the peritoneal cavity after thioglycollate-induced peritonitis, and this appeared to be due to decreased L-selectin function (12).

Neonatal and adult animals received the anti-L-selectin mAb (DREG 200) 4 h after receiving intraperitoneal IL-1 beta  and before skin incision. The animals receiving control antibody (TS2/4) are those previously described in Depressed Leukocyte Emigration in Rabbit Pups in Response to IL-1beta is Primarily Due to Decreased Ability of Immature Leukocytes to Arrest. Treatment of animals with DREG200 had no significant effect on either WBC or ANC in either 1-wk-old or adult animals. L-selectin binding sites were saturated by DREG 200 in animals because adding back additional DREG200 to whole blood in vitro did not result in increased binding compared with samples in which DREG 200 was not added. There was no difference within each age group between the control and DREG 200-treated animals in vessel diameter, VRBC, or Q over time. WSR was significantly less in TS2/4-treated 1-wk-old animals compared with the DREG-200-treated group at T15 only (214 ± 21 vs. 346 ± 23, respectively, P < 0.05) but at no other time. There was also no difference in WSR in the adult animals between the two treatment groups (Table 1 and data not shown).

By T45 and T60, anti-L-selectin mAb inhibited FWBC significantly in the 1-wk-old and adult animals, respectively (Fig. 4A). There was no difference in the leukocyte rolling flux fraction at T60 in the anti-L-selectin mAb-treated 1-wk-old rabbits compared with adult rabbits (7.5 ± 3.3% vs. 7.6 ± 3.7%, respectively). Thus anti-L-selectin mAb decreased rolling flux fraction by 50% in both age groups. Anti-L-selectin mAb had no effect on VWBC in either the 1-wk-old or adult rabbits compared with their TS2/4-treated controls (Table 1 and data not shown).


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  		Fig. 4.   Effect of anti-L-selectin monoclonal antibodies (mAb) on FWBC (A), leukocyte adhesion (B), and leukocyte emigration (C) in 1-wk-old and adult animals treated with intraperitoneal IL-1beta . Plotted here is the arithmetic difference between the mean values for control (TS2/4 treated) and anti-L-selectin (DREG 200)-treated animals for each age and time ± SE of the differences. Statistical differences between the treatment groups at each age at that time point are designated as *P < 0.05 and **P < 0.01. A: anti-L-selectin mAb had no significant effect on FWBC until T45 in 1-wk-old rabbits and T60 in adult animals. B: treatment with anti-L-selectin mAb significantly decreased the number of arrested cells in both adult and 1-wk-old (P < 0.01). Effect on adult was greater than that of 1-wk-old animals (interaction between age and treatment, P < 0.05). C: anti-L-selectin mAb decreased the number of emigrated leukocytes in both adult and 1-wk-old animals; the effect became greater over time (P < 0.001, note positive slope of the line for both ages). Effect on adult animals was greater (i.e., significant interaction between age and treatment group over time, P < 0.01), demonstrated here by increased slope for adult vs. neonatal rabbits; n = 7 and 5 for 1-wk-old and n = 5 and 4 for adult animals treated with control or anti-L-selectin mAb, respectively.

Both 1-wk-old and adult animals treated with anti-L-selectin mAb had significantly fewer adherent leukocytes by T30 (Fig. 4B). The effect on adult animals was greater than that of 1-wk-old rabbits (P < 0.05). Anti-L-selectin mAb treatment also significantly decreased leukocyte emigration in both adult and 1-wk-old rabbits as early as T15 (Fig. 4C). The inhibitory effect of anti-L-selectin on emigration was greater over time in both age groups, although the effect was greater in the adult animals compared with the neonates (P < 0.01, Fig. 4C).

