Alexander Brack

Disease pattern in cranial and large-vessel giant cell arteritis

OBJECTIVE To identify variables that distinguish large-vessel giant cell arteritis (GCA) with subclavian/axillary/brachial artery involvement from cranial GCA. METHODS Seventy-four case patients with subclavian/axillary GCA diagnosed by angiography and 74 control patients with temporal artery biopsy-proven GCA without large vessel involvement matched for the date of first diagnosis were identified. Pertinent initial symptoms, time delay until diagnosis, and clinical symptoms, as well as clinical and laboratory findings at the time of diagnosis, were recorded by retrospective chart review. Expression of cytokine messenger RNA in temporal artery tissue from patients with large-vessel and cranial GCA was determined by semiquantitative polymerase chain reaction analysis. Distribution of disease-associated HLA-DRB1 alleles in patients with aortic arch syndrome and cranial GCA was assessed. RESULTS The clinical presentation distinguished patients with large-vessel GCA from those with classic cranial GCA. Upper extremity vascular insufficiency dominated the clinical presentation of patients with large-vessel GCA, whereas symptoms related to impaired cranial blood flow were infrequent. Temporal artery biopsy findings were negative in 42% of patients with large-vessel GCA. Polymyalgia rheumatica occurred with similar frequency in both patient groups. Large-vessel GCA was associated with higher concentrations of interleukin-2 gene transcripts in arterial tissue and overrepresentation of the HLA-DRB1*0404 allele, indicating differences in pathogenetic mechanisms. CONCLUSION GCA is not a single entity but includes several variants of disease. Large-vessel GCA produces a distinct spectrum of clinical manifestations and often occurs without involvement of the cranial arteries. Large-vessel GCA requires a different approach to the diagnosis and probably also to treatment.

Opioid peptide-expressing leukocytes: Identification, recruitment, and simultaneously increasing inhibition of Inflammatory pain

Background Inflammatory pain can be effectively controlled by an interaction of opioid receptors on peripheral sensory nerve terminals with opioid peptides released from immune cells upon stressful stimulation. To define the source of opioid peptide production, we sought to identify and quantify populations of opioid-containing cells during the course of Freund’s complete adjuvant–induced hind paw inflammation in the rat. In parallel, we examined the development of stress-induced local analgesia in the paw. Methods At 2, 6, and 96 h after Freund’s complete adjuvant inoculation, cells were characterized by flow cytometry using a monoclonal pan-opioid antibody (3E7) and antibodies against cell surface antigens and by immunohistochemistry using a polyclonal antibody to &bgr;-endorphin. After magnetic cell sorting, the &bgr;-endorphin content was quantified by radioimmunoassay. Pain responses before and after cold water swim stress were evaluated by paw pressure thresholds. Results In early inflammation, 66% of opioid peptide–producing (3E7+) leukocytes were HIS48+ granulocytes. In contrast, at later stages (96 h), the majority of 3E7+ immune cells were ED1+ monocytes or macrophages (73%). During the 4 days after Freund’s complete adjuvant inoculation, the number of 3E7+ cells increased 5.6-fold (P < 0.001, Kruskal–Wallis test) and the &bgr;-endorphin content in the paw multiplied 3.9-fold (P < 0.05, Kruskal–Wallis test). In parallel, cold water swim stress–induced analgesia increased by 160% (P < 0.01, analysis of variance). Conclusions The degree of endogenous pain inhibition is proportional to the number of opioid peptide–producing cells, and distinct leukocyte lineages contribute to this function at different stages of inflammation. These mechanisms may be important for understanding pain in immunosuppressed states such as cancer, diabetes, or AIDS and for the design of novel therapeutic strategies in inflammatory diseases.

Glucocorticoid-mediated repression of cytokine gene transcription in human arteritis-SCID chimeras.

