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Early Human Development 58 (2000) 25–39www.elsevier.com/ locate /earlhumdev

The distribution of receptors for thepro-inflammatory cytokines interleukin (IL)-6

and IL-8 in the developing human fetus

*J. Benjamin Dame, Sandra E. Juul

Department of Pediatrics, University of Florida College of Medicine, Gainesville, FL 32610-0296,USA

Accepted 12 January 2000

Abstract

Interleukin-6 receptor a (IL-6R) and interleukin-8 receptor (IL-8RB) are widely expressed inadult human and murine tissues. Little is known about the expression of these receptors and thefunction of their ligands in the developing human fetus. Objectives: To determine the tissuedistribution and cellular expression of IL-6R and IL-8RB in the developing human fetus.Methods: Reverse transcription-PCR and immunohistochemical staining were performed onbrain, spinal cord, eye, heart, lung, liver, spleen, adrenal, kidney, intestine, and placenta fromfetuses of 8 and 1662 weeks post-conception. Results: IL-6R and IL-8RB mRNA weredetected in all tissues tested at both time points. Immunoreactivity to anti-IL-6R antibody waspresent on neurons, and in neuropil of the brain, as well as in bone marrow, bronchi,hepatocytes, zona glomerulosa of the adrenal, glomerular cells in kidney, spleen, and placentaltrophoblasts. Cell-specific expression for IL-8RB in the central nervous system was localized tospecific groups of neurons and astrocytes in the brain and spinal cord, including the neuralretina. In somatic organs IL-8RB was detected in bone marrow, myocardiocytes, bronchiolarepithelial cells, hepatocytes, cells of the zona glomerulosa and the zona fasciculata of theadrenal, the collecting system of the kidney, enterocytes of the bowel and in placental cells.Conclusion: The widespread expression of these cytokine receptors suggests anonhematopoietic role for their ligand in the developing fetus. 2000 Elsevier ScienceIreland Ltd. All rights reserved.

Keywords: Pro-inflammatory cytokines; Interleukin-6; Interleukin-8; Human fetus; Cytokine receptors;Tissue distribution; Cellular expression

*Corresponding author. Tel.: 1 1-352-392-4195; fax: 1 1-352-392-4533.E-mail address: [email protected] (S.E. Juul)

0378-3782/00/$ – see front matter 2000 Elsevier Science Ireland Ltd. All rights reserved.PI I : S0378-3782( 00 )00064-5

26 J.B. Dame, S.E. Juul / Early Human Development 58 (2000) 25 –39

1. Introduction

Interleukin (IL)-6 and IL-8 are important pro-inflammatory regulators. IL-6 is amediator of the acute inflammatory response, functioning in concert with othercytokines to induce differentiation of cytolytic T cells and B cells, and to promotesecretion of immune globulins and other acute phase reactants [1,2]. IL-6 acts bybinding to its high affinity receptor IL-6R (gp80), which can be soluble or cell bound.The IL-6 / IL-6R complex then binds to gp130, which results in signal transductionvia the JAK/STAT system [3]. Only the gp80 portion of the IL-6 receptor complex isunique to IL-6, as the gp130 chain is shared with IL-11, ciliary neurotrophic factor,leukemia inhibitory factor, oncostatin M, and cardiotrophin-1 [4].

IL-8 is a chemotactic factor for neutrophils, and has been implicated in manyinflammatory diseases [5,6]. There are two functional receptors for IL-8; IL-8RA (orCXCR1) is unique to IL-8, whereas IL-8RB (or CXCR2) is shared with melanomagrowth stimulatory activity (MGSA), as well as GROa and GCP-2 [7,8]. Bothreceptors are abundantly expressed on neutrophils.

The expression of IL-6R and IL-8RB are not unique to hematopoietic cells, andhave been demonstrated on a variety of nonhematopoietic cell types, includingmurine and adult human neurons [8–11]. IL-6 and its receptor are expressed inpostnatal rat brain in a developmentally regulated manner [12], and IL-6 may haveneurotrophic and/or neuroprotective effects, while IL-8 may be involved in thedeleterious response of cells during inflammation and aging [8,13–15]. Otherreported effects of IL-6 and IL-8 include a growth-promoting effect on cancerouscells of the lungs and kidneys [16,17]. In addition, IL-8 may promote angiogenesis inastrocytomas and glioblastomas [18].

