Undetectable interleukin (IL)-10 and persistent IL-8 expression early in hyaline membrane disease: a possible developmental basis for the predisposition to chronic lung inflammation in preterm newborns

Abstract

We are interested in determining whether premature birth alters expression of counterregulatory cytokines which modulate lung inflammation. Production of proinflammatory cytokines tumor necrosis factor alpha. IL-1 beta, and IL-8 is regulated in part by the antiinflammatory cytokine IL-10. For preterm newborns with hyaline membrane disease, deficiencies in the ability of lung macrophages to express antiinflammatory cytokines may predispose to chronic lung inflammation. We compared the expression of pro- and antiinflammatory cytokines at the mRNA and protein level in the lungs of preterm and term newborns with acute respiratory failure from hyaline membrane disease or meconium aspiration syndrome. Four sequential bronchoalveolar lavage (BAL) samples were obtained during the first 96 h of life from all patients. All patients rapidly developed an influx of neutrophils and macrophages. Over time, cell populations in both groups became relatively enriched with macrophages. The expression of proinflammatory cytokine mRNA and/or protein was present in all samples from both patient groups. In contrast, IL-10 mRNA was undetectable in most of the cell samples from preterm infants and present in the majority of cell samples from term infants. IL-10 concentrations were undetectable in lavage fluid from preterm infants with higher levels in a few of the BAL samples from term infants. These studies demonstrate that 1) IL-10 mRNA and protein expression by lung inflammatory cells is related to gestational age and 2) during the first 96 h of life neutrophil cell counts and IL-8 expression decrease in BAL from term infants, but remain unchanged in BAL samples from preterm infants.

Figure 1

BAL total cell counts along with cell differentials are shown for the 17 term and 17 preterm patients. The total numbers of cells are higher in the BAL samples from the term newborns at the initial (p = 0.085) and 24-h sample times (p = 0.013). These differences are largely due to the numbers of red blood cells (RBC) found in the BAL from the term newborns, although neutrophil numbers are also higher in the term BAL samples initially (see Fig. 3). These same samples from the term patient group show an increase in the percentages of both neutrophils and macrophages as RBC decrease. In the BAL samples from preterm newborns the percentage of neutrophils remains stable over the 96-h sample period, whereas their BAL cell population becomes enriched with macrophages. The percentage of RBC (p = 0.07) and macrophages(p = 0.03) is higher in the term samples at the initial sample time. There are no other significant differences in the percentages of each cell type at any sample time. Data are expressed as the mean ± SD.

Figure 2

This photomicrograph shows representative BAL derived cell populations from term and preterm newborn lungs. (A) Initial sample from a term newborn. The majority of cells are red blood cells, yet the initial samples from the term newborns contained the highest numbers of neutrophils at any time point. The initial samples from preterm newborn contained fewer red blood cells and about one-half the number of neutrophils compared to samples from term newborns. (B) A 48-h sample from a preterm newborn. By 48 h the BAL cell populations from the term and preterm patients are similar and are predominantly made up of neutrophils and macrophages.

Figure 3

BAL neutrophil and macrophage cell counts are shown over the 96-h sampling period for the term and preterm patient groups. Neutrophil counts are higher in the BAL taken from the term newborns at the initial and 24-hour sample time. These differences do not reach statistical significance (initial, p = 0.22; 24-h, p = 0.09) due to the large intragroup variability to cell counts. The neutrophil counts in the BAL from the term newborns decline, and by 48 h the numbers of neutrophils in the term and preterm BAL samples are similar. Macrophage cell counts are similar in the BAL cell populations from both patient groups over the 96-h sampling period. Data are expressed as the mean ± SD.

Figure 4

The presence of IL-8 and IL-10 mRNA in the BAL-derived inflammatory cell populations is analyzed by RT-PCR. This gel demonstrates a comparison of the ability to detect these mRNA in the cell samples from term and preterm newborns. The mRNA for the potent neutrophil chemo-attractant IL-8 is detectable in the cell populations obtained from both the preterm and term newborns during the entire sampling period. In sharp contrast, mRNA for the anti-inflammatory cytokine IL-10 is detected in the BAL derived cells from term newborns only. The difference in the ability to detect IL-10 mRNA in the BAL cells from term and preterm patients is significant (seeTable 4).

Figure 5

The levels of the proinflammatory cytokines TNF-α, IL-1β, and IL-8, as well as the antiinflammatory cytokine IL-10 were measured in the BAL supernatants from five term and five preterm newborns at all sampling times. IL-8 and IL-1β were initially higher in the term BAL samples and then began to decline by 48 to 96 h, respectively. In contrast to the term BAL samples, levels of IL-8 remained constant in the preterm BAL samples. At the same time, IL-10 levels were undetectable in the BAL supernatants obtained from preterm newborns. Although IL-10 protein levels were low in the majority of samples from both patient groups, elevated IL-10 levels were found in a select few of the BAL supernatants from term newborns at the first three sampling points. There was no significant difference in BAL cytokine protein levels between the term and preterm groups even at the initial and 24-h sample times due to the number of patients studied. It is noteworthy that TNF-α levels were increasing in the term BAL samples at 48-96 h, whereas IL-8 and IL-1β were declining. Data are expressed as mean ± SD.