Giant cell arteritis (GCA) is a vasculitic syndrome that preferentially affects medium and large-sized arteries. Glucocorticoid therapy resolves clinical symptoms within hours to days, but therapy has to be continued over several years to prevent disease relapses. It is not known whether and how glucocorticoids affect the function of the inflammatory infiltrate or why the disease persists subclinically despite chronic treatment. GCA is self-sustained in temporal arteries engrafted into SCID mice, providing a model in which the mechanisms of action and limitations of glucocorticoid therapy can be examined in vivo. Administration of dexamethasone to temporal artery-SCID chimeras for 1 wk induced a partial suppression of T cell and macrophage function as indicated by the reduced tissue concentrations of IL-2, IL-1beta, and IL-6 mRNA, and by the diminished expression of inducible NO synthase. In contrast, synthesis of IFN-gamma mRNA was only slightly decreased, and expression of TGF-beta1 was unaffected. These findings correlated with activation of the IkappaBalpha gene and blockade of the nuclear translocation of NFkappaB in the xenotransplanted tissue. Dose-response experiments suggested that steroid doses currently used in clinical medicine are suboptimal in repressing NFkappaB-mediated cytokine production in the inflammatory lesions. Chronic steroid therapy was able to deplete the T cell products IL-2 and IFN-gamma, whereas the activation of tissue-infiltrating macrophages was only partially affected. IL-1beta transcription was abrogated; in contrast, TGF-beta1 mRNA synthesis was steroid resistant. The persistence of TGF-beta1-transcribing macrophages, despite paralysis of T cell function, may provide an explanation for the chronicity of the disease, and may identify a novel therapeutic target in this inflammatory vasculopathy.

Rapid upregulation of μ opioid receptor mrna in dorsal root ganglia in response to peripheral inflammation depends on neuronal conduction

S.c. painful inflammation leads to an increase in axonal transport of opioid receptors from dorsal root ganglia (DRG) toward the periphery, thus causing a higher receptor density and enhanced opioid analgesia at the injured site. To examine whether this increase is related to transcription, the mRNA of Delta- (DOR) and mu-opioid receptor (MOR) in lumbar DRG was quantified by real time Light Cycler polymerase chain reaction (LC-PCR), and correlated to ligand binding in DRG and sciatic nerve. In normal DRG, DOR mRNA was seven times less abundantly expressed than MOR mRNA. After induction of unilateral paw inflammation, mRNA content for DOR remained unchanged, but a bi-phasic upregulation for MOR mRNA with an early peak at 1-2 h and a late increase at 96 h was found in ipsilateral DRG. As no changes were observed in DRG of the non-inflamed side, this effect was apparently not systemically mediated. A significant increase in binding of the MOR ligand DAMGO was detected after 24 h in DRG, and after early and late ligation in the sciatic nerve, indicating an enhanced axonal transport of MOR in response to inflammation. The early increase in MOR mRNA could be completely prevented by local anesthetic blockade of neuronal conduction in sciatic nerve. These data suggest that mRNA of the two opioid receptors DOR and MOR is differentially regulated in DRG during peripheral painful inflammation. The apparently increased axonal transport of MOR in response to this inflammation is preceded by upregulated mRNA-transcription, which is dependent on neuronal electrical activity.

Pain control by CXCR2 ligands through Ca2+-regulated release of opioid peptides from polymorphonuclear cells

Leukocytes counteract inflammatory pain by releasing opioid peptides, which bind to opioid receptors on peripheral sensory neurons. In the early phase of inflammation, polymorphonuclear cells (PMN) are the major source of opioids. Their recruitment is governed by ligands at the chemokine receptor CXCR2. Here, we examined whether chemokines can also induce opioid peptide secretion from PMN and thus inhibit inflammatory pain. In rats with hindpaw inflammation, intraplantar injection of CXCL2/3, but not of the CXCR4 ligand CXCL12, elicited naloxone‐reversible (i.e., opioid receptor mediated) mechanical and thermal analgesia, which was abolished by systemic PMN depletion. Both CXCR1/2‐ and CXCR4‐ligands induced PMN chemotaxis, but only CXCR1/2 ligands triggered opioid release from human and rat PMN in vitro. This release was unaltered by extracellular Ca2+ chelation, was mimicked by thapsigargin and was blocked by inhibitors of the inositol 1,4,5‐triphosphate receptor (IP3) and by intracellular Ca2+ chelation, indicating that it required Ca2+ from intracellular but not extracellular sources. Furthermore, release was partially reduced by phosphoinositol‐3‐kinase (PI3K) inhibitors. Adoptive transfer of allogenic PMN into PMN‐depleted rats reconstituted CXCL2/3‐induced analgesia, which was inhibited by prior ex vivo chelation of intracellular Ca2+. These findings demonstrate that, beyond cell recruitment, CXCR2 ligands induce Ca2+ ‐regulated opioid release from PMN and thereby inhibit inflammatory pain in vivo.—Rittner, H. L., Labuz, D., Schaefer, M., Mousa, S. A., Schulz, S., Schäfer, M., Stein, C., and Brack, A. Pain control by CXCR2 ligands through Ca2+ ‐regulated release of opioid peptides from polymorphonuclear cells. FASEB J. 20, E2177–E2188 (2006)