The pro-inflammatory system is generally dampened in the fetus, but little isknown about the expression of these receptors during this period. We hypothesizedthat like the erythropoietin receptor [19], the GM-CSF receptor [20], and the G-CSFreceptor [21], IL-6R and IL-8RB are expressed in a variety of human fetal tissues.The specific objectives of this study were to determine the tissue distribution andcellular expression of IL-6R and IL-8RB in the developing human fetus at 8 and 16weeks gestation.

2. Methods

2.1. Human fetal specimens

Brain, spinal cord, eye, heart, lung, giver, spleen, adrenal, kidney, small bowel, andplacenta were collected from fetuses at 8 and 1662 weeks gestation. Tissues wereeither placed in Bouin’s fixative or 10% formalin for immunohistochemistry, orimmediately preserved in liquid nitrogen for RNA extraction. Only fetuses that werenormal by ultrasound examination and underwent elective pregnancy terminationwere studied. Gestational age was determined by fetal foot and long bone length[22,23]. Additional tissues ranging from 8 weeks to adult were obtained from still

J.B. Dame, S.E. Juul / Early Human Development 58 (2000) 25 –39 27

births and autopsy specimens. The studies were approved by the University of FloridaInstitutional Review Board.

2.2. Identification of specific mRNAs

2.2.1. Preparation of total RNATotal RNA was extracted from the washed tissues using the RNeasy elution kit

(Qiagen, Chatsworth, CA). Manufacturer’s directions were followed. Purity andconcentration of extracted RNA was determined by measuring UV absorbance at 260and 280 nm. Total RNA was treated with RNase–free DNase I (Gibco, Rockville,MD) prior to further experimentation.

2.2.2. Reverse transcription of RNA and amplification of cDNAReverse transcription of RNA and amplification of cDNA was performed using a

Thermocycler (Stratagene, La Jolla, CA). Total RNA (2.0 mg) was combined with the2.0 mM oligo (dT) primers (Gibco, Gaithersburg, MD), heated to 708C for 10 minthen placed on ice. The mixture was then combined with 250 mM dNTP (Gibco), 0.01M DTT, 50 mM Tris, pH 8.3, 75 mM KCl, 3 mM MgCl . Following a 428C2

incubation for 2 min, 2 ml of Superscript II (reverse transcriptase, Gibco) was added,and the mixture incubated for 50 min further. The reaction was terminated by heatingto 708C for 15 min. Amplification cycles were carried out under the followingconditions: 10 mM Tris, pH 8.3, 50 mM KCl, 1.5 mM MgCl , 2.0 mM dNTP, 0.22

mM upstream and downstream primers, 5% reverse transcriptase mix, 0.1 units /mlTaq 948C for 3 min followed by 30–35 cycles of 948C for 1 min, 588C for 1 min,728C for 2 min, concluded by 10 min elongation at 728C. Amplification with b-actinprimers was performed using the above procedure to confirm the efficacy of thereverse transcription process for each RNA-extracted tissue. Primer pairs used and thecDNA product length are as follows: IL-6R: 59-GAGGGAGACAGCTCTTTCTAC,59-CCGTTCAGCCCGATATCTGAG, 240 bp; IL-8RB: 59-GGCCGACCTAC-TCTTTGC, 59-CCGCAACACCATCCGCCATTT, 360 bp, b-Actin, 59-TGACGGGGTCACCCACACTGT GCCCATCTA, 59-CTAGAAGCATTTGCGGTGGA CGATGGAGGG, 661 bp.

2.2.3. Specific IL-6R and IL-8RB identificationSpecificity of the RT-PCR products were confirmed by direct sequencing of an

amplified PCR product using pCR vector (Invitrogen, Carlsbad, CA). The plasmidwas extracted and the vector of interest was then sequenced by the ICBR Core labs atthe University of Florida. Sequences were BLAST (NIH) searched and found to beunique to the respective cytokine receptors.