Anti-L-selectin mAb decreased FWBC in 1-wk-old and adult rabbits by 45-60 min after vessel exteriorization, as has been reported previously (42, 43). Although the anti-L-selectin mAb also markedly decreased the number of arrested and emigrated cells, this occurred at earlier times. Whereas effects of L-selectin blockade on FWBC was not different between the 1-wk-old and adult rabbits, its effect on adhesion and emigration was less in the 1-wk-old rabbits.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Observations by our group and others have demonstrated that newborn and infant animals have delays and/or deficits in leukocyte emigration compared with mature animals (8, 12, 23, 32) and that human neonates also have altered leukocyte exudation compared with older children and adults (34). Lacking in these and subsequent studies has been mechanistic insights into leukocyte localization in the developing host in vivo. Using intravital microscopy, we demonstrate here for the first time that at least in rabbit pups there does not appear to be a defect in the ability of the leukocyte to be recruited from the blood stream. Rather, once the neonatal leukocyte is recruited (and rolling), there is a markedly decreased ability to transition from rolling to arrest, a step necessary for emigration (28, 39). This is supported by the following observations. First, in a model of mild tissue trauma, leukocytes of neonatal animals fail to transition from rolling to arrest, despite having the same number of leukocytes that roll as adult animals. Second, in mesenteric venules exposed to intraperitoneal IL-1beta for 4 h, neonatal leukocytes have a diminished capacity to arrest compared with adult even when the effects of increased WBC are taken into account and despite a markedly diminished leukocyte rolling velocity.

That the leukocytes of 1-wk-old rabbits appeared to have little difficulty in initiating rolling in inflamed mesenteric vessels in either model was unexpected in light of previous reports, which suggest that "tethering" functions may be depressed in the immature host. In vitro human cord blood neutrophils demonstrate decreased rolling compared with adult cells under shear stress on IL-1-stimulated HUVEC monolayers, in part due to diminished function of L-selectin (1, 4). Cord blood neutrophils also fail to tether and roll efficiently on histamine-stimulated HUVEC, E-selectin, and P-selectin monolayers (1, 4, 33). In response to intraperitoneal thioglycollate, 1-day-old rabbits had decreased accumulation of leukocytes compared with adults, and treatment with the anti-L-selectin mAb had no effect on leukocyte accumulation in these 1-day-old animals, although it did in adult animals (12). Consistent with data presented here on 1-wk-old rabbits, 1-day-old and preterm rabbits have diminished surface expression of L-selectin (12, 37). Lorant et al. (32) observed that neonatal rats in vivo have diminished localization of leukocytes to sterile-inflamed peritoneum, which coincided with a marked inability of the neonatal rats to express P-selectin on the inflamed mesenteric vessels, although it was present in abundance in adult animals (32). We propose that selectin-dependent and -independent mechanisms downstream of tethering appear to be specifically affected in neonatal rabbits.

The mechanisms responsible for the transition from a rolling to an arrested leukocyte under relevant physiological shear is not fully understood. Clearly, selectin-dependent tethering and CD18-dependent adhesion are involved. Mechanisms that may trigger sufficient CD18 integrin activation to permit cell arrest include signaling through selectin binding, chemotactic factor stimulation, and perhaps through LFA-1 binding itself (reviewed in Ref. 39). Our observations in the 1-wk-old rabbit may reside in differences in one or more of these processes. In the mesenteric vessels of 1-wk-old rabbits that have been subjected to surgical trauma only, leukocytes rolled faster and did not maintain a prolonged interaction with the endothelial surface. If transit time (i.e., the period of time it takes to traverse a vessel segment that is reflected in rolling velocity) is the main determinant of efficiency of adhesion and ultimately migration, as suggested by Ley and co-workers (26, 28), then increased transit time and a diminished ability to maintain a rolling interaction would result in fewer cells arresting. In both mouse cremaster and rat mesentery venules, P-selectin appears to be primarily responsible for leukocyte rolling early after tissue trauma (i.e., <60 min) (14, 29). One-week-old rabbits, like rat pups, may be deficient in endothelial expression of P-selectin (32) or alternatively P-selectin-signaling may be affected. We cannot yet examine P-selectin function directly in rabbits due to a lack of suitable blocking antibodies. Although many studies have used an anti-human P-selectin that cross-reacts with that of other species, the mechanism of this antibody is in doubt. Gibbs et al. (15) provided evidence that it does not block lectin functions of P-selectin but rather instead inhibits complement interaction with P-selectin short consensus repeats.

Selectins themselves may function in the transition of leukocytes from rolling to stable arrest by transducing adhesion-augmenting signals in the absence of a tethering function. In mice in which E-selectin function was eliminated, either with blocking mAbs or due to loss of gene product, rolling remained intact, but the number of accumulated cells in the skin and peritoneum was markedly diminished (35). L-selectin is rapidly shed from the surface of leukocytes upon activation. Inhibition of L-selectin shedding in vivo not only results in dramatically decreased leukocyte rolling velocities but also increased number of arrested and transmigrated leukocytes (19, 20). Human neutrophils may also be stimulated in vitro through selectins (or their leukocyte ligands) resulting in increased Ca2+ flux, release of oxidative products, increased adhesion, and emigration through activation of Mac-1 and LFA-1 (9, 17, 44).