Chronic morphine use does not induce peripheral tolerance in a rat model of inflammatory pain

Although opioids are highly effective analgesics, they are also known to induce cellular adaptations resulting in tolerance. Experimental studies are often performed in the absence of painful tissue injury, which precludes extrapolation to the clinical situation. Here we show that rats with chronic morphine treatment do not develop signs of tolerance at peripheral mu-opioid receptors (micro-receptors) in the presence of painful CFA-induced paw inflammation. In sensory neurons of these animals, internalization of mu-receptors was significantly increased and G protein coupling of mu-receptors as well as inhibition of cAMP accumulation were preserved. Opioid receptor trafficking and signaling were reduced, and tolerance was restored when endogenous opioid peptides in inflamed tissue were removed by antibodies or by depleting opioid-producing granulocytes, monocytes, and lymphocytes with cyclophosphamide (CTX). Our data indicate that the continuous availability of endogenous opioids in inflamed tissue increases recycling and preserves signaling of mu-receptors in sensory neurons, thereby counteracting the development of peripheral opioid tolerance. These findings infer that the use of peripherally acting opioids for the prolonged treatment of inflammatory pain associated with diseases such as chronic arthritis, inflammatory neuropathy, or cancer, is not necessarily accompanied by opioid tolerance.

Control of inflammatory pain by chemokine-mediated recruitment of opioid-containing polymorphonuclear cells.

&NA; Opioid‐containing leukocytes can counteract inflammatory hyperalgesia. Under stress or after local injection of corticotropin releasing factor (CRF), opioid peptides are released from leukocytes, bind to opioid receptors on peripheral sensory neurons and mediate antinociception. Since polymorphonuclear cells (PMN) are the predominant opioid‐containing leukocyte subpopulation in early inflammation, we hypothesized that PMN and their recruitment by chemokines are important for peripheral opioid‐mediated antinociception at this stage. Rats were intraplantarly injected with complete Freunds adjuvant (CFA). Using flow cytometry, immunohistochemistry, and ELISA, leukocyte subpopulations, chemokine receptor (CXCR2) expression on opioid‐containing leukocytes and the CXCR2 ligands keratinocyte‐derived chemokine (KC), macrophage inflammatory protein‐2 (MIP‐2) and cytokine‐induced neutrophil chemoattractant‐2 (CINC‐2) were quantified. Paw pressure threshold (PPT) was determined before and after intraplantar and subcutaneous injection of CRF with or without naloxone. PMN depletion was achieved by intravenous injection of an antiserum. Chemokines were blocked by intraplantar injection of anti‐MIP‐2 and/or anti‐KC antiserum. We found that at 2 h post CFA (i) intraplantar but not subcutaneous injection of CRF produced dose‐dependent and naloxone‐reversible antinociception (P<0.05, ANOVA). (ii) Opioid‐containing leukocytes in the paw and CRF‐induced antinociception were reduced after PMN depletion (P<0.05, t‐test). (iii) Opioid‐containing leukocytes mostly expressed CXCR2. MIP‐2 and KC, but not CINC‐2 were detectable in inflamed but not in noninflamed tissue (P<0.05, ANOVA). (iv) Combined but not single blockade of MIP‐2 and KC reduced the number of opioid‐containing leukocytes and peripheral opioid‐mediated antinociception (P<0.05, t‐test; P>0.05, ANOVA). In summary, in early inflammation peripheral opioid‐mediated antinociception is critically dependent on PMN and their recruitment by CXCR2 chemokines.