2.2.4. ImmunohistochemistryTissues were fixed in Bouin’s fixative for 6 h, removed to 70% alcohol for

overnight incubation, then paraffin-embedded, or fixed in 10% buffered formalinovernight and paraffin embedded. Six-mm sections were deparaffinized in xylene andrehydrated through a graded series of alcohols. A 30-min incubation in 10 mg/ l


Saponin at room temperature was used for antigen retrieval. Tissues were stainedusing the Ventana NEXUS Automated Immunohistochemical staining system (Ven-tana, Tucson, AZ) [20]. Primary antibody reactions were done for 38 min at 378C.Sections were lightly counterstained with hematoxylin and bluing reagent for 2 mineach. Antihuman IL-6R antibody (Santa Cruz Biotechnology, Santa Cruz, CA) andanti-human IL-8RB antibody (Santa Cruz) were used at 1:25 and 1:50 dilution,respectively. Phosphate-buffered saline containing 0.1% Triton X-100 was used as thediluent for all antibody reactions. Tonsil and placenta were used as positive controls.Absence of primary antibody and incubation with preimmune serum were used asnegative controls.

3. Results

Expression of mRNA for IL-6R (gp80) and IL-8RB (CXCR2) was detected byRT-PCR in eye, heart, lung, liver, spleen, adrenal, kidney, intestine, and placenta atboth gestational periods. The top panel of Fig. 1A shows IL-6R cDNA fragmentsamplified from organs at 1662 weeks gestation. CNS tissue including brain, brainstem, and spinal cord at gestational ages ranging from 6 to 18 weeks also expressed

Fig. 1. RT-PCR results for IL-6R and IL-8. This figure shows a representative ethidium bromide-stained2% (w/v) agarose gel showing RT-PCR cDNA fragments for IL-6R (upper panels) and IL-8R (bottompanels) from somatic organs (A) and from CNS tissues (B). Lane 1 of (A) shows the 100-bp ladder, whilelanes 2–9 show RT-PCR fragments amplified from heart, lung, liver, spleen, adrenal, kidney, stomach, andplacenta, respectively. Lane 10 shows the positive control (T cells for IL-6R, neutrophils for IL-8R), andlane 11 shows the negative control; RNA which was not reverse transcribed. (B) Brain from 8- and18-week gestation brain, 8- and 9-week brain stem, and 8- and 17-week gestation spinal cord.


mRNA for IL-6R (see Fig. 1B). cDNA fragments representing IL-8RB mRNAexpression are shown in the bottom panels of Fig. 1A,B. Expression of each receptormRNA did not change with increasing gestational age; thus, only the 16-week resultsare shown.

As RT-PCR is an extremely sensitive technique, it is possible that hematopoieticcell contamination could account for the detection of IL-6 and IL-8 receptor in thetissues tested. We therefore used immunohistochemistry as a complementary tech-nique to specify the cellular expression of these receptors. Table 1 contains asummary of the specimens studied, and the RT-PGR and immunohistochemicalfindings at each gestational age.

The distribution of IL-6R was more limited in cell specificity than predicted byRT-PCR. Cell-specific reactivity was detected in selected neurons of the CNS. Forexample, pyramidal cells of layer III, V and VI of the cerebral cortex were stronglyimmunoreactive (Fig. 2A), while only a select population of neurons in the basalbrain stained (Fig. 2B). In the cerebellum, Purkinje cells were strongly reactive, aswere basket cells in the molecular layer (Fig. 2C). The developing neural retina alsoshowed moderate reactivity (Fig. 2D); however, no reactivity was noted in spinalcord neurons, nor were astrocytes observed to be immunoreactive.