As has been been reported by von Andrian and colleagues (43), anti-L-selectin mAb inhibited the tethering of leukocytes in IL-1beta -stimulated mesenteric vessels by 45-60 min after exteriorization was begun. However, significant inhibition of arrest and emigration commenced much earlier than the effects on leukocyte rolling. Thus it appears that for both neonatal and adult rabbit leukocytes, L-selectin not only serves a tethering function but also appears to support the transition from rolling to arrest. In addition, L-selectin-dependent arrest is diminished in younger age animals. It is unclear whether this is due to decreased expression of L-selectin and/or a decreased ability to respond to the L-selectin-dependent signal.

We think it is unlikely that we might we have "missed" a large proportion of fluxing leukocytes, thus underestimating differences between adult and newborn animals or alternatively the effect of L-selectin inhibition on leukocyte tethering. Previous studies in rabbits have used fluorescent techniques combined with stroboscopic epiillumination to determine leukocyte rolling (30, 42, 43). Maximum rolling velocities obtained by this method are considerably higher than those obtained under transmitted light (30). However, with the use of fluorescent techniques, the most frequent rolling velocity class was 20-40 µm/s, well within the range of the cell velocities reported here with transmitted light (31). In addition, in models where the vessels are inflamed with cytokines, such as tumor necrosis factor, rolling velocity is much lower (25, 29).

Recent studies support that factors produced by the endothelium, such as IL-8 and platelet-activating factor, are involved in the process of transition from rolling to arrest because these factors result in CD18-dependent adhesion (36, 45). It is unknown whether there is developmental regulation of these or similar chemokines at the inflammatory site in the immature host.

Stationary adhesion requires activation of CD18 integrins. For most adhesive functions of neutrophils, LFA-1 (CD11a/CD18) and Mac-1 (CD11b/CD18) are predominantly involved. Studies of neutrophil emigration through endothelial monolayers in vitro reveal a significantly greater contribution of LFA-1 than Mac-1 (13). Either integrin, however, is sufficient for stopping rolling cells, although both are required for prolonged stable adhesion under shear conditions (16, 21). In vivo, leukocytes roll for prolonged periods and distances before becoming adherent (28). The gradual decrease in rolling velocity before arrest is CD18 dependent, although the specific contribution of Mac-1 or LFA-1 is unknown (28). In the present studies 1-wk-old rabbits had diminished leukocyte arrest in response to IL-1beta despite a diminished rolling velocity compared with adults. This may be due to deficiencies in one or more critical CD18 function. In the neonatal rabbit model of thioglycollate-induced peritonitis, blocking CD18 had less inhibitory effect on peritoneal leukocyte accumulation in 1-day-old rabbits compared with 14-day-old and adult rabbits (12). Neutrophils from human cord blood have diminished emigration under static conditions due to decreased Mac-1 function, but it is unclear whether this also contributes to decreased arrest and emigration under shear conditions (7). Further studies will be necessary to elucidate the contribution of LFA-1 and Mac-1 to leukocyte arrest in vivo both in adult animals and in the developing host.


    ACKNOWLEDGEMENTS

The authors acknowledge the kind gifts of the antibodies from Drs. Takashi Kishimoto and Robert Rothlein and the contributions of Celetta Callaway for production of monoclonal antibodies and the technical and clerical assistance of Maria Hong, Karmen Howard, and Michelle Thomas Sturm. We also thank Drs. Paul Kubes and Samina Kanwar for assistance in establishing the protocol and thoughtful insights.


    FOOTNOTES

* M. M. Mariscalco and W. Vergara contributed equally to this paper.

This study was supported by National Institutes of Health Grant NIH-AI-19031.

Address for reprint requests and other correspondence: M. M. Mariscalco, Texas Children's Hospital, Children's Nutrition and Research Center, 1100 Bates St., Rm. 6014, Houston, Texas 77030-2600 (E-mail: marym{at}bcm.tmc.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

10.1152/ajpheart.00090.2001

Received 16 February 2001; accepted in final form 26 October 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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Am J Physiol Heart Circ Physiol 282(2):H636-H644
0363-6135/02 $5.00 Copyright © 2002 the American Physiological Society



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