Tissue-Destructive Macrophages in Giant Cell Arteritis

Giant cell arteritis (GCA) is an inflammatory vasculopathy in which T cells and macrophages infiltrate the wall of medium and large arteries. Clinical consequences such as blindness and stroke are related to arterial occlusion. Formation of aortic aneurysms may result from necrosis of smooth muscle cells and fragmentation of elastic membranes. The molecular mechanisms of arterial wall injury in GCA are not understood. To identify mechanisms of arterial damage, gene expression in inflamed and unaffected temporal artery specimens was compared by differential display polymerase chain reaction. Genes differentially expressed in arterial lesions included 3 products encoded by the mitochondrial genome. Immunohistochemistry with antibodies specific for a 65-kDa mitochondrial antigen revealed that increased expression of mitochondrial products was characteristic of multinucleated giant cells and of CD68+ macrophages that cluster in the media and at the media-intima junction. 4-Hydroxy-2-nonenal adducts, products of lipid peroxidation, were detected on smooth muscle cells and on tissue infiltrating cells, in close proximity to multinucleated giant cells and CD68+ macrophages. Also, giant cells and macrophages with overexpression of mitochondrial products were able to synthesize metalloproteinase-2. Our data suggest that in the vascular lesions characteristic for GCA, a subset of macrophages has the potential to support several pathways of arterial injury, including the release of reactive oxygen species and the production of metalloproteinase-2. This macrophage subset is topographically defined and is also identified by overexpression of mitochondrial genes. Because these macrophages have a high potential to promote several mechanisms of arterial wall damage, they should be therapeutically targeted to prevent blood vessel destruction.

Endogenous peripheral antinociception in early inflammation is not limited by the number of opioid-containing leukocytes but by opioid receptor expression

&NA; Endogenous inhibition of inflammatory pain is mediated by leukocytes that secrete opioid peptides upon exposure to stress (cold water swim stress, CWS) or after local injection of corticotropin releasing factor (CRF). Since in early inflammation few opioid‐containing leukocytes are detected and since peripheral opioid‐mediated antinociception is low we examined whether antinociception could be augmented by increased recruitment of opioid‐containing polymorphonuclear cells (PMN). Rats were intraplantarly (i.pl.) injected with Freunds complete adjuvant (FCA) and with the PMN‐recruiting chemokine macrophage inflammatory protein‐2 (MIP‐2, 1–10 &mgr;g; control: saline) for 2 h. Intraplantar leukocytes were quantified by flow cytometry. Paw pressure threshold (PPT) was determined before and after exposure to CWS, i.pl. injection of CRF and opioid peptides. Opioid receptors (OR) were measured by binding studies in dorsal root ganglia (DRG) and by immunohistochemistry in the paw. Our studies showed that (i) MIP‐2 injection dose‐dependently augmented recruitment of PMN and opioid‐containing leukocytes (5‐fold increase in cells/paw, P<0.05), (ii) PPT was not different between groups at baseline and after CWS or CRF (maximum MPE: 20±2.3–29±7.2%, P<0.05), (iii) injection of opioid peptides dose‐dependently increased the PPT (P<0.05, maximum MPE: and 18±2.6–21±3.6%), (iv) MOR (&mgr; OR, MOP) binding sites in the ipsilateral DRG were unchanged (24±2–22±1.2 fmol/mg protein, P>0.05, ANOVA) and (v) the number of MOR and DOR (&dgr; OR, DOP) stained nerve fibers in peripheral tissue were unaltered (both P>0.05, t‐test). In summary, antinociception during early inflammation is apparently not limited by the number of opioid‐containing leukocytes but by OR availability.

Pain and the immune system

In inflammation, leucocytes containing opioid peptides migrate into the tissue. Opioid peptides can be released and bind to opioid receptors on peripheral nerve terminals, which counteracts inflammatory pain. Migration of opioid peptide-containing leucocytes is controlled by chemokines and adhesion molecules. Neurokinins, such as, substance P also contribute to the recruitment of these cells. Opioid peptide release from granulocytes can be stimulated by chemokines, such as, CXCR2 ligands. The release is dependent on intracellular calcium and activation of phosphoinositol-3 kinase and p38 mitogen activated kinase. Endogenous opioid peptides produced by leucocytes not only confer analgesia but recent evidence supports the concept that they also prevent the development of tolerance at peripheral opioid receptors. This review presents the discoveries that led to the concept of analgesia produced by immune-derived opioids.