The distribution of IL-8R in the central nervous system differed somewhat fromthat of IL-6R. Fig. 3A shows IL-8RB reactivity on pyramidal neurons in the cerebralcortex. In contrast to the absence of IL-6R staining in the spinal cord, IL-8Rreactivity was present on anterior horn cells (Fig. 3B), and in neurons of Clarke’scolumn. Cells of astrocytic morphology in the brain white matter were alsoimmunoreactive (Fig. 3C), as was the neural retina (Fig. 3D).

In somatic tissues, strong IL-6R immunoreactivity was identified in osteoclasts,osteocytes and osteoblasts of the developing bone marrow (Fig. 4A), on placentalsyncytiotrophoblasts (Fig. 4B), and in renal glomeruli, (particularly the least matureglomeruli) in the outer cortex (Fig. 4C). The proximal collecting tubules showed noimmunoreactivity, while the distal convoluted tubules and the collecting ducts wereweakly reactive. Moderate reactivity was noted in cells of the adrenal zonaglomerulosa (Fig. 4D), in cells lining the bronchi of 9-week lung (Fig. 4E),hepatocytes (Fig. 4F), with diffuse weak reactivity noted in the red pulp region of thespleen (Fig. 4G). No immunoreactivity was observed in myocardial tissues (Fig. 4H)or fetal bowel (Fig. 4I), although in postnatal bowel, immunoreactivity was present inenterocytes (not shown).

Strong IL-8RB immunoreactivity was detected in osteoclasts, osteoblasts andosteocytes, as well as in cells which appear to be of granulocytic lineage in the bonemarrow (Fig. 5A). Placental syncytiotrophoblasts and cytotrophoblasts were alsostrongly reactive, as were placental Hofbauer cells (Fig. 5B). The pattern ofimmunoreactivity in other somatic organs differed for IL-8R as compared to IL-OR.Moderate immunoreactivity was noted in myocardiocytes at both 9 and 17 weeks ofgestation (Fig. 5C). Moderate immunoreactivity was also present at the apical surfaceof bronchial epithelial cells (Fig. 5D), and in the collecting ducts and distal collectingtubules of the developing kidney, with only weak staining of glomeruli (Fig. 5E).Cells in the adrenal zona glomerulosa and zona fasciculata (Fig. 5F), enterocytes of


Table 1IL-6R and IL-8R evaluation in fetal tissues

Tissue GA PCR Immuno PCR Immunoa(weeks) IL-6R IL-6R IL-8R IL-8R

Brain / 8–9 Positive Weakly positive Positive Weakly positive

Brainstem undifferentiated neurons undifferentiated neuronsa10–11 Positive ND Positive ND

16–18 Positive ND Positive ND

32 ND Strongly immunoreactive ND Strongly immunoreactive

neurons neurons and astrocytes

Adult ND Strongly immunoreactive ND Strongly immunoreactive

neurons neurons and astrocytes

Cerebellum 29–32 Positive Positive Purkinje cells Positive Negative

Spinal cord 8–9 Positive ND Positive ND

16–18 Positive ND Positive ND

Adult ND Negative ND Positive neurons

Eye 8–10 Positive Positive neural retina Positive Positive neural retina

16 Positive ND Positive ND

Lung 8–9 Positive Positive bronchial epithelial Positive Positive bronchial epithelial

cells (basolateral surface) cells (apical surface)

16–17 Positive ND Positive Positive bronchial epithelial

cells (apical surface)

Heart 8 Positive Neg Positive Positive myocardiocytes

17 Positive Neg Positive Positive myocardiocytes

Liver 8 Positive ND Positive Positive hepatocytes

16 Positive positive hepatocytes Positive Positive hepatocytes, and

hematopoietic elements

Spleen 8 Positive ND Positive ND

16 Positive Diffuse reactivity Positive ND

Adrenal 8 Positive Negative Positive Diffuse, weak staining

16 Positive Positive cells in zona Positive Positive cells in zona

glomerulosa glomerulosa and

zona fasciculata

Kidney 8 Positive Positive early glomeruli Positive Positive early glomeruli

16 Positive Positive undifferentiated Positive Positive collecting system with

epithelial cells, and weaker staining of glomeruli

immature glomeruli

Bowel 8 Positive ND Positive ND

16 Positive Negative Positive Positive enterocytes

Term ND Positive enterocytes Positive ND

(3 months)

Placenta 8 Positive Positive Positive Positive syncytiotrophoblasts,

syncytiotrophoblasts cytotrophoblasts and Hofbauer cells

16 Positive Positive Positive Positive syncytiotrophoblasts

syncytiotrophoblasts

Term ND ND ND Positive syncytiotrophoblasts

Clavicle 8 Positive ND Positive ND

14–16 Positive Positive osteoblasts, Positive Positive osteoblasts,

osteocytes, osteoclasts osteocytes, osteoclasts,

granulocytes

a GA, gestational age; ND, not done.


Fig. 2. IL-6R (gp80) immunoreactivity in the CNS. DAB was used as the chromogen (brown). (A)Immunoreactive pyramidal neurons from layer III of the adult cerebral cortex (original magnification,3 400). An arrow indicates a neuronal cell body. (B) Selective staining of neurons (arrow) in the basalbrain (original magnification, 3 400). (C) Immunoreactive Purkinje cells and basket cells from acerebellum of 30 weeks gestation ( 3 400); (D) the immunoreactive neural retina (arrow) at 10 weeksgestation ( 3 200).

the developing bowel (Fig. 5G) and hepatocytes (Fig. 5H) also showed moderateimmunoreactivity.

4. Discussion

Many cytokines have now been identified which show a wider spectrum of activitythan was first appreciated. Among these are erythropoietin [19,24–26], GM-CSF[20], G-CSF [21], vasointestinal peptide [27,28], and many interleukins[13,15,29,30]. These cytokines have typically been functionally identified in onesystem, only to have further roles delineated later. IL-6 and IL-8 appear to be furtherexamples.

IL-6 was first identified as a product of monocytes, but has since been shown to beproduced by a variety of somatic and CNS cell types, including fibroblasts,endothelial cells, astrocytes, and microglia, as well as by cells from human


Fig. 3. IL-8RB (CXCR2) Immunoreactivity in the CNS. (A) Immunoreactive pyramidal neurons of thecerebral cortex (original magnification, 3 400). An arrow indicates a neuronal cell body. (B) Selectivestaining of an anterior horn cell (arrow) and a neuron in Clarke’s nucleus of the spinal cord (originalmagnification, 3 400). (C) An immunoreactive astrocyte in the deep white matter ( 3 400); (D) theimmunoreactive neural retina (arrow) at 10 weeks gestation ( 3 200).

astrocytomas [9,31]. IL-6 mRNA production in brain has been localized to Purkinjecells and hippocampal neurons [12,32], and functional receptors have been demon-strated on PC12 cells, sympathetic neurons, cholinergic neurons, and culturedastrocytes [33,34]. Tumor necrosis factor-a is one cytokine involved in the upregula-tion of IL-6R expression in neurons, while corticosteroids downregulate receptorexpression [10].

Increasing evidence supports an essential role for IL-6 in the development anddifferentiation of neurons and astrocytes in the CNS. Studies involving knockoutmice have shown that IL-6 is important for the modulation of neurosensory functionsin vivo, and for survival of subgroups of differentiated motor and sensory neurons aswell as for the differentiation of major populations of astrocytes in vivo [33–35] theseeffects may, in part, be mediated by IL-6 induction of neurotrophin expression [33].

There is also convincing evidence that it plays a role in the modulation ofinflammatory or pathological events. For example, elevated IL-6 concentrations havebeen measured in the brains of Alzheimer patients, and IL-6 immunoreactivity hasbeen identified at the site of primitive plaques, raising the question as to whether IL-6


Fig. 4. IL-6R (gp80) immunoreactivity in somatic organs. (A) Strong immunoreactivity of selected cells ina 14-week gestation bone marrow. (B) The strong cell-specific staining of placental syncytiotrophoblasts inthe mid-trimester placenta. In the 15-week kidney (C), immunoreactivity is noted in those glomeruli closestto the cortical rim (arrow head). Moderate immunoreactivity of cells in the zona glomerulosa of the16-week adrenal, and of cells lining the bronchi in a 9-week gestation lung are shown in (D) and (E),respectively. Moderate staining is also present in hepatocytes at 16 weeks (F) (original magnification,3 200), with absence of staining in the hematopoietic islands. Diffuse reactivity was noted in the red pulpof an 18-week spleen (G), and no immunoreactivity is present in a 16-week gestation myocardium (H) orin 16-week gestation small bowel (I) (original magnification, 3 200). All photomicrographs were taken atoriginal magnification of 3 100, except where noted.

contributes to brain injury in this disease [15,36,37]. Transgenic mice, in whichhuman IL-6 expression has been targeted to neurons, develop reactive astrocytosisand an increase in ramified microglial cells, but do not show evidence of neurondamage [38]. There is also an association with intrauterine infection; elevatedamniotic fluid concentrations of IL-6 correlate with neonatal white matter damage inhuman infants [39]. One mechanism by which IL-6 may affect the susceptibility ofcertain neurons to damage is by altering the intracellular calcium responses to NMDA[40]. Finally, IL-6 also may affect emotional lability, as null mutations of thiscytokine result in ‘increased emotionality’ of affected mice [41]. Thus it is clear thatIL-6 can have positive or harmful effects in the CNS, depending on the circum-stances.


Fig. 5. IL-8RB (CXCR2) immunoreactivity in somatic organs. (A) Strong immunoreactivity of selectedcells in a 14-week gestation bone marrow (original magnification, 3 200). Strong cell-specific staining ofplacental syncytiotrophoblasts and cytotrophoblasts are shown in the pre-term placenta in (B). (C)Immunoreactivity in a 17-week gestation myocardium; (D) immunoreactivity of bronchial epithelial cellsin a 16-week gestation lung. In the 15-week kidney (E), immunoreactivity is noted primarily in thecollecting tubules (arrow), less so in the glomeruli. (F) Specific reactivity in the zona glomerulosa and zonafasciculata of the 16-week adrenal ( 3 200). Moderate reactivity was identified in enterocytes of the16-week gestation small bowel villi (G) ( 3 200), and in hepatocytes at 16 weeks (H). (I) A negativecontrol placenta. All photomicrographs were taken at original magnification of 3 100, except where noted.

We have shown expression of IL-6R mRNA, and the local cellular expression ofthis receptor, on subsets of CNS neurons and in Purkinje cells in the developinghuman fetus. We also demonstrated the fetal neural retina expressed IL-6R. It hasbeen reported that retinal pigmented epithelial cells are capable of producing IL-6[42], suggesting a possible interaction of these cells and the neural retina.

In addition to CNS expression, we identified IL-6R expression in a wide spectrumof somatic organs. As expected, cell-specific staining was observed in osteoclasts,osteoblasts and osteocytes of bone marrow. IL-6 has an important role in theregulation of bone absorption. This cytokine appears to mediate the effects of PTH aswell as the upregulation of osteoclastogenesis caused by androgen deficiency orestrogen deficiency [43–46]. Although IL-6 is known to induce B cell differentiatIonand other hematopoietic progenitors, immunoreactivity in hematopoietic elements


was weak at 14–16 weeks gestation. We did not detect IL-6R expression in thedeveloping heart, although IL-6 has previously been reported to interact withmyocardial gp130 to cause myocardial hypertrophy in mice [47]. IL-6R expressionwas detected in bronchial epithelial cells, which have previously been shown toconstitutively express functional IL-6R, and IL-6 stimulation increases IL-6R mRNAproduction as well as IL-8 release [48].

Murine studies have shown that IL-6R mRNA is expressed in liver and spleen,with upregulation by IL-6, dexamethasone, and LPS administration [49,50]. We alsoobserved moderate expression of IL-6R in liver hepatocytes, and in the red pulpregion of the spleen. The role of IL-6 in these developing organs is not clear.

IL-6 is a potent activator of the human hypothalamic–pituitary–adrenal axis, andthe administration of IL-6 can moderate the secretion of cortisol and ACTH [30,51].In the adrenal gland, IL-6 regulates adrenal synthesis of mineralocorticoids, glucocor-ticoids, and androgens in vitro, suggesting that IL-6 can act as a direct regulator ofstress response [51,52]. We identified IL-6R expression in the fetal adrenal,corroborating these animal data. We also found the undifferentiated epithelium in theoutermost immature layer of the renal cortex to be highly immunoreactive. The mostimmature layer of glomeruli were highly reactive with IL-6R antibody, suggesting apossible role for this cytokine in renal development. The IL-6 knock-out mouse,however, has normal development of glomerular architecture and mesangium, thusthe role of this cytokine in renal development is likely redundant [53]. Glomerularepithelial cells have previously been reported to express IL-6 and IL-6R, both ofwhich are up-regulated by stimulation with LPS, IL-1b or TNFa.

We did not detect IL-6R expression in fetal small bowel, although it was present inpostnatal bowel. This may reflect a change in receptor expression with ontogeny, ormerely a lack of immune challenge in the fetal bowel. Adults show constitutiveexpression of IL-6R in normal bowel, with enhanced expression of IL-6R after LPSand IFN-g stimulation [54].

Placental syncytiotrophoblasts showed robust staining for IL-6R. This confirmsprevious studies indicating that placenta expresses IL-6R, as detected by flowcytometry and immunohistology [55].

As with IL-6R, IL-8RB mRNA and protein expression showed a wide distributionin the in the CNS and in somatic tissues. Immunostaining allowed us to delineate thedistribution of these receptors in the developing fetus. In the CNS, pyramidal neuronsof the cerebral cortex showed specific staining, as did neurons in the spinal cord.Purkinje cells in the cerebellum were not immunoreactive. Although never previouslyreported in the fetus, this distribution of IL-8R has been reported in adult brain [8,11].We also identified IL-8R reactivity in astrocytes. The potential for local production ofIL-8 by astrocytes and the presence of functional receptors for IL-8 on neuronssuggests a possible paracrine interaction between neurons and astrocytes and anautocrine function for astrocytes [56,57]. This may be an important pathway in thebrain’s response to infection. The developing neural retina also showed reactivity toanti-IL-8 RB antibody. Many cells types in the eye produce IL-8 in response toinflammatory cytokines, LPS, or UV insult. Some of these including retinalpigmented epithelial cells [58], orbital fibroblasts [59], and corneal stromal cells [60].


Although the IL-8RB (CXCR2) is not unique to IL-8, it is responsive to IL-8. Thelocal production of IL-8 suggests a role for IL-8 in the eye during development.

Less is known about the developmental role of IL-8 than IL-6. We have identifiedmoderate IL-8R immunoreactivity in several organs, including lung, liver, adrenal,kidney and bowel. Although the receptor has been identified in these tissues in adults,it is thought primarily to be a mediator of inflammatory response [17,61,62], ratherthan a trophic factor. Our findings raise the possibility that IL-8 may play apreviously unidentified, but important role.

IL-8R staining was very prominent in the placenta in the syncytiotrophoblast layeras well as the cytotrophoblast layer. Hofbauer cells, which have a phagocyticfunction, were also immunoreactive. It is known that the placenta produces IL-8 in aregulated manner, and it is hypothesized that this cytokine is important in theinitiation of labor [63–65].

In conclusion, we have shown that receptors for IL-6 and IL-8 are widelydistributed in the developing human fetus. Our findings, along with the previouslyknown distribution and function of IL-6R and IL-8RB support possible developmen-tal roles for these receptors in the fetus. Further studies are needed to define thesefunctions.

Acknowledgements

This work was supported by MCAP award RR-00083, by the Howard HughesMedical Institute. Research Resources Program of the University of Florida Collegeof Medicine, and by a Stop Cancer grant. We would like to thank Dr RobertChristensen for his guidance and editorial aid, and Dr Anthony Yachnis for sharinghis expertise in histology.

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